Immunochemical Techniques: Immunochemical Techniques Part A

Contributors to V o l u m e 70 Article numbers are in parentheses following the names o f contributors. Affiliations lis...

Author: Nathan P. Kaplan | Nathan P. Colowick | Helen Van Vunakis | John J. Langone

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Contributors to V o l u m e 70 Article numbers are in parentheses following the names o f contributors. Affiliations listed are current.

FRANK L. ADLER (30), Division of Immu-

BERNARD F. ERLANGER (4), Department of

nology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 LOUISE T. ADLER (30), Division of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 AUGUSTIN BAER (31), Station .fdddrale de Recherches Laitikres, 3097 LiebefeldBern, Switzerland

W1GGO FISCHER-RASMUSSEN (22), Depart-

SARA BAUMINGER (7), Institute of Repro-

WARREN D. GEHLE (27), Litton-Bionetics,

Microbiology, Columbia University Health Sciences Center, New York, New York 10032 ment of Obstetrics and Gynecology, KObenhavns Kommunes, Hvidovre Hospital, University of Copenhagen, DK2650 Copenhagen-Hvidovre, Denmark Kensington, Maryland 20795

ductive Endocrinology, Municipal Governmental Medical Center, Tel Aviv, and Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel

C. N. HALES (24), Department of Clinical

Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QR, England

HUGH J. CALLAHAN (2), Department of BiD-

H. J. HANSEN (20), Roche Research Center,

chemistry, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Hoffmann-La Roche Inc., Nutley, New Jersey 07110

SYLVIA R. CHALKLEY (21), Department of

MA~OR1E R. HEPBURN (16), Department of

Child Health, Westminster Children's Hospital, London SWIP 2NS, England

Pathology, University of Michigan, Ann Arbor, Michigan 48109

M. CHANTLER (5), Wellcome Reagents Limited, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, England T. CHARD (18), Department of Reproductive Physiology, Joint Unit of Obstetrics and Gynaecology and Reproductive Physiology, St. Bartholomew's Hospital Medical College and The London Hospital Medical College, London ECI, England J. W. COEEEY (20), Department of Pharmacology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 S. L. COMMEREORD (14), Medical Department, Brookhaven National Laboratory, Upton, New York 11973 FRANK J. DIXON (ll), Department o f l m munopathology, Scripps Clinic and Rd'search Foundation, La Julia, California 92037

B. A. L. HURN (5), Wellcome Reagents

SHIREEN

Limited. Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, England W|LLIAM P. JENCKS (31), Department of

Biochemistry, Brandeis University, Waltham, Massachusetts 02154 ELVIN A. KABAT (1), Departments of Mi-

crobiology, Human Genetics and Development, Neurology and the Cancer Center, Columbia University, New York, New York 10032 JOHN J. LANGONE (13, 25), Laboratory of

lmmunobiology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 JENS LARSEN (22), Department of Obstetrics and Gynecology, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark

EVA ENGVALL (28), La Julia Cancer Re-

LAWRENCE LEVINE (31), Department of

search Foundation, La Julia, California 92037

Biochemistry, Brandeis University, Waltham, Massachusetts 02154 ix

X

CONTRIBUTORS

MICHAEL G. MAGE (6), Protein Chemistry

Section, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2O205

TO V O L U M E 70 A. RENSHAW (21), Division of Clinical

Chemistry, Clinical Research Centre, Harrow, Middlesex HAl 3UJ, England ROBERT T. RUBIN (23), Division of Biological Psychiatry, Department of PsychiaH. L. J. MAK1N (19), Steroid Laboratory, try, Harbor-UCLA Medical Center, Department of Chemical Pathology, The Torrance, California 90509 London Hospital Medical College, LonE. R. SAUERZOPE (20), Roche Research don El 2AD, England Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 PAUL H. MAURER (2), Department of BiDchemistry, Thomas Jefferson University, SEYMOUR I. SCHLAGER (15), Laboratory of Philadelphia, Pennsylvania 19107 Immunobiology, National Cancer Institute, National Institutes of Health, BePATRICIA J. McCONAHEY (I1), Department thesda, Maryland 20205 of Immunopathology, Scripps Clinic and Research Foundation, La Julia, Cali- MORTON B. SIGEL (23), Lutcher Brown fornia 92037 Center for Diabetes and Endocrinology, Scripps Clinic and Research Foundation, J. MERRETT (26), RAST Allergy and Research La Julia, California 92037 Unit, Benenden Chest Hospital, Cranbrook, Kent TNI7 4AX, England KENDALL O. SMITH (27), Department of Microbiology, The University of Texas Health T. G. MERRETT (26), RASTAllergy and ReScience Center, San Antonio, Texas 78284 search Unit, Benenden Chest Hospital, B. DAVID STOLLAR (3), Department of BioCranbrook, Kent TN17 4AX, England chemistry and Pharmacology, Tufts UniM. E. MEYERHOFF (29), Department of versity School of Medicine, Boston, MassaChemistry, University of Michigan, Ann chusetts 02111 Arbor, Michigan 48109 BARBARA B. TOWER (23), Division of BioA. REES MIDGLEY, JR. (16), Department of logical Psychiatry, Department of PsyPathology, University of Michigan, Ann chiatry, Harbor-UCLA Medical Center, Arbor, Michigan 48109 Torrance, California 90509 MARTIN MORRISON (12), Department of BiDD. J. H. TRAEEORD (19), Steroid Laborachemistry, St. Jude Children's Research tory, Department of Chemical Pathology, Hospital, Memphis, Tennessee 38101 The London Hospital Medical College, WlLLIAM D. ODELL (17), Department of London El 2AD, England Medicine, University of Utah College of HELEN VAN VUNAKIS (10), Department of Medicine, Salt Lake City, Utah 84132 Biochemistry, Brandeis University, WalJACQUES OUDIN (9), Institut Pasteur, tham, Massachusetts 02154 28 rue du Docteur Roux, 75015 Paris, W. P. VANDERLAAN (23), Lutcher Brown France Center for Diabetes and Endocrinology, RUSSELL E. POLAND (23), Division of BioScripps Clinic and Research Foundation, logical Psychiatry, Department of PsyLa Julia, California 92037 chiatry, Harbor-UCLA Medical Center, J. P. VANDEVOORDE (20), Roche Research Torrance, California 90509 Center, Hoffmann-La Roche Inc., Nutley, - New Jersey 07110 G. A. RECHNITZ (29), Department of Chemistry, University of Delaware, ME1R WILCHEK (7), Department of BioNewark, Delaware 19711 physics, The Weizmann Institute of Science, Rehovot, Israel MORRIS REICHLIN (8), Veterans Administration Medical Center, Departments of J. S. WOODHEAD (24), Department of MediMedicine and Biochemistry, State Unical Biochemistry, Welsh National School versity of New York at Buffalo School of of Medicine, Heath Park, Cardiff CF4 Medicine, Buffalo, New York 14215 4XN, Wales

Preface Immunochemical procedures provide an important supplement to the battery of available chemical and instrumental methods and can often yield new information not readily obtainable in other ways. Antibodies are extraordinary analytical reagents since they can have specificity for macromolecules (proteins, nucleic acids, and polysaccharides) as well as for small molecules belonging to almost every chemical class. In a field that is moving so rapidly, an exhaustive compilation of immunochemical techniques is neither possible nor practical. Our purpose is to provide the investigator with significant examples and sufficient background information so that he can properly assess and adapt these techniques to his research. This is the first of several volumes to be devoted to the description and application of immunochemical techniques. It deals with the basic principles of antigen-antibody reactions, production of reagent antibodies, as well as with purification and characterization of antibodies and antigens. Antibodies to individual compounds can be used with tracer molecules to develop sensitive, specific, rapid, and reproducible immunoassays. General procedures required to develop suitable radioimmunoassays and solid phase immunoradiometric assays are described in detail. Immunoassays that utilize means of detection other than radioactivity are included. Such assays are becoming increasingly popular because they possess the desirable attributes of good analytical methods yet avoid the generation of radioactive waste. Already in progress is a second volume that will supplement the topics presently covered and include other important techniques. Innovative and practical applications of immunochemical methods have been described in other volumes of this series. We have avoided duplication so far as possible, and have included a cross-reference bibliography for each section (see pp. 481-484) to direct the reader to related papers in other volumes. Subsequent volumes will be involved with the development and application of immunoassays for specific compounds as well as for different classes of compounds. We are grateful to the authors for their contributions and to the staff of Academic Press for invaluable assistance. Carla Langone is to be commended for her competent handling of the correspondence. Timely advice and constructive criticism were given by Nathan O. Kaplan and Sidney P. Colowick. Although this is the seventieth volume in the Methods in Enzymology Series, their enthusiasm, interest, and concern remain undiminished. HELEN VAN VUNAKIS JOHN J. LANGONE

xi

METHODS IN ENZYMOLOGY EDITED BY Sidney P. Colowick and Nathan O. Kaplan VANDERBILT UNIVERSITY

DEPARTMENT OF CHEMISTRY

SCHOOL OF MEDICINE

UNIVERSITY OF CALIFORNIA

NASHVILLE, TENNESSEE

AT SAN DIEGO LA JOLLA, CALIFORNIA

I. II. III. IV. V. VI.

Preparation and Assay of Enzymes Preparation and Assay of Enzymes Preparation and Assay of Substrates Special Techniques for the Enzymologist Preparation and Assay of Enzymes Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques VII. Cumulative Subject Index

xiii

METHODS IN E N Z Y M O L O G Y EDITORS-IN-CHIEF S i d n e y P. C o l o w i c k

N a t h a n O. K a p l a n

VOLUME VIII. Complex Carbohydrates

Edited by ELIZABETHF. NEUFELD AND VICTOR GtNSBURG VOLUME IX. Carbohydrate Metabolism

Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation

Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure

Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B) Edited by LAWRENCEGROSSMAN AND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle

Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids

Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids

Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions

Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALD B. MCCORMICKAND LEMUEL D. WRIGHT XV

xvi

METHODS IN ENZYMOLOGY

VOLUME XIX. Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND

VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques

Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A)

Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)

Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B)

Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXlI. Biomembranes (Part B)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

METHODS IN ENZYMOLOGY

xvii

VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX

Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAMB. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B)

Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C)

Edited by W. A. WOOD VOLUME XLIII. Antibiotics

Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes

Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B)

Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling

Edited by WILLIAM B. JAKOBY AND MEre WILCHEK

xviii

METHODS

IN E N Z Y M O L O G Y

VOLUME XLVII. Enzyme Structure (Part E)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFE VOLUME XLIX. Enzyme Structure (Part G)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C)

Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A. HOFEEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations)

Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME LIII. Biomembranes (Part D: Biological Oxidations)

Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME LV. Biomembranes (Part F: Bioenergetics)

Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics)

Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence

Edited by MARLENEA. DELUCA VOLUME LVIII. Cell Culture

Edited by WILLIAM B. JAKOBY AND IRA H. PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)

Edited by KIV1E MOLDAVE AND LAWRENCE GROSSMAN VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

METHODS IN ENZYMOLOGY

VOLUME 61. Enzyme Structure (Part H)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanisms (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanisms (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E)

Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA

Edited by RAY Wu VOLUME69. Photosynthesis and Nitrogen Fixation (Part C)

Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME 71. Lipids (Part C) (in preparation)

Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D) (in preparation)

Edited by JOHN M. LOWENSTEIN

xix

[1]

PRINCIPLES

[1]

OF

Basic Principles

ANTIGEN--ANTIBODY

REACTIONS

of Antigen-Antibody

3

Reactions

By ELVlN A. KABAT Definitions

Antigen: An antigen is any substance that, when introduced parenterally into an animal, will induce the formation of antibodies. The antibody formed is generally found in serum or other biological fluids and should react with the antigen used to induce its formation. The term immunogen is often used to apply to substances that will induce a state of cellmediated immunity as well as the formation of antibodies regardless of their specificity. It is possible to increase immunogenicity by introducing groups onto proteins that do not alter their specificity or become part of an antigenic determinant. Antibody: A serum protein belonging to the family called immunoglobulins. There are five classes of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. These are all built of two chains--heavy and light chains (see Figs. 1 and 4). Antibodies may be found in the serum of normal individuals, presumably as a consequence of contact with antigens by the oral, respiratory, enteric, or parenteral routes. Many antibodies result from apparent or inapparent infections with microorganisms. It is generally thought that all immunoglobulin molecules in normal serum are antibodies resulting from contact with unknown antigens. The capacity to form the repertoire of 105 to 107 different antibody specificities is generally considered to be genetically determined in all vertebrates. During embryonic development lymphocyte precursors differentiate, the process ending with each lymphocyte having the capacity to form one kind of antibody combining site. Synthesis and secretion of antibody on contact with antigen is a highly regulated process involving interactions between different types of lymphocytes that may enhance or suppress their maturation into antibody-secreting plasma cells. Hapten: A substance, generally of low molecular weight, that, when injected, does not induce the formation of antibodies; it can react with antibodies induced to it when it is coupled to a protein, polypeptide, or other substance to form an antigen. It is also used to describe an antigenic determinant, that portion of a complete antigen which enters into the combining site of antibodies, the formation of which the antigen containing it has triggered. Lectin: A plant or animal protein having a receptor site specific for a sugar or oligosaccharide unit. METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181970-1

4

PRINCIPLES AND METHODS

[1]

General Considerations It was only when a basic understanding of the nature of antigen-antibody interaction became clear in studies begun over half a century ago by Michael Heidelberger and his school, 1-e that it became possible to apply these insights in a conceptual manner to the development of the highly sensitive array of analytical immunochemical methods now available to molecular and cellular biologists, clinicians, and scientists in other disciplines. Although the principles originally involved quantitative studies of the precipitin and agglutination reactions using the heterogeneous populations of antibodies generally produced in animals by hyperimmunization, 5-1° they have since found direct application to the study of the interactions of lectins 1H3 with polysaccharides and glycoproteins, of immunoglobulins with protein A of staphylococcus 14 and to the monoclonal antibodies present in sera of humans with multiple myeloma or Waldenstr6m's macroglobulinemia ~5 and of BALB/c and NZB mice with plasmacytomas induced with mineral 0iU6-2°; they will soon be applied to

1 M. Heidelberger, Chem. Rev. 24, 323 (1939). 2 M. Heidelberger, Bacteriol. Rev. 3, 49 (1939). 3 M. Heidelberger, "Lectures in Immunochemistry." Academic Press, New York, 1956. 4 E. A. Kabat and M. M. Mayer, "Experimental Immunochemistry," 1st ed. Thomas, Springfield, Illinois, 1948. 5 E. A. Kabat, "Kabat and Mayer's Experimental Immunochemistry," 2nd ed. Thomas, Springfield, Illinois, 1961. e E. A. Kabat, "Structural Concepts in Immunology and Immunochemistry," 2nd ed. Holt, New York, 1976. 7 M. Sela, ed., "The Antigens," Vol. l (1973); Vol. 2 (1974); Vol. 3 (1975); Vol. 4 (1977). Academic Press, New York. 8 C. A. Williams and M. W. Chase, eds., "Methods in Immunology and Immunochemistry," Vol. 1 (1967); Vol. 2 (1968); Vol. 3 (1971); Vol. 4 (1977); and Voi. 5 (1976). Academic Press, New York. a j. Garvey, N. E. Cremer, and D. H. Sussdoff, "Methods in Immunology," 3rd ed. Benjamin, Reading, Massachusetts, 1977. 10 D. M. Weir, "Handbook of Experimental Immunology" Vol. 1, "Immunochemistry," 3rd ed. Blackwell, Oxford, 1978. 11 I. J. Goldstein and C. E. Hayes, Adv. Carbohydr. Chem. Biochem. 35, 127 (1978). 12 E. A. Kabat, J. Supramol. Struct. 8, 79 (1978). 13 M. E. A. Pereira and E. A. Kabat, Crit. Rev. Immunol. 1, 33 (1979). 14 G. Mota, V. Ghetie, and J. Sj6qvist, Immunochemistry 15, 639 (1978). 15 R. J. Slater, S. M. Ward, and H. G. Kunkel, J. Exp. Med. 101, 851 (1955). 18 M. Potter, Adv. Immunol. 25, 141 (1977). 17 E. A. Kabat, Adv. Protein Chem. 32, 1 (1978). 18 M. Leon, N. M. Young, and K. R. Mclntyre, Biochemistry 9, 1023 (1970). 19 j. Cisar, E. A. Kabat, J. Liao, and M. Potter, J. Exp. Med. 139, 159 (1974). zo C. P. J. Glaudemans, Adv. Carbohydr. Chem. 31, 313 (1975).

[1]

PRINCIPLES OF A N T I G E N - A N T I B O D Y

REACTIONS

5

hybridomas (pages 34-35). 2m~ Quantitative precipitin reactions and inhibition by haptens of quantitative precipitin reactions and more sensitive methods, such as competitive binding assays, have become indispensable to the elucidation of the topology of the specific receptor sites on monoclonal antibodies, lectins, and other biologically active proteins. Indeed, in the absence of X-ray crystallographic data they offer perhaps the only approach to the understanding of the specificities of antibodies and lectins. To the extent that X-ray crystallographic s t u d i e s ~3-32, ec 16,17 have been carried out on these substances, they corroborate fully the inferences as to site size and structure made from the immunochemical studies. The earliest analyses of washed specific precipitates of hemoglobin and its antibody were carried out in Peking by H. Wu et al.,33 who showed that both hemoglobin and antibody were contained in the precipitate. A thorough examination of the course of the precipitin reaction was made by Heidelberger and Kendall,34-36 who first studied the reaction of the nitrogen-free specific capsular polysaccharide of the type III pneumococcus with horse type III antipneumococcal sera and subsequently several protein-antiprotein reactions 37,3s,el. 1-6 and established that antigen and anti21 G. K6hler and C. Milstein, Eur. J. lmmunol. 6, 511 (1976). 22 F. Meichers, M. Potter, and N. L. Warner, eds., "Lymphocyte Hybridomas. Second

Workshop on Functional Properties of Tumors ofT and B Lymphocytes." Springer-Verlag, Berlin and New York, 1978. 23 M. Schiffer, R. L. Girling, K. R. Ely, and A. B. Edmundson, Biochemistry 12, 4260 (1973). 2~ A. B. Edmundson, K. R. Ely, R. L. Girling, E. E. Abola, M. Schiffer, F. A. Westholm, M. D. Fausch, and H. F. Deutsch, Biochemistry 13, 3816 (1974). 25 R. J. Poljak, L. M. Amzel, H. P. Avey, B. L. Chen, R. P. Phizackerly, and F. Saul, Proc. Natl. Acad. Sci. U.S.A. 70, 3305 (1973). 2e D. M. Segal, E. A. Padlan, G. H. Cohen, S. Rudikoff, M. Potter, and D. R. Davies, Proc. Natl. Acad. Sci. U.S.A. 71, 4298 (1974). 2r O. Epp, P. Colman, H. Fehlhammer, W. Bode, M. Schiffer, and R. Huber, Eur. J. 'Biochem. 45, 513 (1974). 2s D. R. Davies, E. A. Padlan, and M. Segal, Contemp. Top. Mol. Immunol. 4, 127 (1975). 29 E. A. Padlan, Q. Rev. Biophys. 10, 35 (1977). 30 F. A. Saul, L. M. Amzei, and R. J. Poljak, J. Biol. Chem. 253, 585 (1978). 31 K. W. Hardman and C. F. Ainsworth, Biochemistry 15, 1120 (1976). 32 j. W. Beeker, G. N. Reeke, Jr., B. A. Cunningham, and G. M. Edelman, Nature (London) 259, 406 (1976). 3a H. Wu, L. H. Cheng, and C. P. Li, Proc. Soc. Exp. Biol. Med. 25, 853 (1927). M. Heidelberger and F. E. Kendall, J. Exp. Med. 50, 809 (1929). 35 M. Heidelberger and F. E. Kendall, J. Exp. Med. 55, 555 (1932). 36 M. Heidelberger and F. E. Kendall, J. Exp. Med. 61,563 1935). 37 M. Heidelberger and F. E. Kendall, J. Exp. Med. 62, 697 (1935). 3a E. A. Kabat and M. Heidelberger, J. Exp. Med. 66, 229 (1937).

6

PRINCIPLES AND METHODS

[1]

body combine in multiple proportions. Unlike the usual reactions of simpler compounds that combine in multiple proportions, forming complexes of defined composition, the multivalence of the antigen and the bior multivalence of the antibody or lectin resulted in a smooth curve when increasing quantities of antigen (or macromolecule) were added to a given quantity of antibody (or lectin, etc,). This is the typical quantitative precipitin curve.37, cf. 1-6 It should be remembered that these studies were carried out before anything was known of the valence of antibody and antigen and that the bi- or multivalence of antibody and the multivalence of antigen were key assumptions. Multivalence of antigen is now recognized as a consequence of the occurrence of 1. Repeating units of linear polysaccharides 2. Multiple terminal nonreducing sequences in branched polysaccharides 3. Projecting sequences of amino acids in synthetic polypeptides or of nucleotides in polynucleotides, DNA, or RNA ct 7 4. Various groups of known structure introduced chemically onto polysaccharides, polypeptides, or proteins cf. 5-10 5. Surface patches arising conformationally in various molecules, notably polypeptides39-42 and p r o t e i n s , 43,44 a s helices,/3 sheets, turns, and connecting random coils el"45 Also of importance in understanding antigen-antibody interations are 6. The existence of several different antigenic determinants on the surface of macromolecules such as proteins 43"44 and on complex glycoproteins such as the water-soluble blood group glycoproteins6"46"4r; these often are found in more than a single copy per molecule. Antibodies may be formed to the various antigenic determi39 E. A. Kabat, J. lmmunol. 97, 1 (1966). 4o j. W. Goodman, Immunochemistry 6, 139 (1969). 41 j. W. Goodman, in "The Antigens" (M. Sela, ed.), Vol. 3, p. 127. Academic Press, New York, 1975. 42 M. Sela, Science 166, 1365 (1969). 43 M. J. Crumpton, in "The Antigens" (M. Sela, ed.), Vol. 2, p. 1. Academic Press, New York, 1974. 44 M. Z. Atassi, ed., "Immunochemistry of Proteins," VoE 1 (1977); Vol. 2 1978. Plenum, New York. 45 R. E. Dickerson and I. Geis, "Structure and Action of Proteins." Harper, New York, 1969. 46 E. A. Kabat, in "Chemistry of Carbohydrates in Solution" (H. S. Isbell, ed.), Am. Chem. Soc. Adv. Chem. Ser. 117, 334 (1973). 4r T. Feizi, E. A. Kabat, G. Vicari, B. Anderson, and W. L. Marsh, J. lmmunol. 106, 1578 (1971).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

7

nants giving rise to complex populations of antibodies of different specificities (heterogeneity of antibodies). 7. The occurrence on cells, cell membranes, and liposomes of glycolipid, glycoprotein, and lipoproteins, which provide multiple repeats of a given antigenic determinant and lead to aggregation reactions such as agglutination ,6 and of movement in the cell membrane on interaction with antibody with formation of patches and caps of antigen-antibody aggregates on individual cells. 4a It should be remembered that accessibility to the antibody or lectin combining site is an absolute requirement for an antigenic determinant or for the carbohydrate moiety to react with the lectin and that they must therefore be at the surface of the molecule. Denaturation of proteins, uncoiling of helical polypeptides, proteins, and glycoproteins, unwinding of double helices or of synthetic polynucleotides, dissociation of quaternary structure or partial enzymic digestion 4a'5° often expose new antigenic 51,52 determinants. Much useful information is obtainable from such studies. Determinants inaccessible 51-53 in the native structure are often termed hidden antigenic determinants. The assumption of bi- or multivalence of antibodies has been amply verified. 6'~ IgG, I g E ) s and presumably IgD are bivalent; electron micrographs show that IgA may exist as a bivalent molecule or as a tetravalent dimer, and IgM in various species may be a tetramer, 56'5Tpentamer) 5 or hexamer 5s with valences of 8, 10, and 12, respectively, although all of these may not be available for reaction with antigen simultaneously. Bivalence of IgG has been established by equilibrium dialysis, 59,6° fluores-

4s R. B. Taylor, P. H. Duffus, M. C. Raft, and S. de Petris, Nature (London) New Biol. 233, 225 (1971). 49 C. Lapresle, Ann. Inst. Pasteur 89, 654 (1955). 5o M. Raynaud and E. H. Relyfeld, Ann. Inst. Pasteur 97, 636 (1959). 51 C. Lapresle and J. Durieux, Ann. Inst. Pasteur 92, 62 (1957); 94, 38 (1958). 52 C. Lapresle and J. Durieux, Bull. Soc. Chim. Biol. 39, 833 (1957). 5a C. K. Osterland, M. Harboe, and H. G. Kunkel, Vox Sang. 8, 133 (1963). D. Beale and A. Feinstein, Q. Rev. Biophys. 9, 135 (1976). 55 S.-E. Svehag, in "Specific Receptors, Antibodies, Antigens, and Cells" (Third Int. Convocation on Immunol. Buffalo, N.Y., 1972), p. 80. Karger, Basel, 1973. 5~ E. M. Shelton and M. Smith, J. Mol. Biol. 54, 615 (1970). 57 R. T. Action, P. F. Weinheimer, S. J. Hall, W. F. Niedermeyer, E. Shelton, and J. C. Bennett, Proc. Natl. Acad. Sci. U.S.A. 68, 107 (1971). 58 R. M. E. Parkhouse, B. A. Askonas, and R. R. Dourmashkin, Immunology 18, 575 (1970). 5g H. N. Eisen and F. Karush, J. Am. Chem. Soc. 71, 363 (1949). •0 F. Karush, J. Am. Chem. Soc. 79, 3380 (1957).

8

PRINCIPLES AND METHODS

[1]

PAPAIN SPL|TS

F~G. 1. Schematic view of four-chain structure of human IgGx molecule. Numbers on right side are actual residue numbers in protein Eu. eT,eaNumbers of Fab fragment on left side are aligned for maximum homology; light chains are numbered as in Wu and Kabat e9 and Kabat and Wu. TM Heavy chains of Eu have residue 52A and 3 residues 92A, B, C; they lack residues 100A, B, C, D, E, F, G, H, and 35A, B. Thus residue 110 (end of variable region) is 114 in actual sequence. Hypervariable regions or complementarity-determining segments or regions (CDR) are shown by heavier lines. VL and Va: light chain and heavy chain variable region; Cnl, Ca2, and C,3: domains of constant region of heavy chain; CL: constant region of light chain. Hinge region, in which two heavy chains are linked by disulfide bonds, is indicated approximately. Attachment of carbohydrate is at residue 297. Arrows at residues 107 and l l0 denote transition from variable to constant region s. Sites of action of papaln and pepsin and locations of a number of genetic factors are given. Modified from Kabat 7~ in Kabat.17

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

9

cence quenching, el ultracentrifugation,~ enzymic cleavage with papain ~ and pepsin ~ and by X-ray crystallographice5 and electron microscopic ~ studies. These together with sequencing studies have led to an understanding of the three-dimensional structure of IgG (Figs. 1-3).°7 - n Figure 4 73-75 shows that all classes of immunoglobulins are built with a structure similar to that of human IgG, the multimeric forms of IgA and IgM having additional SH units and a third chain, the J chain, TM which serves to link them.

Sizes and Shapes of Antigenic Determinants A n t i b o d y c o m b i n i n g sites c o m p r i s e o n e o f t h e m o s t u n i q u e r e c o g n i t i o n s y s t e m s k n o w n . T h e t o t a l i t y o f t h e d a t a i n d i c a t e t h a t an a n t i b o d y c o m b i n ing site m a y b e o b t a i n e d t h a t c a n r e c o g n i z e a n y k i n d o f o r g a n i c c o m p o u n d r a n g i n g in size f r o m a l o w e r limit o f a b o u t 4 - 6 / ~ , to an u p p e r limit o f a b o u t 3 4 / ~ , in m o l e c u l a r w e i g h t f r o m p e r h a p s 200 to a b o u t 1000, a n d in a n y s h a p e o r c o n f o r m a t i o n t h a t s u c h an a m o u n t o f m a t t e r c a n a s s u m e . 6,39-41 A n t i b o d y c o m b i n i n g sites as d e t e r m i n e d f o r a n t i p o l y s a c c h a r i d e anti-

8~ S. F. Velick, C. W. Parker, and H. N. Eisen, Proc. Natl. Acad. Sci. U.S.A. 46, 1470 (1960). 62 H. K. Schachman, L. Gropper, S. Hanlon, and F. Putney, Arch. Biochem. Biophys. 90, 175 (1963). R. R. Porter, Biochem. J. 73, 119 (1959). A. Nisonoff, F. C. Wissler, and D. L. Woernley, Biochem. Biophys. Res. Commun. 1, 318 (1959). e5 V. R. Sarma, E. W. Silverton, D. R. Davies, and W. D. Terry, J. Biol. Chem. 246, 3753 (1971). R. C. Valentine and N. M. Green, J. Mol. Biol. 27, 615 (1967). e7 G. M. Edelman, B. A. Cunningham, W. E. Gall, P. D. Gottlieb, U. Rutishauser, and M. J. Waxdai, Proc. Natl. Acad. Sci. U.S.A. 63~ 78 (1969). ea G. M. Edelman, Biochemistry 9, 3197 (1970). eg T. T. Wu and E. A. Kabat, J. Exp. Med. 132, 211 (1970). 70 E. A. Kabat and T. T. Wu, Ann. N.Y. Acad. Sci. 190, 382 (1971). rl E. A. Kabat, in "Specific Receptors, Antibodies, Antigens, and Cells" (Third Int. Convocation Immunol., Buffalo, N.Y., 1972))' p. 4. Karger, Basel, 1973. T~E. M. Silverton, M. A. Navia, and D. R. Davies, Proc. Natl. Acad. Sci. U.S.A. 74, 5140 (1977). 7s j. A. Gaily, in "The Antigens" (M. Sela, ed.), Vol. 1, p. 161. Academic Press, New York, 1973. 74 B. Frangione, in "Immunogenetics and Immunodeficiency." Univ. Park Press, Baltimore, Maryland, 1975. 75 F. W. Putnam, G. Florent, C. Paul, T. Shinoda, and A. Shimizu, Science 182, 287 (1973). r6 M. E. Koshland, Adv. Immunol. 20, 41 (1975).

FIG. 2. Stereoview of the three-dimensional structure of human IgG myeloma protein Dob. The smaller circles represent a-carbon atoms; the larger circles represent carbohydrate hexose units. The Fab arms of the molecule are aligned vertically, and a horizontal twofold axis of symmetry bisects the molecule through the Fc. In this view the light chain is in the foreground of the upper Fab, and the heavy chains in the foreground of the lower Fab compare with Fig. 1. From Silverton et al. TM

FIG. 3. Space-filling view of the Dob IgG molecule. One complete heavy chain is white, and the other is clark gray; the two light chains are lightly shaded. The large black spheres represent the individual hexose units of the complex carbohydrate. In this view the twofold axis of symmetry is vertical. A crevasse is seen between Ca2 of the white heavy chain and the CL domain of the Fab on the left. From Silverton et al. TM

[1]

PRINCIPLES OF A N T I G E N - A N T I B O D Y REACTIONS Human IgG z Mouse Ig2b

Human IgG 1

Human IgG4, IgAt IgAz, A2m(2 )

Human IgG 3

Guinea pig IgG2 Mouse IgGz,

Rabbit IgG

Human IgA2, A2m(I ) Balb/c Mouse IgA

Human IgD

11

Human IgM Human IgA Dimer

,$

I

~

I-I

t

~

FIG. 4. Chain structure and disulfide bonding patterns in immunoglobulins. IgG, IgD, and IgA monomers are from Gaily73; IgM and IgA dimers are based on Frangione74; - - S - - S - bonds of IgM after Putnam et al. 75 From Kabat. a

bodies may be grooves or cavities 77-sz depending upon whether terminal nonreducing ends or internal linear sequences are recognized. Binding of the antigenic determinant is noncovalent and involves hydrophobic and hydrogen bonding, charge interaction, etc., the total binding energy being due to the sum of such interactions and the fit of the antigenic determinant r7 j. Cisar, E. A. Kabat, M. M. Dorner, and J. Liao, J. Exp. Med. 142, 435 (1975). 7a K. Takeo and E. A. Kabat, J. lmmunol. 121, 2305 (1978). 79 A. Wu, E. A. Kabat, and M. G. Wiegert, Carbohydr. Res. 66, 243 (1978). so G. Schepers, Y. Blatt, K. Himmelsbach, and I. Podit, Biochemistry 17, 2239 (1978). al L. G. Bennett and C. P. J. Glaudemans, Carbohydr. Res. 72, 315 (1979). 8, W. Schalch, J. K. Wright, L. S. Rodkey, and D. G. Braun, J. Exp. Med. 149, 923 (1979).

12

PRINCIPLES AND METHODS

[1]

in the antibody combining site. e,83"~ Binding is usually measured as an association constant, Ks, the values varying over a wide range for different antigenic determinants and even for a single antigenic determinant; association constants tend to be of the order of about 104 to 108 M -1 for carbohydrate d e t e r m i n a n t s 77,Ta,8°-a2,84-s7 although a proportion of the antibody in antisera to the group-specific carbohydrate of the streptococcus has been found to have a Ka of 109.82,88,89The Ka value may reach 10l° or more for hydrophobic structures, such as the dinitrophenyl group, 84 for digoxin and fluorescein, and for proteins, such as insulin?° Antigenic determinants of proteins generally have Ka values in the range of 105 to 10s.91-94 For a detailed analysis and additional references, see Karush. s4 To the extent to which they have been compared, enzyme sites and antibody combining sites cover comparable ranges of sizes and shapes. The relative contribution of each sugar to the binding has been measured for the lysozyme site. 95,96 The lysozyme site has been found by X-ray crystallography97 to be a groove accommodating the hexasaccharide of the bacterial cell wall built on alternating N-acetylmuramic acid and Nacetyl-D-glucosamine residues; the myeloma antidextran QUPC52 has also been found to be a groove complementary to an internal chain of six a-(I-->6) linked glucoses, isomaltohexaose. 1s,77 Both these sites are at about the upper limit in size. At the other end of the scale, glycosidases may split a terminal sugar, and antibodies 9s,99,ef-e,39-41 and lectinsef. H-13.100.10~ may react with a single sugar unit plus a portion of the second sugar. s3 F. Karush, Adv. Immunol. 79, 3380 (1962). 84 F. Karush, in "Immunoglobulins" (S. Litman, G. Ward, and R. A. Good, eds.), p. 85. Plenum, New York, 1978. a5 j. W. Kimball, lmmunochemistry 9, 1169 (1972). a6 J.-C. Jaton, H. Huser, W. F. Riesen, J. Schlesinger, and D. Givol, J. Immunol. 116, 1363 (1976). a7 D. G. Strcefkirk and C. P. J. Glaudemans, Biochemistry 16, 3760 (1977). s s j. K. Wright, W. Schalch, L. S. Rodkey, and D. G. Braun, FEBS Lett. 93, 317 1978). s9 W. Schalch, J. K. Wright, L. S. Rodkey, and D. G. Braun, Fur. J. Immunol. 149, 923 (1979). 9o S. Berson and R. S. Yalow, J. Clin. Invest. 38, 1996 (1956). 91 D. H. Sachs, A. N. Schechter, A. Eastlake, and C. B. Anfinsen, Biochemistry 11, 4268 (1972). W. B. Dandliker and S. A. Levison, lmmunochemistry 1, 165 (1968). 93 N. Sakato, H. Fujio, and T. Amano, Biken J. 14, 405 (1971). 94 I. Pecht, E. Maron, R. Arnon, and M. Sela, Fur. J. Immunol. 19, 368 (1971). 95 j. A. Rupley, Proc. R. Soc. London Ser. B 167, 416 (1967). D. M. Chipman, V. Grisaro, and N. Sharon, J. Biol. Chem. 242, 4388 (1967). 97 D. C. Phillips. Sci. Am. 215 (11), 78 (1966). a s y . Arakatsu, G. Ashwell, and E. A. Kabat, J. Immunol. 97, 858 (1966). A. M. Staub and R. Tinelli, Bull, Soc. Chim. Biol. 42, 1637 (1960).

[1]

PRINCIPLES OF A N T I G E N - A N T I B O D Y

REACTIONS

13

T h e Quantitative Precipitin Curve At the time that the quantitative precipitin curve was originally developed, 1-6,34-3s analyses of the washed specific precipitates were carried out by the micro-Kjeldahl procedure and the working range was about 0.10-1.0 mg of total N. Improvements in analytical methods in the intervening half century have reduced the quantities needed per sample to about 1-6/.~g of total N in the precipitate, 1°2 analyses on the washed precipitate being carried out after a Kjeldahl-type digestion followed by the ninhydrin reaction. 1°3 It is doubtful whether smaller amounts can be used, since the "solubility"l°3~ of p01ysaccharide-antibody precipitates at 0 ° is appreciable, values being 0.6-1.0/~g of N per milliliter for horse; 0.71.8/~g of N per milliliter for human, and 3-7/~g of N per milliliter for rabbit antibodies; for protein-antiprotein precipitates, values are 3-10 ftg of N per milliliter. 5 Polysaccharide-lectin precipitates 1°4,1°5 fall into the same range. Thus, in carrying out quantitative precipitin determinations with an upper range of 6 - 8 ftg of N of Ab, lectin, etc., per determination, it is important to work in total volumes of 0.5 ml or less to minimize these effects and to wash the precipitates with small volumes of saline at 0°. Since the reaction mixtures are left at 0 - 4 ° for 5 - 7 days for equilibrium to be reached, and since for most purposes comparative data are needed, results tend to be quite reproducible, but they may be low by an amount corresponding to the "solubility" effect plus small losses in washing due to solubility of the specific precipitate. Quantitative

Precipitin

Curves

Procedure. 5,102,103Determinations on a micro scale are performed in 3ml conical Pyrex centrifuge tubes. A volume of antiserum (containing antibody or myeloma antibody or crude seed extract or purified lectin) previously centrifuged until it no longer deposits sediment and containing about 6 - 8 ~g of antibody N (AbN) or lectin N in a volume of about 50100/.d is added to tubes containing a suitable range of accurately mea-

K. D. Hardman, in "Carbohydrate-Protein Interaction" (I. J. Goldstein, ed.), Am. Chem. Soc. Syrup. Ser. 88, 12 (1979). ~o~ M. Sarkar, J. Liao, E. A. Kabat, T. Tanabe, and G. Ashwell, J. Biol. Chem. 254, 3170 (1979). 102 E. A. Kabat and G. Schiffman, J. lmmunol. 88, 782 (1962). loa G. Schiffman, E. A. Kabat, and W. Thompson, Biochemistry 3, 113 (1964). ~oza ,, Solubility" refers to the effects of carrying out the quantitative precipitin assays with the same amounts of antigen and antibody but varying the total volume with saline. ~04 M. E. Etzler and E. A. Kabat, Biochemistry 9, 869 (1970). ~o5 M. E. A. Pereira, E. A. Kabat, and N. Sharon, Carbohydr. Res. 37, 89 (1974). 1oo

14

PRINCIPLES AND METHODS

[1]

sured quantities of the antigen or substance reacting with lectin. The total volume is adjusted with saline. The contents of each tube are mixed, and the tubes are placed at 37° for 1 hr (or at room temperature with lectins) and then at 4°. The contents of the tubes are mixed twice daily. After 5-7 days they are centrifuged in a refrigerated centrifuge, the supernatants are decanted, and the precipitates are allowed to drain thoroughly with the tubes inverted on a towel and leaning against a rack. The precipitates are washed with 0.5-ml portions of saline (0.9% NaCI solution) at 0°. They are then analyzed for N by the ninhydrin method after a Kjeldahl-type digestion with H2SO4. l°a For work with cold agglutinins47'1°~ all reagents are chilled; the setup is made in an ice bath, and the tubes are kept in ice water for the entire time before washing the precipitates; washing of the precipitates is performed in a cold room. This is also necessary when carrying out quantitative precipitin determinations using horse antisera and cross-reacting polysaccharides s because of their much higher solubility with increasing temperature. Ninhydrin Procedure for Quantitative Precipitin and Quantitative Inhibition Determinations l°a

Reagents 1. Digestion mixture: 1 ml of concentrated H2SO4 diluted to 20 ml 2. H~O2, 30% 3. Standard (NH4)2SO4 solution: 30.0 mg of (NH4)2SO4 in 3.00 ml of distilled water. This stock solution when diluted 1 : 10 for routine use contains 212 ~g of N per milliliter. 4. Sodium acetate buffer, 4 M pH 6.5:136 g of NaAc.3H~O are dissolved in 100 ml of H20 in a hot water bath and allowed to cool; 25 ml of glacial acetic acid are added, and the volume is made to 250 ml with distilled H~O. Adjust to pH 6.5 with NaOH. Store at 4° without preservative. The saturated solution must be warmed to dissolve NaAc before use. 5. KCN, 10 mM 6. Ninhydrin solution: 160 mg of ninhydrin are dissolved in 3 ml of ethylene glycol monomethyl ether plus 1 ml of 4 M acetate buffer, pH 6.5. 7. Prepared just before use by adding 100/~1 of reagent 5 to 4 ml of reagent 6. 1oe M. Heidelberger and A. C. Aisenberg, Proc. Natl. Acad. Sci. U.S.A. 39, 453 (1953).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

15

Procedure. Add 25/zl of reagent 1 to each washed specific precipitate in the conical 3-ml centrifuge tubes. (NI-L)2SO4 standards containing 2, 3, and 4 p,g of N are set up at the same time. The samples are digested in a sand bath at 160° for 90 min. Cool, add 15/zl of 30% H20~ to each tube, and redigest the samples in the sand bath at 160°C for an additional 90 minutes. Cool, add 200/~1 of distilled water and 100 tzl of reagent 7 to each tube. Mix gently and place tubes in a water bath at 95° for 20 min. Cool. Add 1.5 ml of 50% ethanol and transfer to a 10-ml volumetric flask; wash tube twice with 1.5 ml of 50% ethanol and bring to the mark with 50% ethanol. Read absorption at 570 nm. The conditions of digestion and assay are chosen so that equal absorption values on a molar basis are given by (NH4)2SO4 and amino acids. Figure 5 and the table give representative data on the precipitation reaction of crystalline hen egg albumin with rabbit anti-egg albumin as studied in 1935 by Heidelberger and Kendall. 3r They provide more insight into the general course of the precipitin reaction than is often seen with the currently used, more micro, procedure given in the preceding paragraph. Not only was the precision substantially greater, solubility effects generally being negligible, but it was standard practice to examine supernatants from each of duplicate determinations for the presence of antigen or antibody. This was generally accomplished by adding antibody or antigen to a portion of each supernatant and examining the tubes for precipitate. Three zones, were recognized: a zone of antibody excess, an equivalence zone in which neither antigen nor antibody was detectable, and a zone of antigen excess. The point on the curve at which free antigen was first seen corresponded to the point of maximum precipitation: with larger excesses of antigen, the amount of antigen-antibody precipitate decreased owing to the formation of soluble complexes, and this was termed the inhibition zone. Tests on supernatants are often carried out by gel diffusion techniques that correlate well with the usual tests and may give improved sensitivity.5,1°7 With the more micro method using 6 - 8 / z g of antibody N per determination and the keeping of tubes in the refrigerator for a week, supernatant tests are often not practical, since the small quantities of precipitate would not be seen. For most purposes one is interested in determining specificity differences by comparing various precipitin curves one with another. With polysaccharide antigens of high molecular weight, and with systems involving IgM antibody, the inhibition zone is reached only with amounts of antigen many times those required for maximum precipitation, whereas with protein antigens, with carbohydrate antigens of lower molecular weight, and with IgG antibodies, inhibition by 1o7 j. Munoz and E. L. Becker, J. lmmunol. 65, 47 (1950).

16

PRINCIPLES AND METHODS

[1]

Ratio AbN: EaN in precipitate

,.=

o 0

< p~ 0 ,.G

:o ~

If

///,, E

o

.-,_

A

a~

~f f '6

z

\ol!

°°"

d

/

m

iV

g~

0 o

--

o

0,/, ~~\. oo

o

~

(6#) peze|!d!0eJd N

o o~=

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

17

ADDITION OF INCREASING AMOUNTS OF EGG ALBUMIN TO 1.0 ML OF A 1:2 DILUTION OF RABBIT ANTISERUM TO EGG ALBUMIN AT 0 °a'b

EaN added

(~g) 9.1 15.5 25 40 50 65 74 82 90 98 124 135 195 307 490

Antibody Total N by Ratio AbN: EaNpptd Npptd difference EaN in (~g) (p.g) (/~g) precipitate Total Total Total Total Total Total Total Total 87 89 87 [72]~ [48] [4]

156 236 374 526 632 740 794 830 826 820 730 610 414 106 42

147 220 349 486 582 675 720 748 739 731 643 [538] [366]

Antibody Npptd calculated from equation Tests on supernatant (p.g)

16.2 14.2 14.0 12.2 11.6 10.4 9.7 9.1 8.5 8.2 7.4 7.5 [7.6]

137 225 343 499 582 677 714 738 746

Excess Ab Excess Ab Excess Ab Excess Ab Excess Ab Excess Ab No Ab or Ea No Ab, 1/zg of EaN Excess Ea Excess Ea Excess Ea Excess Ea Excess Ea Excess Ea Excess Ea

a Data from M. Heidelberger and F. E. KendallY 0 Ab, antibody; Ea, egg albumin. c Values in brackets are considered to be uncertain.

excess antigen occurs much more rapidly. Further details, representative data, curves, and procedures may be found in Kabat 5 and in Maurer. l°s This review will concentrate on more recent studies using quantitative precipitin data, especially those leading to insights about structure and to the development of more sensitive methods o f assay using immunochemical reagents. With certain horse antitoxins and antiprotein sera '°a-m and in some patients with Hashimoto's thyroiditis 112 who have antibodies to thyroglobulin, one finds a different type o f quantitative precipitation curve, termed a flocculation curve, of"5.6 Precipitation occurs only o v e r a narrow range, and soluble a n t i g e n - a n t i b o d y complexes are formed in the region o f antibody excess as well as of antigen excess. Flocculation curves have 10s p. H. Maurer, in "Methods in Immunology and Immunochemistry" (C. A. Williams and

M. W. Chase, eds.), Vol. 3, p. 1. Academic Press, New York, 1971. los A. M. Pappenheimer, Jr. and E. S. Robinson, J. I m m u n o l . 32, 291 (1937). Ho D. Gitlin,C. S. Davidson, and L. H. Wetterlow, J. Immunol. 63, 291 (1949). m E. H. Relyveld and M. Raynaud, Ann. Inst. P a s t e u r 96, 537 (1959). n2 I. M. Roitt, P. N. Campbell, and D. Doniach, Biochem. J. 69, 248 (1958).

18

PRINCIPLES AND METHODS

[1]

not been used for investigations of antigenic structure and will not be considered. With various kinds of antisera, the course of the precipitin reaction has been described by the equation AbN precipitated =

ax-

(1)

bx 2

where x is the amount of antigen or antigen N added and a and b are constants that differ from one antiserum to another. This equation was found empirically by Heidelberger and Kendall, 34 who subsequently derived it from the law of mass action? e' of. 1-3.H4 Dividing both sides by x gives (AbN)/x in the precipitate

=

a

-

bx

(2)

This is the equation of a straight line in which the ratio of AbN to antigen or antigen N in the precipitate is plotted against x; a is the intercept on the Y axis and - b is the slope (Fig. 5). The equation generally holds throughout the antibody excess region and equivalence zone up to the point of maximum precipitation and was quite useful, especially in comparing various antisera to the same antigen in view of the heterogeneous populations of antibody molecules formed. For systems involving a single antigen and its homologous antibodies, it has been shown that all the added antigen is precipitated throughout the antibody excess and equivalence zones and up to the point of maximum precipitation. 1-e With some homogeneous myeloma proteins and with lectins, one often finds a straight line, but with others the lines are not straight. The need for the equation for homogeneous antibodies from myelomas or hybridomas and for lectins is reduced, since if one uses a given amount of different preparations of the same myeloma antibody or of the same lectin the precipitin curves with the same antigen or glycoprotein are identical. IgA monomeric antibodies often do not precipitate with macromolecular antigens, apparently because of limited flexibility of their hinge, and thus may be effectively monovalent. As such they may under suitable circumstances attach to specific precipitates, and under other conditions they may inhibit precipitation of IgA polymer or of IgG antibodies; these phenomena were described H~,~6 long before the classes of immunoglobulins were recognized. The antibody was termed nonprecipitable antibody and was separated from the precipitating antibody by successive small additions of antigen, a procedure that competitively favored removal of precipitating antibody leaving the nonprecipitating antibody in the superna113 L, Pauling, D. H. Campbell, and D. Pressman, Physiol. Rev. 23, 203, (1943). 114 F. E. Kendall, Ann. N.Y. Acad. Sci. 43, 85 (1942). 115 A. M. Pappenheimer, Jr., J. Exp. Med. 71, 263 (1940). l~e M. Heidelberger, H. P. Treffers, and M. M. Mayer, J. Exp. Med. 71, 271 (1940).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

19

tant. 4,5,3s Nonprecipitable antibody was initially hypothesized to be univalent; subsequent studies showed that the nonprecipitable fraction of equine antibody to p-azophenylactoside, which migrated as an IgA, had a valence of two, as determined by equilibrium dialysis against a small hapten, and a sedimentation constant of 7 S; the binding constants of the precipitating and nonprecipitating antibodies were of comparable affinities. 117 It was therefore proposed that the two sites were so distributed that when one site was occupied by a large antigen molecule the other site was sterically unable to react. The 7 S subunit of IgA mouse myeloma MOPC315 did not precipitate with DNP coupled to protein, nor did it agglutinate sheep erythrocytes to which DNP groups had been coupled.llS,119

More recent studies ~2° have shown that the 7 S subunit of MOPC315 would bind only one molecule of DNP-dextran of molecular weight 44,000, containing but one DNP group, but would bind two molecules of DNP coupled to ~-aminocaproic acid. Affinity labeling of varying proportions of DNP sites, measurement of residual sites by equilibrium dialysis, calculation of the theoretical valence for binding to the mw 44,000 univalent DNP dextran, and comparison with the valence observed, satisfied a steric model but not the alternative possibilities of an asymmetric T M or an allosteric model. Similar conclusions had also been drawn ~22for the IgM subunit (IgMs) of a human Waldenstrtm macroglobulin, Lay, which bound only one molecule of IgG. The observed valence of an IgM antibody decreased from 10 to 5 as the molecular weight of the antigen used to measure it increased from 342 to 7100 while the valence of IgG remained unchanged. 12z It is of interest that an IgG1K protein, Dob, TM had a 15-residue deletion in its hinge region, but from the X-ray data this did not seem to affect its flexibility; an explanation for the inability of the 7 S IgA and IgM subunits to bind more than a single macromolecule must await X-ray crystallographic studies on one of these proteins. Estimation of the Total Antibody Content of Antisera For some purposes, one's primary interest may not be in the quantitative precipitin curves as such, but one may wish merely to determine the 117 N. Klinman, J. H, Rockey, and F. Karush, Science 146, 401 (1964). 11s H. N. Eisen, E. S. Simms, and M. Potter, Biochemistry 7, 4126 (1968). 119 M. Potter, Physiol. Rev. 52, 631 (1972). 120 R. Eisenberg and P. Plotz, Biochemistry 17, 480 (1978). m S. I. Chavin and E. C. Franklin, Ann. N.Y. Acad. Sci. 168, 84 (1969). 122 M. J. Stone and ~ . Metzger, J. Biol. Chem. 243, 5977 (1968). 123 S. C. Edberg, P. M. Bronson, and C. J. Van Oss, lmmunochemistry 9, 273 (1972).

20

PRINCIPLES AND METHODS

[1]

antibody content of the antisera. This was originally done by locating the point of m a x i m u m precipitation (Fig. 5) and setting up one or m o r e determinations in this range (for details see K a b a t and MayerS). In practice, h o w e v e r , it is easier to couple the antigen to an i m m u n o a d s o r b e n t , 124"~2~ to add an amount sufficient to r e m o v e all the antibody, mix thoroughly, centrifuge, wash, elute the antibody with acid, and determine the quantity spectrophotometrically. This minimizes solubility effects and permits assays on a much smaller scale; using 100-/zl samples of serum, satisfactory results were obtained with antisera containing about 25 ~g of antib o d y per milliliter (6/~g o f A b N per milliliter); care must be taken to use excess i m m u n o a d s o r b e n t . E s t i m a t i o n of A n t i g e n F r o m Fig. 5 it m a y be seen that with a given antiserum the total N in the precipitate is a function of the quantity o f antigen N added. This relationship for each antiserum serves as a calibration curve, which can be used to determine the quantities of antigen in biological fluids. Thus the antiserum in Fig. 5 could be used to determine quantitatively the a m o u n t of egg albumin in egg white. It is only n e c e s s a r y to prepare and add to the m e a s u r e d volume o f antiserum a suitable dilution of the egg white such that the egg albumin it contains is sufficient to give an amount o f washed specific precipitate N falling in the antibody excess region o f the curve. The total N found is interpolated on the c u r v e to obtain the a m o u n t of egg albumin N in the volume of egg white dilution used. It is evident from examining the curve in Fig. 5 that two points would be found, one in the inhibition zone and one in the antibody excess zone. To be sure that one is getting the correct results, one must be certain that the determination was set up in the region of excess antibody; this was routinely established by tests on the supernatants as described above. On the microscale now used it would be necessary to set up several points to be sure that one was in the zone of antibody excess. Determinations o f antigen by the quantitative precipitin method were used routinely for m a n y years in s o m e institutions for the estimation of IgG in human cerebrospinal fluid, increases in cerebrospinal fluid IgG being found in multiple sclerosis and in neurosyphilis. 126-12s In recent 124A. E. Gurvich, R. B. Kapner, and R. S. Nezlin, Biokhimiya 24, 144 (1959); English translation, p. 129. 125T. J. Gill and C. F. Bernard, lmmunochernistry 6, 567 (1969). 126E. A. Kabat, M. Glusman, and V. Knaub, A m . J. Med. 4, 653 (1948). 12~E. A. Kabat, D. A. Freedman, J. P. Murray and V. Knaub, A m . J. Med. Sci. 219, 55 (1950). 128M. D. Yahr, S. S. Goldensohn, and E. A. Kabat, Ann. N . Y . Acad. Sci. $8, 613 (1954).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

21

years more rapid micro procedures, such as competitive binding and fluorometric methods, have essentially replaced quantitative precipitin assays.

Use of Quantitative Precipitin Curves for Structural Insights Figure 6 shows results of quantitative precipitin determinations carried out under these conditions with four antidextran myelomas--three specific for a-(1-~6) linked dextrans W3434, W3129, and QUPC52 and the fourth, UPC102, for a-(l-*3) linked dextrans. 19 The various proportions of a-(1--~6), a-(1-~3)-like, and a-(1--~4)-like linkages in the dextrans are given129,la°; the terms (1-~3)-like and (1--,4)-like are used, since a glucose residue substituted on C-2 and C-4 will not take up periodate and will behave as though it were (1---3) linked and glucoses substituted either on C-2 or on C-4 will behave equivalently, each taking up 1 mol of periodate. When the actual proportions of each linkage are known from methylation T M or other s t u d i e s , 1a°,132,133 these values are often revised. The curves in Fig. 6 for the different myeloma proteins provide certain important insights into the uses of such quantitative data. The analytical precision of the method is clearly seen, in that several of the different dextrans give the same curve. In Fig. 6A for W3434, it is clear that the values for B1299 Fr.S, Bl141, B512, and B1424 all fall on one curve and values for B1498 Fr.S and B1355 Fr.S fall on a second curve. The better reacting groups are those with no or with low a-(1---~3)-like linkages, whereas the poorer groups are high in a-(1---~3)-like linkages. N150N, with a much lower molecular weight than B512 but of the same general structure, reacts much more poorly. The best dextran, B1399, gives somewhat higher results than the first group; this dextran is like the group reacting next best and having high a-(1--~4)-like linkages; these linkages have been shown to be largely a - ( l ~ 2 ) from measurements of optical rotation of the cuprammonium complexes 13zand by the isolation of kojibiose, ~33o-Glc-c~(1---~2)-D-Glc. With W3129, the difference between B1399 and the better reacting group of dextrans is not seen; N150N precipitates about 50% of the total ~9 A. Jeanes, W. C. Haynes, C. A. Wilham, J. C. Rankin, E. H. Melvin, M. J. Austin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya, and C. E. Rist, J. Am. Chem. Sco. 76, 5041 (1954). ~30 R. L. Sidebotham, Adv. Carbohydr. Chem. Biochem. 30, 371 (1974). ~3~ F. R. Seymour, M. E. Slodki, R. D. Plattner, and A. Jeanes, Carbohydr. Res. 53, 153 (1977). ~3~ T. A. Scott, N. N. Hellman, and F. R. Senti, J. Am. Chem. Soc. 79, 1178 (1957). ~zz H. Suzuki and E. H. Hehre, Arch. Biochem. Biophys. 104, 305 (1964).

22

[1]

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[1]

PRINCIPLES OF ANTIGEN--ANTIBODY

REACTIONS

23

AbN and the poorer reacting group of dextrans are more effective in precipitating relative to the better group than was seen with W3434. These differences suggest that the two myeloma antidextrans differ somewhat in their combining sites. QUPC52 can readily be seen to be quite different (Fig. 6C) from the other two myeloma antidextrans in that B512 and B1255, which have low proportions of non or-(1---->6)linkages, are very much better in precipitating than are the dextrans with higher proportions of non-a-(1---~6) linkages. QUPC52 differs from W3129 in that it precipitates well TM with a synthetic linear dextran T M whereas W3129 does not. The ability to precipitate with a linear dextran means that the dextran is multivalent and thus that internal sequences of several sugars must constitute an antigenic determinant. 77,Ta,la5 This, plus additional competitive binding data with oligosaccharide inhibitors that will be discussed later, led to the conclusion that the antibody combining site of QUPC52 must be a groove into which internal chains of a-(1--~6) linked glucoses fit. 77 Two other NZB myeloma antidextrans, PC3858 and PC3936, TM also precipitate with the synthetic linear dextran and thus also have groove type sites although their sites appear to be smaller than that of QUPC52. Fluorescence quenching measurements by Bennett and Glaudemans 81 have shown that monovalent Fab fragments of W3129 bind only to the terminal nonreducing ends of dextran molecules of molecular weight 36,000 and give the same Ka per terminal nonreducing end, as does isomaltopentaose. 77 The degree of precipitation with the synthetic linear dextran as compared with the usual native dextrans has been of value in examining antidextrans 77 produced in humans, 'zT"or. ~,6 and in rabbits 98''a6 and has shown that the heterogeneous populations of antidextran may be mixtures of molecules with specificities directed toward internal chains of a-(1---~6) linked residues as well as those with specificities directed toward terminal nonreducing ends of chains. Fractions of such antibodies separated by isoelectric focusing were found to differ in the proportions of their antibodies precipitable by the synthetic linear dextran. 77 Antibodies specific for the terminal nonreducing ends as well as for internal sequences of the group-specific A variant carbohydrate of the streptococcus, a polymer of L-rhamnose with ct-(1---~2) and a-(1---~3) linkages, have also been found in rabbit antisera. 82 Unlike the findings with human antidextran, the binding constants of the antibodies specific for internal sequences were of higher Ka than those directed toward the termi,34 E. R. Ruckel and C. S c h u e r c h , Biopolymers 5, 515 (1967). ,35 W. Richter, Int. Arch. Allergy 46, 438 (1974). ,3s I. M. O u t s c h o o r n , G. Ashwell, F. Gruezo, and E. A. K a b a t , J . lmmunol. 113, 896 (1974). ,37 E. A. Kabat and D. Berg, J. lrnmunol. 70, 514 (1953).

24

PRINCIPLES AND METHODS

[1]

hal nonreducing ends, and this was ascribed to hydrophobic interactions with the CH3 group of C-6 of rhamnose. It is of interest that the terminally specific antidextran ~7'7s and antistreptococcal group A variant molecules s2 had similar Ka for their respective determinants. The existence of linear polysaccharides that are antigenic, such as pneumococcal SIII, S V I I I , sS's6,138-14° S V I a , 141'142 essentially demand the existence of groove-type sites. The fourth myeloma antidextran (Fig. 6D) has an entirely different specificity. The precipitin curves show that it reacts best with three dextrans, B1498, B1355 FR.S, and B1501 FR.S, high in a-(1--*3)-like linkages. A second group, B1399 FR.S., B1255, B742 FR.C, precipitated about half as well, whereas a third group gave very little precipitate. This, plus inhibition data (see later), indicates that the specificity involves a(1-->3) linkages. Another a-(1---~3) specific IgM myeloma, MOPC104E, TM was found not to react at all with dextran B512 with 96% a-(1---~6) linkages, to react best with dextran B1355 FR.S, and to react less well with dextrans with smaller proportions of a-(1---~3) linkages; however, it also reacted with dextrans having high o~-(1---~2) and o~-(1---~4)linkages (Fig. 7). All of these precipitated the same maximum quantity of antibody, unlike the curves in Fig. 6D. Myeloma antibodies to fructosans could be divided into two groups: those specific for/3-(2--->1)-linked fructosans as evidenced by the ability of inulin, a linear/3-(2--> 1) linked polymer, to precipitate, and those specific for/3-(2--->6) linkages as seen from the reaction with ryegrass levan, a linear polymer of/3-(2-->6) linkages. The/3-(2--* 1) specific myeloma proteins did not react with ryegrass levan and the fl-(2--,6) specific myeloma proteins did not react with inulin. '9,a7,79 If one compares Figs. 6 and 7, it is apparent that with the IgA myelomas individual dextrans precipitate different total quantities of antibody N, whereas with the IgM antidextran myelomas all dextrans that can react, regardless of structure, will precipitate all the myeloma antidextran. This difference is ascribable to the presence.in the IgA myelomas of monomers and polymers, the monomers partially inhibiting precipitation as discussed earlier. This distinctive behavior of IgA myelomas holds for antifructosans.~7 With the IgG2a antifructosan myeloma UPC 10, all levans tested, including ryegrass levan, precipitated all the antibody when added ,as R. G. Mage and E. A. Kabat, Biochemistry 2, 1278 (1963). ,3a j. K. N. Jones and M. B. Perry, J. Am. Chem. Soc. 79, 2787 (1957). ~4o A. M. Pappenheimer, Jr., W. P. Reed, and R. Brown, J. Immunol. 150, 1237 (1968). ~41 p. A. Rebers and M. Heidelberger, J. Am. Chem. Soc. 81, 2415 (1959). m M. Heideiberger and P. A. Rebers, J. Bacteriol. 80, 145 (1960).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY I

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in suitable amounts, but with an IgA antifructosan myeloma Y5476, ryegrass levan precipitated but half of the maximum antibody; the patterns of precipitation of other levans fell into groups with different maximum amounts of precipitable antibody. 77 However, with IgA NZB antifructosan myeloma 3660, all levans tested including ryegrass levan precipitated about the same amount TM of N --- 15%; this result would be explainable if the proportion of IgA monomers in this myeloma was relatively small. Although myeloma antibodies provide a substantial advantage for structural studies of antigenic determinants in that they contain homogeneous combining sites, nevertheless the specificities available are limited. Only one myeloma with antiprotein specificity has been found 143,el. 16; it is 14a M. Smith and M. Potter, J. I r n m u n o l . 114, 1847 (1975).

26

PRINCIPLES AND METHODS

[1]

specific for flagellin, but it has not been studied immunochemically. Also the preponderance of IgA myelomas in the mouse may make for complications in interpretation due to IgA monomer and polymer mixtures in varying proportions. Most of the structural studies on antigenic determinants have been carried out with antisera produced by immunization and so have been with heterogeneous populations of antibodies. This does not pose problems for structural studies by quantitative precipitin determinations, since precipitin curves for cross-reacting antigens essentially resemble those of Fig. 6 in that most frequently only a fraction of the antibody is precipitable, and such findings are interpreted in terms of structural similarities and differences of the cross-reacting as compared with the homologous antigen. Moreover, most of the antibodies produced by hyperimmunization are mixtures of IgG with some IgM and IgA, the quantity of the IgA antibodies especially in rabbit antisera often being insufficient to inhibit precipitation or distort the shape of the quantitative precipitin curve. One may thus interpret the precipitin curve of a cross-reaction as being due essentially to the presence of a proportion of antibodies having a site capable of accommodating the homologous as well as the crossreacting determinant, the remainder of the antibodies being directed toward determinants that do not cross react. Studies on many types of antigens have amply justified this type of analysis. ~-7 Structural Insights from Quantitative Precipitin Inhibition Assays Much additional understanding of the nature of antigenic determinants has been gained by integrating experimental data obtained by quantitative precipitin assays, as previously described, with quantitative assays of inhibition by low molecular weight haptens of precipitation of antibody by antigen. This procedure was first introduced by Landsteiner, of-144 who showed with azoproteins and their antibodies that the haptenic group and its analogs would, in suitable concentration, enter the antibody combining site and competitively block access of the antigenic determinant. Landsteiner carded out all his assays qualitatively, comparing visually the amount of precipitate in the presence and in the absence of the hapten; varying quantities of hapten were employed, and a qualitative or semiquantitative estimate of relative inhibiting power could be made. Current practice, however, is to utilize the quantitative precipitin technique, 1-e selecting a quantity of antigen and antibody close to the point of maximum 144K. Landsteiner, "The Specificity of Serological Reactions," 2nd ed. Harvard Univ. Press, Cambridge, Massachusetts, 1943. Paperback reprint Dover, New York, 1962.

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY

REACTIONS

27

precipitation and using these quantities plus varying amounts of the inhibitor to be tested. Inhibition assays have been shown to be reversible equilibria in the thermodynamic sense in that the same degree of inhibition is obtained when antibody and antigen are mixed and inhibitor is added as compared with addition of antibody to a mixture of inhibitor and antigen. 14~The latter procedure is the method of choice for routine assays as well as for competitive binding assays. Continuing to use the homogeneous myeloma dextran-antidextran systems as a model, Fig. 8 shows inhibition curves obtained ~9 with various oligosaccharides and the four myeloma antidextrans shown in Fig. 6. It is evident that with W3434 (Fig. 8A) and W3129 (Fig. 8B) IM5 is the best inhibitor, IM6 and IM7 being equal to IM5 on a molar basis, but that IM4 is less potent. The two antidextrans show differences in that with W3434 the tetrasaccharide is definitely better than the trisaccharide while with W3129 they are equal. With the myeloma antidextrans, IM2 is much less active than IM3 and is given on a curve with an abscissa 10-fold greater. The shapes of the inhibition curves are atypical in that they are curves up to about 40% inhibition and are then essentially linear. Such behavior is associated with IgA immunoglobulins that are mixtures of IgA monomer and polymer; separation of the polymer from the monomer fraction (Fig. 8C) results in typical inhibition curves. The other curves in Figs. 8A, B, and C provide additional data as to the site specificity of the myeloma antidextrans. Thus, with W3129, it may be seen that methyl t~-D-Glc is much poorer than IM2 but much better than methyl/3-D-Glc (W3434 was not studied). The a-(I--->6) linkage is essential for the specificity since maltose, D-GIc-a-(1-*4)-D-GIc, and kojibiose, D-Glc-a-(1-->2)-D-GIc, are essentially inactive whereas nigerose, o-Glc-t~-(1-->3)-o-Glc, is much less active than methyl t~-o-Glc. With QUPC52, with a groove-type site, IM6 and IM7 are equal as inhibitors and are better than IM5, with IM4, IM3, and IM2 successively poorer as inhibitors. With the antidextran of o~-(1--~3) specificity UPC102 (Fig. 8E), nigerotriose, D-Glc-a-(1-->3)-o-Glc-a-(1-~3)-o-Glc, is the best inhibitor with nigerotetraose, nigeropentaose, and a mixture of nigerohexa- and heptaoses all being equal to nigerotriose, thus establishing the site size as complementary to the trisaccharide. Again nigerose is much better than maltose, isomaltose, or kojibiose and methyl et-o-Glc is better than methyl/3-0Glc. Two other ot-(1--~3) specific myeloma antidextrans, J55814~ and t4s E. A. Kabat, J. Am. Chem. Soc. 76, 3709 (1954). ~4e A. Lundblad, R. Steller, E. A. Kabat, J. W. Hirst, M. G. Weigert, and M. Cohn, Immunochemistry, 9, 535 (1972).

28

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PRINCIPLES AND METHODS

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[1]

PRINCIPLES OF A N T I G E N - A N T I B O D Y REACTIONS

29

MOPC104E, TM have also been studied. With J558 nigeropentaose and the mixture of nigerohexa- and heptaoses were equal and better than nigerotetraose, which in turn was better than nigerotriose, whereas with MOPC104E nigerotriose was the best inhibitor, the tetra- and pentasaccharities being of equal potency. The principles thus far outlined for the myeloma antidextrans are directly applicable to the exploration of other myeloma antibody and of lectin combining sites. 6,mla Since the specificity of these sites is generally not known, the precision of site mapping is necessarily limited by the available oligosaccharides. These are generally obtained as a by-product of structural investigations on polysaccharides and glycoproteins and to a limited extent by synthesis, el 147.148but such studies are increasing and one hopes that a broader spectrum of oligosaccharides will become available. The quantitative precipitin and quantitative inhibition assays may be carried out on whole ascitic fluid or serum containing the myeloma antidextrans, and in one instance with a crude seed extract containing the lectin of Euonymus europaeus. 149 One must be certain that one is not examining a mixture of lectins; thus seeds of Bandeiraea simplicifolia contain lectins of three distinct specificities. One of these, BS-II, is most specific for D-GIcNAc linked a but also reacts well with N,N'-diacetylchitobiose, 15° whereas the other, BS-I, is a mixture of two isolectins, which can combine in various proportions to give five tetramers: A4, AaB, A2B,, AB3, and B4151; A 4 and B 4 show distinct blood group-related specificities. A4 reacts best with terminal D-GalNAc linked a but also reacts with terminal D-Gal, whereas B4 is strictly specific for D-Gal linked a. For studies of the specificities of these lectins, the lectins obviously must be purified, and indeed the site studies must necessarily be carried out on A4 and B 4 separated from the other isomers. 152 It is of interest that in studies based on a competitive binding assay using various oligosaccharides and with a mixture of the five isolectins, carried out 15a before the existence of the multiple forms were known, TM gave semilog plots of two different slopes, certain oligosaccharides giving one slope and others a different slope. 147 E. A. Kabat, J. Liao, and R. U. Lemieux, lmmunochemistry 15, 727 (1978). 14s T. Feizi, E. Wood, C. Aug6, S. David, and A. Veyfi6res, lmmunochemistry 15, 733 (1978). 149 j. Petryniak, M. E. A. Pereira, and E. A. Kabat, Arch. Biochem. Biophys. 178, 118 (1977). 150 C. Wood, E. A. Kabat, S. Ebisu, and I. J. Goldstein, Ann. lmmunol. (Inst. Pasteur) 129C, 143 (1978). 151 L. A. Murphy and I. J. Goldstein, J. Biol. Chem. 252, 4739 (1977). 152 C. Wood, E. A. Kabat, L. A. Murphy, and I. J. Goldstein, Arch. Biochem. Biophys. 198, 1 (1979). 153 E. C. Kisailus and E. A. Kabat, Carbohydr. Res. 67, 255 (1978).

30

PRINCIPLES AND METHODS

[1]

Such behavior may indicate the possibility that one is dealing with a mixture of lectins. Lectins from the snails Axinella polypoides 'r~ and Aaptos papillata 155each were found to be mixtures of three lectins, two of which were obtained in purified form; the sites of the two lectins from each species showed some differences in specificity, and one of the lectins from Axinella (lectin I) was strongly mitogenic for human peripheral blood T cells and to a lesser extent for human B cells, lr~ Association Constants of Antigen-Antibody, H a p t e n - A n t i b o d y and L i g a n d - L e c t i n Interactions Three procedures are among those most frequently used for determining association constants ee"e for hapten-antibody and ligand-lectin interactions: equilibrium dialysis, la'e°'157 fluorescence quenching or enhancem e n t , 19,15s-16° and affinity electrophoresis. 7s,161-164 Equilibrium dialysis is the most reliable from the thermodynamic standpoint but requires considerable quantities of purified antibody or lectin. Fluorescence quenching or enhancement requires much smaller quantities of purified protein, but determination of the maximum degree of quenching or enhancement is often difficult. Affinity electrophoresis offers the advantage that it may be used with protein mixtures, such as ascitic fluid containing myeloma proteins or crude extracts of lectin, as long as the protein being studied is present in sufficient proportions to be visible as a distinct band in acrylamide gels. It is, however, less precise than the other methods and is usually applied to systems in which the antigen or substance reacting with the lectin is uncharged. It has the additional advantage that it is directly applicable to determination of the binding constant of precipitating macromolecular antigens such as dextran, whereas equilibrium dialysis requires that the ligand be dialyzable and fluorescence quenching may only be used with macromolecular antigens or ligands in systems in which precipitation does not occur as, for example, with the Fab fragment of antibodies. 81 It would 1~ H. Bretting and E. A. Kabat, Biochemistry 15, 3228 (1976). 155 H. Bretting, E. A. Kabat, J. Liao, and M. E. A. Pereira, Biochemistry 15, 5029 (1976). 156 S. G. Phillips, H. Bretting, and E. A. Kabat, J. Immunol. 117, 1226 (1976). 157 I. M. Klotz, in "The Proteins" (H. Neurath and K. Bailey, eds.), 1st ed., Vol. 1B, p. 727. Academic Press, New York, 1953. 158 S. F. Velick, C. W. Parker, and H. N. Eisen, Proc. Natl. Acad. Sci. U.S.A. 46, 1470

(1960). 15, V. Harisdangkul and E. A. Kabat, J. lmmunol. 108, 1232 (1972). l e o D. G. Streefkirk and C. P. J. Glaudemans, Biochemistry 16, 3760 (1977). 161 K. Takeo and S. Nakamura, Arch. Bioehem. Biophys. 153, 1 (1972). lnu V. Horesjf, M. Tich~, and J. Kocourek, Bioehirn. Biophys. Acta 499, 290 (1977). le~ S. Sugii, K. Takeo, and E. A. Kabat, J. lmmunol. 17,3, 1162 (1979). ~ V. Horesjf, M. Tich~, and J. Kocourek, Trends Bioehem. Sei. 4, 6 (1979).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

31

probably be difficult to use affinity electrophoresis with heterogeneous mixtures of antibodies. None of the three methods is useful to determine the quantity of antigen or hapten. In principle, affinity electrophoresis is carded out in polyacrylamide gels. The receptor protein moves to a given position relative to a tracking dye or other protein. Addition of the macromolecular ligand in various concentrations in the gel retards the movement of the receptor protein. If in addition, a small haptenic molecular oligosaccharide is added to the gel, the mobility is restored. From either set of measurements the K a of the receptor-macromolecular ligand or receptor-hapten interaction can be calculated.~61-~ Competitive Binding Assays: Radioimmunoassay and E n z y m e Immunoassay This technique is the most widely used for the estimation of antigens (or antibodies) in biological fluids and often yields precise data at the nanogram or picogram level. In principle, all that is needed is a labeled ligand to be assayed, a protein containing the specific receptor for the ligand, and a way of separating bound from free ligand without disturbing the equilibrium. The ligand may be an antigen, hapten, or glycoprotein reacting with a lectin, a hormone reacting with a receptor protein, etc., and is labeled with a radioactive isotope 165-m or with an enzyme, such as alkaline phosphatase or/3-galactosidase.17a-~76 To standardize and evaluate the system, increasing quantities of the antibody, lectin, or specific receptor macromolecule are added to a constant quantity of labeled ligand

la5 R. Yalow and S. Berson, J. Clin. Invest. 39, 1157 (1960). le~ W. D. Odell and W. H. Daughaday, "'Principles of Competitive Protein-Binding Assays." Lippincott, Philadelphia, Pennsylvania, 1971. 1e7 K. E. Kirkham and W. M. Hunter, "Radioimmunoassay Methods." Williams & Wilkins, Baltimore, Maryland, 1971. l~a R. M. Yalow, Pharmacol. Rev. 25, 161 (1973). la~ C. W. Parker, "Radioimmunoassay of Biologically Active Compounds." Prentice-Hall, New York, 1976. 17o W. M. Hunter, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), Vol. i, Ch. 17. Davis, Philadelphia, 1973. 1rl C. D. Hawker, Anal. Chem. 45~ 878A (1973). 17~ E. Haber and K. Paulsen, in "The Antigens" (M. Sela, ed.), Vol. 2, p. 249. Academic Press, New York, 1974. 1T3 E. Engvall and P. Perlmann, lmmunochemistry 8, 871 (1971). 174 E. Engvall, K. Jonsson, and P. Perlmann, Biochim. Biophys. Acta 251, 427 (1971). 1r5 E. Engvall and P. Perlmann, J. Immunol. 109, 129 (1972). 17e D. R. Hoffman, J. Allergy Clin. lmmunol. 51, 303 (1973).

32

PRINCIPLES

[1]

AND METHODS

(4000-8000 cpm) and allowed to come to equilibrium; the bound ligand is separated from the free ligand, and the proportion of free or of bound ligand is determined. For the system to be suitable, the receptor protein if added in sufficient quantity should precipitate 90-100% of the added ligand as determined by counting or enzyme assay. Once this has been established, the actual estimation of unknown quantities of the substance present in biological systems is by competition of unlabeled and labeled ligand for the receptor sites. This competition follows the equation S* + Ab ~:~ S*Ab + (bound) S SAb

where S* and S represent labeled and unlabeled ligand, and Ab represents antibody or other specific receptor macromolecules. In practice the quantities of S* and Ab corresponding to the point at which 50% of S* would be bound are chosen; increasing quantities of S are added to this quantity of S*, followed by the chosen amount of Ab; the mixture is allowed to come to equilibrium, bound and free ligand are separated, and the quantity of bound or free label is measured. From the quantity of label bound in the presence and the absence of competitor, the percentage of inhibition is calculated according to the formula 1

total cpm added - cpm in supernatant with inhibitor to ic - ads witu¥ or j

×

100

Percentage of inhibition is plotted against log amount of competitor added. This serves as a calibration curve by which the amounts in unknown mixtures may be determined. Suitable quantities of each unknown are added to the quantities of labeled ligand and receptor molecule used to obtain the calibration curve; bound or free label is determined, percentage of inhibition is calculated, and the quantity of ligand is read off from the calibration curve. There are extensive discussions of the theory of protein ligand interaction, 168,169"177-179 and other methods of plotting data have been decribed. 179,1s° Most important for successful assays is the method of separating bound from free ligand. In the original study of Yalow and Berson using human anti-insulin, 165this was accomplished by electrophoresis on paper, ~77 D. 17s D. 17a D. ~s0 F.

Rodbard and H. A. Feldman, this series, Voi. 36, p. 3. N. Orth, this series, Vol. 37, p. 22. Rodbard and G. R. Frazer, this series, Vol. 37, p. 3. W. Dahlquist, this series, Vol. 48, p. 270.

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

33

since it had been noted that free insulin was bound to the paper at the site of application whereas insulin-antibody complexes migrated in an electric field. Numerous other methods have been used, including precipitation by salts and adsorption of free ligand on charcoal. The most widely used and most generally applicable methods for antigen-antibody and ligand-lectin interactions are (a) coupling the receptor macromolecules, antibody, lectin, etc., to an insoluble particle, such as Sepharose; (b) adsorption of the receptor macromolecule at the bottom of a plastic tube, in which the reaction is then carried out; (c) precipitation of the macromolecule with its bound ligand with an antiserum prepared to the macromolecule itself. When the labeled ligand in competitive binding assays is multivalent-for example, is a macromolecule with several determinants--the method is largely independent of the intrinsic binding constant, Ka, for an individual binding site since attachment to the receptor protein will generally involve more than a single determinant, the total binding being expressed as an exponential function of Ka times the number of valences involved. Because of a statistical effect, TM assays with unlabeled monovalent ligands will require relatively higher concentrations for a given degree of inhibition with multivalent, as compared with monovalent, labeled ligands.101 In such systems the increase in sensitivity obtainable by competitive binding as compared with quantitative inhibition of precipitation using oligosaccharides as inhibitors is much smaller than is usually observed when the labeled ligand and the inhibitor have the same valence. 1°~,1s2 Competitive binding assays with monovalent labeled ligands and monovalent inhibitors are extremely dependent upon the Ka of the ligand-antibody bond. If one begins with an initial ratio of bound (B) to unbound (F) of 1, the effective range of sensitivity will be 1/Ka; with Ka of 105 and a ligand of molecular weight 100, B/F of 1 would be at about 1/zg/ml, making the working range in micrograms. Thus, for many lectins with Ka of 10a or 104, competitive binding assays necessarily require the use of multivalent ligands. In competitive binding assays involving monovalent labeled ligands and monovalent inhibitors the slope on a semilog plot is linear and covers a 2-log range between about 10% and 90% inhibition if binding at one site does not affect binding at a second site; such behavior is termed noncooperative. In both competitive binding assays and in inhibition by monovalent 181 E. A. Kabat, J. Immunol. 77, 377 (1956). 182 M. E. A. Pereira, E. C. Kisailus, F. Gruezo, and E. A. Kabat, Arch. Biochem. Biophys. 185, 108 (1978).

34

PRINCIPLES A N D M E T H O D S

[1]

haptens of precipitation of antibody or lectin by multivalent antigens, polysaccharides, glycoproteins, etc., many of these semilog plots give a comparable range of inhibition over a 1-10g 77'1°1'1°5'154"15~'181-186 rather than over a 2-10g range. Only occasional lectins such as Ulex iectin II ls7 are inhibited by low molecular weight ligands over a 2-10g range. In contrast, however, with multivalent macromolecular inhibitors, such as blood group substances and asialo-orosomucoid, 1°1 inhibition is seen over a 2log range. The basis for these differences is not clear; such differences are usually attributed to cooperative effects, filling of one site facilitating the filling of another. However, there is no independent evidence to support this inference with respect to these systems. The interaction of the multivalent macromolecular antigens may, for steric reasons, not result in all receptor sites being occupied. This has been clearly shown for the interaction of tobacco mosaic virus and its antibody.lSS Moreover, in precipitin inhibition studies, as the hapten displaces more and more antigen from antibody sites, the antigen itself will begin to contribute to the formation of the soluble complexes. When competitive binding radioimmunoassay or enzyme immunoassay are carried out with antisera, which almost always contain a heterogeneous spectrum of antibodies, the dilutions used are generally so high that one is working with only the fraction of antibodies of highest affinity. This usually does not influence the precision of the results. However, if one is studying a group of inhibitors to determine structural relationships, one may find that results would not be identical with respect to relative inhibiting power for a given set of compounds if the same set of assays were carried out using different dilutions of antiserum. Such differences may be difficult to interpret. The use of several hybridomas with monoclonal antibodies of different specificities and Ka to a given antigen would eliminate this difficulty and would contribute to structural analyses of the kinds of antibodies formed to individual antigenic determinants. Hybridomas are obtained z1"22by fusing spleen cells of immunized mice with a myeloma cell line defective in hypoxanthine guanine phosphoribosyltransferase or thymidine kinase, culturing the cells in hypoxanthine-aminopterin-thymidine, conditions under which unfused myeloma cells will die; unfused spleen cells also die off and hybrid clones grow. These are screened, el-23. 1s3 G. Nicolson, J. Blaustein, and M. E. Etzler, Biochemistry 13, 196 (1974). 184 R. A. Poretz and I. J. Goldstein, Biochemistry 9, 2890 (1970). 1s5 I. J. Goldstein, C. E. Hollerman, and E. E. Smith, Biochemistry 4, 876 (1965). ~s6 M. E. A. Pereira, E. A. Kabat, R. Lotan and N. Sharon, Carbohydr. Res. $1, 107 (1976). ~sr M. E. A. Pereira, F. Gruezo, and E. A. Kabat,Arch. Biochem. Biophys. 194, 511 (1979). ~ s s I. Rappaport, J. lrnmunol. 82, 526 (1959).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

35

xs9 and those that are producing antibody are cloned. Each cloned cell line will produce homogeneous antibody; if such cells are injected into a mouse, tumors will grow and secrete antibody. Large quantities of the desired antibody may be obtained from ascitic fluid or from serum. Each hybridoma may produce an antibody of different specificity toward the antigen; a set of different hybridomas will give an estimate of the repertoire of antibody-forming cells to the antigen or antigenic determinant for the conditions of immunization employed. If the myeloma parent itself produces an immunoglobulin, the individual chains of the myeloma and the antibody globulin may associate to form mixed immunoglobulins, many of which will not have antibody activity, then the antibody desired could constitute only a fraction of the total. To avoid this, myeloma lines are being used that express only light chains (cf. M. Scharff 22, ls9) or do not secrete immunoglobulin.190 The latter variety will be most useful if they do not shut off antibody synthesis when hybridized and if they have a high frequency of fusion. A drug-susceptible cell line from the rat has also been used; it secretes only light chains. TM Antidextran antibodies of a-(1---~6) and a-(l-*3) specificity may be assayed in cell lysates by their ability to compete with an ot-(l--->3) myeloma antidextran J558 or the anti-ct-(1---~6) myeloma antidextran W3129 for the respective 14C-labeled a-(1--~3) linked dextrans B1355 and the or-(1-*6) linked dextran B512.192 Nonradioactive dextrans of the appropriate specificity competed with the radioactive dextran for the a-(l---~3) or a-(1---~6) antidextran myelomas over a l-log range although both the labeled and unlabeled dextrans were multivalent. Ka of such antibodies may be determined by affinity electrophoresis.163 Immunochemical Approaches to Structural Studies of Complex Polysaccharides The power of the immunochemical methodologies outlined thus far may best be illustrated by several examples showing how they have been used to define antigenic determinants. One of these is the blood group I - i system. Individuals with a disease termed chronic cold agglutinin disease generally have in their serum a monoclonal IgM autoantibody, active at 4 °, that agglutinates human adult erythrocytes much more strongly than erythrocytes from the umbilical 189 j. Sharon, S. L. Morrison, and E. A. Kabat, Proc. Natl. Acad. Sci. U.S.A. 76, 1420 (1979). 1~0 M. Shulman, C. D. Wilde, and G. Ki~hler, Nature (London) 276, 269 (1978). 191 G. Galfrr, C. Milstein, and B. Wright, Nature (London) 277, 131 (1979). 1~ L. Matsuuchi and S. L. Morrison, J. Immunol. 121,962 (1978).

36

PRINCIPLES AND METHODS

[1]

cord.Cf. 6.193-195 This antibody has been termed anti-I. The blood group I antigen is widely distributed, being present on all but 1 in 10,000-15,000 individuals; the rare individuals lacking this antigenic determinant have another termed i. 195 Water-soluble human ovarian cyst fluids that do not show blood group A, B, H, or Le specificity show high I and i activity. 196-19aThe I and i activities are present in the interior of the water-soluble blood group A, B, H, and Le active substances, from which they can be exposed by removal of the outer tiers of sugars, generally by periodate oxidation and Smith degradation .20oSince the biosynthesis of blood group glycoproteins is generally not complete, substances with A, B, H, and Le activity may also have small numbers of I and i determinantsY Figure 9 shows the latest 13 composite proposed structure 2°1 for the water-soluble A, B, H, and Le a and Le b glycoproteins, and Figure 10 shows the precursor structure 2°2 for the ovarian cyst glycoprotein showing I and i specificity and lacking the other blood group determinants. Anti-I and anti-i sera from different individuals show different specificities and have made possible classification of blood group I antigenic determinants into six groups and blood group i antigenic determinants into four groups. 2°3 The I determinant reacting with anti-I Ma (group 1) has been characterized most extensively 47"147,14sand will be discussed in detail. Another determinant showing some activity with anti-I Step (group 3), anti-I Da (group 5), and anti-I Phi (group 6) and anti-i activity of several groups has also been associated 2°4 with the glycolipid: D-Gal-/3(1--->4)-D-GlcNAc-/3-(1--~3)-D-Gal-fl-(l~4)-D-GlcNAc-fl-(l~3)-D-Gal-fl(1---~4)-D-Glc-fl-(1---~1)-ceramide, termed lacto-N-norhexaosylceramide, when incorporated on liposomes, but the structures involved in each specificity have not been further defined. Activity specific for anti-I Step 193 D. Roelcke, Clin. lmmunol, lmmunopathol. 2, 266 (1974). ~ T. Feizi, in "Human Blood Groups" (Fifth Int. Convocation Immunol.) p. 164. Karger, Basel, 1977, 195 R. R. Race and R. Sanger, "Blood Groups in Man," 6th ed. Blackwell, Oxford, 1975. ~9s W. T. J. Morgan, Proc. R. Soc. London Ser. B 151, 308 (1959). ~a7 W. M. Watkins, in "Glycoproteins" (A. Gottschalk, ed.), 2nd ed., p. 830. Elsevier, New York, 1972. 198 K. O. Lloyd, in MTP Int. Rev. Sci. Org. Chem. Ser. 2, Vol. 7. "Carbohydrates" (G. O. Aspinall, ed.), p. 251. Butterworth, London, 1975. ~aa G. Vicari and E. A. Kabat, J. Immunol. 102, 821 (1969). 2 0 o T. Feizi, E. A. Kabat, G. Vicari, B. Anderson, and W. L. Marsh, J. Exp. Med. 133, 39 (1971). 201 K. O. Lloyd and E. A. Kabat, Proc. Natl. Acad. Sci. U.S.A. 61, 1470 (1968). 202 G. Vicari and E. A. Kabat, Biochemistry 9, 3414 (1970). ~03 T. Feizi and E. A. Kabat, J. Exp. Med. 135, 1247 (1972). 2o4 H. Neimann, K. Watanabe, S. Hakomori, R. Childs, and T. Feizi, Biochem, Biophys. Res. Commun. 81, 1286 (1978).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

37

q ; ¢.~ :t

,1~ ¢¢

T

i

~02

O

e'~

e.. r3

02 t"

0

.6o=.~ 02

v

~02~

•~

02

"O

02

v-~

:¢~02 02

e'~

~

I

l

, ~ 7 .0~2' oo ~ ~33_ I

-.~ eL 02 ~-.

02

e~

~3. I

e~

/

\ o~ ~

'~

02

02 ,.-

02

02 ,.. t..,

v-4 "F

v.-t v i

"7~ ~q7 O~ 02

O'O

02

¢~

e~

02~

38

PRINCIPLES AND METHODS

o

!

t

~

t

~, ~

.~ .~

[l]

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

39

has been associated with band 3, the major membrane protein of erythrocytes, z°5 and various other I and i activities have been associated with certain gangliosides of human erythrocytes. 206 The determinant reacting with anti-I Ma (group 1) is one of the most extensively characterized, using not only oligosaccharides isolated by degradation of the precursor OG substance (Fig. 10), which reacted with all anti-I and anti-i sera, but also with oligosaccharides of various possible alternative structures prepared by chemical synthesis in two laboratories.el. 147,148 Figures 1 1 and 12 show the results obtained in two laboratories, one by inhibition of precipitation 147and the other by radioimmunoassay. 148 In each instance all oligosaccharides containing the structure D-Gal-/3(1-~4)-D-GIcNAc-/i-(1---~6)-CHz- were equally active on a molar basis in reacting with anti-I Ma. The oligosaccharide D-Gal-/3-(1-~4)-D-GIcNAc was about one-tenth as active. Changes in linkages of the above structure all reduced the inhibitory activity. As long as the fl-(1-~6)-CH2- portion was present, reduction of the sugar of which it was a part did not affect the activity. Results by radioimmunoassay and by inhibition of precipitation were identical. The evidence that the determinant is no larger than that given above is indicated by the finding that D-Ga/-~- ( 1 ~ 4)-D-GIcHAc- ~- ( I ~ 6)-v- galactitoI v-Ga1-~- ( 1 ~ 4)-D-GleNAc-~- (I~6)-D-Gal D'GaI"~'(I~4)'D'GIcNAc'~'(I"~6)D Gal or v-ga/actltol D_Ga1.~. (i ~ 3)_v. GIcNAe.¢_ ( I j 3 ) "

and v-Gal-/3-(1--~4)-D-GlcNAc-fl-(l-~6)-N-acetyl-D-galactosaminitol all have the same inhibiting activity on a molar basis. Figure 12 shows that a second anti-I serum of group 1, Woj, gave identical results. Recently synthesis of D-Gal-fl-(1-~4)-D-GlcNAc-fl-(1---~ 6)-D-GaINAc-aO-(CH~)sCOOCH3 D-GaI-fl-(I---~4)-D-GIcNAc-fl-(1 x~

6) 4)D-Gal-/i-O-(CHs)sCOOCHa

and

/1 o-Gal-/3-(1--*4)-o-GlcNAc-~8-(1

showed each to be equivalent in activity to the others in Fig. 11 whereas D-GIcNAc-/3-(1-~6)-D-GaINAc-a-O-(CH2)aCOOCHa was inactive) °°a thus, defining the site unequivocally as complementary to D-Gal-/i-(1--~4)D-GIcNAc-/3-(1-o6)-O-CH~-. Radioimmunoassay required about 4~ as z05 R. A. Childs, T. Feizi, M. Fukuda, and S.-I. Hakomori, Biochem. J. 173, 333 (1978). z06 R. A. Childs, S.-I. Hakomori, and M. E. Powell, Biochem. J. 173, 245 (1978). ~06a E. A. Kabat, J. Liao, R. U. Lemieux, and M. H. Burzynska, in preparation.

40

PRINCIPLES

[1]

AND METHODS

100

z

80

y

6O '1I-Z w

40

20

0

• 0.5

,& 1

2

~

I

I

I

3

4

5

6

MICROMOLES INHIBITOR ADDED

FIG. 11. Plots of the inhibition of the precipitation observed on mixing 15/zl of anti-I Ma (group 1) (30 p,l ofa 1:2 dilution) with 14.5/~g of glycoprotein OG (20% from 10%) in a total volume of 400/zl and using the following inhibitors (Kabat e t a1.147): Compound n u m b e r Symbol

1 2 3 4 5 6 7

[] • A • @ ~ O

n-Gal-fl- ( 1 ~ D-Gal-/3- (I ~ D-Gal-/3- (I ~ o-Gal-/3- ( 1 ~ D-Gal-/3- ( 1 ~ D-Gal-/3- ( i ~ D-Gal.~ - ( 1 ~

4)-D- GIeNAe-fl- ( 1 ~ 6)-D-Gal 4)-D- GIeNAc-/3- (I ~ 3)-D-GaI 3)- D-GIcNAc-fl- ( I ~ 6)-D-Gal 3)-o-GIcNAc-fl- ( l ~ 3 ) - n - G a l 4)-D-GlcNAe-/3- ( 1 ~ 6)-D-Gal-fl- 1- O- (CH~)aCOOCHs 4)-D-GIeNAc-/3- (I ~ 3 ) - o - G a l - f l - I- O- (CH~)aCOOCH~ 3)- D- GIeNAc-/3- ( 1 ~ 6) - D-Gal-/3-1- O--(CHz)aCOOCI-Ia

8

•

9

× O +

D-Gal-/3- ( 1 ~ 3)-o-GlcNAc-fl- ( 1 ~ 3)-D-Gal-/3-1- O- (CH2)aCOOCHa OG RLI. 1 D-Gal-/3-(I~4)-D-GlcNAc-/3(I~6)-3, 4-dideoxyhex-3-enitols n-Gal-~- ( 1 ~ 4)- n- GIcNAc D-Gal-~- ( 1 ~ 6 ) - D-GlcNAc

much material as did inhibition o f precipitation 147 •148 ; with the lectin s y s t e m the c o m p e t i t i v e binding a s s a y s required about ~o as m u c h inhibitor. 18z Immunochemical of Proteins

Studies on the Antigenic Determinants

B e c a u s e o f the predominant contribution o f c o n f o r m a t i o n to the tertiary structure o f protein antigens and b e c a u s e protein antigens m a y contain several antigenic determinants, the p r e v i o u s l y described approaches

[1]

PRINCIPLES

OF

ANTIGEN-ANTIBODY

(a) Anti-I Ma1:4000 I00

/

o -20

¢ i I

! / f" o

i I0

41

(b) Anli- I W0j 1:500

~'o

/

O-

REACTIONS

i I00

o o "1

-

I

I

1.000 1 10 01ig05accharideaddedInto01)

t= o

o 1

100

o I

1,000

Fro. 12. Inhibition of binding of anti-] sera Ma and Woj to nSl-labeled blood group l-active glycoprotein by synthetic oligosaccharides. Symbols for synthetic oligosaccharides: D'GlcNAc-fl" (l"~6)D Gal

[]

D-GIcNAc-~3- (1/3) -

D'GaI'/3"(I~4)'D'GIcNAc-~"(I"~6)DGal

v

D-GRI-#3- ( 1 ~ 3) - -GleNAe-/3- (1

From T. Feizi

et

•

/3) "

D-Gal-fl-(I~ 3)-D-GIcNAc-13-(I~ 3)-D-Gal

A

D- Gal-/3- (1 ~ 4) -D- GlcNAc-/3- ( 1 ~ 6)-D- Gal

•

o-Gal-fl- ( 1 ~ 3)-D-GlcNAc-/3- ( 1 ~ 6)-D-GaJ.

O

al. 148

to the location of antigenic determinants must be supplemented by basic chemical procedures. Moreover, protein antigens also generally involve cellular immune responses and contain determinants that act on the thymus-derived lymphocytes and produce delayed-type hypersensitivity. A detailed examination of this vast body of material ef' 207 and the technologycf. 2o8 is outside the scope of this volume, and only a brief outline will be given of the principles applied in localization and identification of antigenic determinants on proteins and polypeptides. Two general approaches are employed. One involves fragmentation of the protein by defined methods so as to obtain fractions containing different antigenic determinantsCC6'7'49-sa; these may be coupled to insoluble adsorbents, to separate the heterogeneous populations of antibodies with a view to obtaining antibodies to the individual determinants. The other 207 M. Z. Atassi and A. B. Stavitsky, eds., "Immunology of Proteins and Peptides I . " Plen u m , N e w Y o r k , 1978. 2o8 I. Lefkovits and B. Pernis, "Research Methods in Immunology." Academic Press, N e w Y o r k , 1978.

42

PRINCIPLES AND METHODS

[1]

involves inhibition assays with oligo- and polypeptides of known structure and increasing size to delineate the determinants in the manner already described for oligosaccharide determinants. Many of the oligopeptides become available as by-products from the determination of the. primary structure of the protein. 43"9~Individual amino acids in these peptides may be modified chemically, and the effect on their capacity to inhibit or to precipitate can be assayed. 2°9 Such studies make it possible to decide whether a given residue in the peptide is or is not reacting with the antibody combining site. The ease with which peptides of any desired structure may be synthesized makes it possible to evaluate the relative contributions of any amino acid to a given antigenic determinant by synthesizing a series of peptides in which one amino acid has been replaced by another. 21° With the heterogeneous populations of antibodies to protein antigens usually found in antisera, antibody to a given antigenic determinant may only comprise 10-15% or even less of the total antibody. 2°9-211,el. 7,91 If inhibition assays were to be carried out with the whole antiserum and the intact antigen as precipitant, the maximum degree of inhibition obtainable with the determinant in question would be only 10-15%, and this could create substantial uncertainty. It is for this reason that whenever fragments containing different populations of antigenic determinants are obtainable, the antiserum should be absorbed to remove antibodies to antigenic determinants other than the one that is being studied; in this way the maximum degree of inhibition and the precision obtainable is increased. Even when an antigenic determinant is defined, antibodies to it produced in individual animals may not be equivalent; for example, with the C-terminal antigenic determinant of myoglobin, in some antisera the Cterminal hexa- and heptapeptides were equal in inhibiting on a molar basis, whereas in others the heptapeptide was more active. T M It may often be difficult to evaluate the contribution of conformation as compared with chain length in defining an antigenic determinant. If the crystallographic structure is available, one may arrive at a decision by seeing which residues of the inhibiting peptide would be at the surface of the molecule and able to react or whether they are internal and are increasing inhibiting power by favoring the conformation present in the intact molecule. Thus Crumpton T M found that in myoglobin a peptide molecule of residues 15-33 was much more active than one with residues 15-29, although both were reacting with the same fraction of the anti2og M. Z. Atassi, Immunochemistry 15, 909 (1978). 210 M. J. Crumpton, Biochem. J. 116, 923 (1970). 21~ M. J. Crumpton and J. M. Wilkinson, Biochem. J. 94, 545 (1965).

[1]

PRINCIPLES OF A N T I G E N - A N T I B O D Y

REACTIONS

43

FIG. 13. The conformation of polypeptide chain of hen egg white lysozyme as deduced from X-ray studies at 2/~ resolution. The loop region is indicated by a circle. From Blake et a / . $12

body; from the three-dimensional structure, residues 30-33 were internal and not able to function as contacting residues, and they were inferred to be contributing to the enhanced binding efficiency by stabilizing the conformation in the intact protein. A very good example is the study of the extent to which residues of the loop polypeptide of lysozyme contributes to specificity. The loop peptide comprises residues 64-80 in which Cys-64 and Cys-80 form a disulfide bond, and thus this structure is highly conformational because opening the disulfide bond destroyed immunological activity. From the X-ray crystallographic study (Fig. 13),212 the loop peptide occurs in an exposed position. Figure 14 shows the sequence of the synthetic loop peptide zla and the capacity of synthetic loop peptides containing residues modified 2~ C. C. F. Blake, D. F. Koenig, G. A. Mair, A. C. T. North, D. C. Phillips, and V. R. Sarma, Nature (London) 206, 757 (1965). 213 R. Arnon, E. Maron, M. Sela, and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 68, 1450 (1971).

44

PRINCIPLES AND M E T H O D S

~

7

[1]

0

H2N'-~E ~N/)

Synthetic loop

~' i

HOOC82

80 ~

75

Intactloop,. 100

00¢,

/ Pro~.7//

i 0.1

/

l

I

I

I

1.0

10

100

100(}

P

Syntheticloopderivativesadded(pg) FIG. 14. Effects of replacement by Aia of the indicated residuesin the synthetic loop peptideof lysozymeon ability to inhibit inactivation by antiioop antibodiesof bacteriophageloop conjugate. From Arnon e t al. 213 and Teicher et al. =~4

at different positions to react with antiloop antibodies. 214 In those studies, assay of inhibiting power was based on the ability of the synthetic loop peptides to inhibit inactivation by antiloop antibodies of a conjugate to bacteriophage 215"21e of the loop peptide. 21r A synthetic loop peptide was prepared in which Cys-76 was replaced by Ala. When it was coupled through the COOH of Ala-82 to a poly(DL-Ala)-poly(Lys), and used for 214 E. Teicher, E. Maron, and R. Arnon, Immunochemistry 10, 265 (1973). =15 O. M~ikel~t, Immunology 10, 81 (1966). 2~e j. Haimovitch and M. Sela, J. lmmunol. 97, 338 (1966). 21~ j. Haimovitch, E. Hurwitz, N. Novik, and M. Sela, Biochim. Biophys. Acta 207, 115 (1970).

[1]

PRINCIPLES OF ANTIGEN--ANTIBODY REACTIONS

45

immunization, the antibodies produced reacted with native lysozyme. Also, the loop peptide coupled to Sepharose specifically absorbed from antisera to lysozyme the antiloop antibodies, which could then be eluted. The occurrence of cross-reactions with lysozymes of various species may contribute substantially to the precise delineation of the antigenic determinants. Turkey and bobwhite quail lysozymes differ from that of chicken in that Arg replaces Lys at positions 73 and 68, respectively. Turkey lysozyme reacted identically but quail lysozyme was weaker, thus indicating that Arg-68 makes a greater contribution to the interaction with the antibody combining site. The predominant contribution of the folded native structure is amply illustrated by the findings that complete reduction of disulfide bonds and S-carboxymethylation completely alters the immunochemical reactivity of protein antigens, such as ribonuclease, 218 lysozyme, and other antigens. 2t9,221 However, reduction of but two of the four disulfide bonds in ribonuclease had a negligible effect~2zon the ability to precipitate with antibody to native ribonuclease. Preparation of various derivatives effecting substitution on the protein antigen at known positions and evaluating the effects on reactivity with antisera have also been extensively used, especially by Atassi and coworkers, z°7,2°9 These include modifications or substitutions on tyrosines, tryptophans, methionines, arginines, and free amino or carboxyl groups. An unusual approach to mapping surface antigenic determinants if a three-dimensional structure for the protein is available has been introduced by Atassi 2°7'~°9and termed "surface simulation synthesis." In principle, it consists in measuring distances between the a-carbons of the hypothesized contacting residues of the antigenic determinant on the surface of the molecule and synthesizing a linear peptide with amino acid residues whose side chains would correspond in position to those of the model. Diglycine was used to approximate the - - S - - S bond distances for those determinants with the hypothesized contacting residues from two noncontiguous regions brought into proximity by - - S - - S bonds. Several alternative peptides were also synthesized whose a-carbon distances did not match those of the three-dimensional structure. The proportion of the total lysozyme-antilysozyme precipitate that the peptide was capable of 21s R. K. Brown, R. Delaney, L. Levine, and H. Van Vunakis, J. Biol. Chem. 234, 2043 (1959). 219 j. Gerwing and K. Thompson, Biochemistry 7, 538 (1968). 2zo j. Young and C. Y. Leung, Biochemistry 9, 2755 (1970). ~1 R. Arnon, in "The Antigens" (M. Sela, ed.), Vol. 1, p. 88. Academic Press, New York, 1973. 2~2 H. Neumann, I. Z. Steinberg, J. B. Brown, R. F. Goldberger, and M. Sela, Eur. J. Biochem. 3, 171 (1967).

46

PRINCIPLES

AND METHODS

[1]

inhibiting and the molar ratio of peptide to lysozyme at 50% inhibition were assayed. The most active peptide of minimal size was considered to mimic the surface structure of the antigenic determinant. The lysozyme molecule was determined to have three antigenic sites with residues coming from widely separated portions of the polypeptide chain; the residues proposed as contacting and those synthesized to produce a linear sequence considered as best simulating the active site are seen in Fig. 15. In some instances the peptide synthesized in the "reverse direction"--for example, using the C-terminal amino acid of the hypothesized determinant as the amino terminus--was used as a control. In some instances, the sequence was considered to have directionality whereas in others it did not. One of the uncertainties of the surface simulation method is that all the synthetic peptides proposed are rather short and each contains three highly charged residues. The inferences are based on a limited number of model polypeptides, mostly directional peptides; it would be desirable to make substitutions in the determinants in which the charged groups remain in position, but other presumed contacting residues are changed as well as those in which one charged amino acid is replaced by another. This could provide insight into the specificity of the method. Another question that arises is whether the simulation peptide is indeed reacting with the active site to prevent lysozyme from precipitating; it is conceivable that it might be reacting at another site and causing a conformational change at the site or that it might be reacting around the site to prevent lysozyme from entering rather than interacting by complementarity in the site itself. Changes in the spacing and substitution of other residues in the simulation peptides could increase the weight of the evidence favoring specific vs nonspecific interaction. The antigenic determinants proposed by Atassi 2°7,2°9do not include the loop peptide 64-82 (Fig. 13) to which Maron e t a l . 223 found 8-10 mg of antibody per 100 mg of goat antilysozyme ct.z23a; these antibodies were more restricted in heterogeneity than the total antilysozyme. The problem of antibody heterogeneity may well be complicating the precise delineation of antigenic determinants of proteins. Thus far all efforts to locate the antigenic determinants have been made with heterogeneous antibody populations. The subject must be reexplored with antiprotein antibodies obtained from hybridomas, which would provide monoclonal antibody populations. In this way one cannot have uniform reactivity with only the antigen itself, but must have it also with the vari223 E. Maron, C. Shiozawa, R. Arnon, and M. Sela, Biochemistry 10, 763 (1971). 223a I. M. Ibrihimi, J. Eder, E. M. Prager, A. C. Wilson, and R. Arnon, Mol. lrnmunol. 17, 37 (1980).

[I]

PRINCIPLES OF ANTIGEN--ANTIBODY

REACTIONS

47

Site 1 Constituent

residues:

125

5

Arg

Arg

i

(~C-to-crC, in n m )

0.93

~

_L~

'

O. 58

Distances:

(aC-to-c~C, in n m )

I. 05

'

3.01

Arg--Gly

Gly

13 Lys i

>',= 0.45-DH

'

i*

site:

i

~'~

'

i

The synthetic

14 Arg

i

~

Distances:

7 Glu

'

]

Arg--Gly--Glu--Gly

Gly--Arg

i

Lys

I

Site 2 62 Constituent

residues:

Distances:

(c~C-to-c~C, in rim)

97 Lys

Trp

0.71

i

96 Lys

site:

Distances: (aC-to-c~C, in nm)

89

87

Thr

Asp

=I: 0 . 4 1 ~ - 0 . 5 6 - * / ~ - 0 . 5 1 - ~ 0 . 5 4 I I r I

i.i The synthetic

93 Asn

i

2.73

Phe - -

Gly

Lys - -

,i i

Lys--

i,

Asn - -

Thr--

Asp

2.16

-[

Site 3 Constituent r e s i d u e s : Distances:

(~C-to-~C, in nm)

116 Lys

i

113 Asn - 0.5

~-I~0.4 i

114 Arg

site:

Distances: (c~C-to-c~C, in nrn)

Lys

[I

0.8

~-',~ I

l, The synthetic

34 Phe i

~',~ O. 4--~ I

2.1

Ash

Arg

33 Lys

i

~i i

Gly--Phe--Lys 1.8 p

FiG. 15. Three antigenic sites oflysozyme. The diagram shows the spatially adjacent residues constituting each antigenic site and their numerical positions in the primary structure. The distances separating the consecutive residues and the overall dimension of each site (in its extended form) are given, together with the dimension of each "surface-simulation" synthetic site. The latter assumes an ideal C~-to-C ~ distance of 0.362 nm. From M. Z. Atassi and C.-L. Lee, Biochern. J. 171,429 (1978).

ous constituent polypeptides, simulated peptide stretches, chemically modified antigens, etc. If a sufficient representation of the repertoire of antibody combining sites to the various determinants is obtained, it will be possible not only to localize precisely the individual sequences making up the determinants, but also to ascertain whether individual clones making antibody to a given determinant do or do not see it in different aspects and make antibodies directed against different residues. Various clones may see different residues as immunodominant in the same determinant or may be specific for different lengths of a given determinant, as already

48

PRINCIPLES AND METHODS

[1]

noted for the antisera reacting differently with the C-terminal hexa- and heptapeptide of myoglobin, zH Such studies might help to clarify the recent findings of Hurrell et al.,224 who noted that myoglobins of different species did not cross react even when complete sequence homology existed for the residues comprising a proposed antigenic determinant and suggested the hypothesized determinants may be larger or may be influenced conformationally in their reactivity by other segments of the molecule. Uncertainties that complicate studies on antigenic determinants of proteins are seen in the findings of Benjamini et al. 2zs and of Schechter et al.226 Benjamini et al.225 noted that attachment of a hydrophobic octanoyl group to the N terminus of a tripeptide making up part of a pentapeptide antigenic determinant of tobacco mosaic virus protein made it more active. Schechter et a/. 226 obtained antibodies by immunization to (o-Ala)2Gly-e-aminocaproic acid coupled to protein and found that with some antisera o-Ala4 and o-Ala~ were better than the determinant group of the antigen itself. Heteroclitic antibodies, 227 those that react better with a cross-reacting antigen than with the homologous antigen used for immuninization, are puzzling with respect also to mapping combining sites of protein antigens. Even with respect to carbohydrate determinants, problems in the identification of antigenic determinants remain. Zopfet a/. 22s have demonstrated that antibodies to a tetrasaccharide determinant may be directed toward one side of the molecule whereas others can be of entirely different specificity directed toward the opposite side. A sugar residue substituted on one side can block reactivity with one specificity without influencing reactivity to the antibodies of the other specificity. A n t i g e n - A n t i b o d y Reactions in Gels Since the classical studies of Oudin of"229 and Ouchterlonyof.230 on antigen and antibody interactions in gels and by Grabar and Williamsct. 231 on zz4 j. G. R. Hurreil, J. A. Smith, P. E. Todd, and S. J. Leach, Immunochemistry 14, 283 (1977). 225 E. Benjamini, D. Michaeli, and J. D. Young, Curt. Top. Microbiol. Immunol. ~ , 85 (1972). 2~6 B. Schechter, I. Schechter, and M. Sela, J. Biol. Chem. 245, 1438 (1970). ~27 T. Imanishi and O. M~ikel~i, J. Exp. Med. 1411, 1498 (1974). 228 D. A. Zopf, C.-M. Tsai, and V. Ginsburg, Arch. Biochem. Biophys. 185, 61 (1978). 229 j. Oudin, Methods Med. Res. 5, 335 (1952). ~a00. Ouchterlony, Prog. Allergy, 5, 1 (1958); 6, 3 (1962). 231 p. Grabar and P. Burtin, *'Immunoelectrophoretic Analysis." Elsevier, Amsterdam, 1964.

[2]

PROTEINS AND POLYPEPTIDES AS ANTIGENS

49

immunoelectrophoresis, these have become indispensable tools of the immunologist and immunochemist and have been applied in almost all fields o f biological science. They are invaluable for analyzing complex mixtures o f antigens and antibodies, for establishing that monospecific antisera contain only antibodies to the desired antigen, for detecting antibodies formed to impurities in the antigen used, and for qualitative detection and quantitative estimation of antigens and antibodies. Details are given by Oudin (this volume [9]). Acknowledgments Work of the laboratories is supported by grants from the National Science Foundation PCM 76-81029and a grant to the Cancer Center CA 13696from the National Institutes of Health.

[2] P r o t e i n s a n d P o l y p e p t i d e s

as Antigens

By PAUL H. MAURER and HUGH J. CALLAHAN During the past two decades there has been a tremendous increase in the realization o f the utility of antibodies directed against enzymes as tools in biochemical studies, l'z Antibodies against enzymes can be used (a) to detect and assay quantitatively the concentration of enzymes; (b) to concentrate and purify enzymes from dilute solutions and mixtures; (c) to study the active catalytic sites, multimolecular forms, and conformational structures of enzymes; (d) to localize enzymes in sectioned cells; (e) to study the appearance and modification of enzymes in the course of embryonic and phylogenetic development. Concomitantly in immunology there has been increasing knowledge concerning the many factors that can influence the multifaceted and complex sequence of events of the immune response beginning with the introduction of an antigen into a host (immunogen) to the formation of humoral antibody, a The goal of this chapter is not only to present information about techniques that have become available for producing antibody, but in so doing to make the invesi (B. Cinader, ed.), Ann. N. Y. Acad. Sci. 103, 493-1154 (1963). 2 "Antibodies to Biologically Active Molecules" (B. Cinader, ed.). Pergamon, Oxford, 1967. a See "Essential Concepts," discussed in: L. E. Hood, I. L. Weissman, and W. B. Wood, "Immunology," pp. 1-74. Benjamin-Cummings, Menlo Park, California, 1978.

METHODS IN ENZYMOIX~Y, VOL. 70

Copyright(~)igBOby Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-1gl970- I

[2]

PROTEINS AND POLYPEPTIDES AS ANTIGENS

49

immunoelectrophoresis, these have become indispensable tools of the immunologist and immunochemist and have been applied in almost all fields o f biological science. They are invaluable for analyzing complex mixtures o f antigens and antibodies, for establishing that monospecific antisera contain only antibodies to the desired antigen, for detecting antibodies formed to impurities in the antigen used, and for qualitative detection and quantitative estimation of antigens and antibodies. Details are given by Oudin (this volume [9]). Acknowledgments Work of the laboratories is supported by grants from the National Science Foundation PCM 76-81029and a grant to the Cancer Center CA 13696from the National Institutes of Health.

[2] P r o t e i n s a n d P o l y p e p t i d e s

as Antigens

By PAUL H. MAURER and HUGH J. CALLAHAN During the past two decades there has been a tremendous increase in the realization o f the utility of antibodies directed against enzymes as tools in biochemical studies, l'z Antibodies against enzymes can be used (a) to detect and assay quantitatively the concentration of enzymes; (b) to concentrate and purify enzymes from dilute solutions and mixtures; (c) to study the active catalytic sites, multimolecular forms, and conformational structures of enzymes; (d) to localize enzymes in sectioned cells; (e) to study the appearance and modification of enzymes in the course of embryonic and phylogenetic development. Concomitantly in immunology there has been increasing knowledge concerning the many factors that can influence the multifaceted and complex sequence of events of the immune response beginning with the introduction of an antigen into a host (immunogen) to the formation of humoral antibody, a The goal of this chapter is not only to present information about techniques that have become available for producing antibody, but in so doing to make the invesi (B. Cinader, ed.), Ann. N. Y. Acad. Sci. 103, 493-1154 (1963). 2 "Antibodies to Biologically Active Molecules" (B. Cinader, ed.). Pergamon, Oxford, 1967. a See "Essential Concepts," discussed in: L. E. Hood, I. L. Weissman, and W. B. Wood, "Immunology," pp. 1-74. Benjamin-Cummings, Menlo Park, California, 1978.

METHODS IN ENZYMOIX~Y, VOL. 70

Copyright(~)igBOby Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-1gl970- I

50

PRINCIPLES AND METHODS

[2]

tigator aware not only of factors that might enhance antibody formation, but also of factors that might be operative in suppression of the immune response. 4 As a rule biochemists and enzymologists work with limited amounts of purified materials. In addition to the usual biochemical methods, it should be realized that immunochemical techniques exist that allow one (a) to determine whether an enzyme preparation is indeed "pure" and (b) to use antibody against the enzyme to further purify the enzyme preparation via immunoadsorbent techniques) Knowledge of some immunological generalizations may help the investigator before he proceeds with the preparation of antibody. The ability of an animal to elicit an immune response depends upon complex interactions between the specific immunogen being presented to the host, the properties of the antigen, and the physiological state of the specific animal of choice. It is known that not all proteins or polypeptides can be immunogenic, i.e., can elicit an immune response under a standard set of conditions, and that the method by which the antigen is presented can influence the response. There also can be major differences in responses to the same macromolecule from species to species and among strains (or animals) within a specific species. 6 Nevertheless, it is known that by employing the correct "carrier" and conjugation procedure for the "nonimmunogen" there are ways of eliciting a response to almost any macromolecule. The molecular weight and the complexity in structures of the macromolecule influence the nature of the immune response. In general, the greater the molecular weight and the more complex the protein or polypeptide structure, the greater the response that can be expected. From studies with synthetic polymers of amino acids, it has been learned that ordinarily one does not elicit significant responses against homopolymers of amino acids, and that decreased responses are obtained against high molecular weight polymers containing a-D- or y-D-amino acidsY ,8 Determination of the antigenic structures of proteins has posed a chemical challenge of enormous proportions for years. ° Many investigators, therefore, have employed synthetic polymers of amino acids in im4 D. H. Katz, "Lymphocyte Differentiation, Recognition, and Regulation." Academic Press, New York, 1977. I. Parikh and P. Cuatrecasas, in "'Immunochemistry of Proteins" (M. Z. Atassi, ed.), Vol. 2 pp. 1-44. Plenum, New York, 1977. 8 Reviewed in: "Immunogenicity--Physico-Chemicai and Biological Aspects" (F. Borek, ed.). North Holland/American Elsevier, Amsterdam, 1972. 7 p. H. Maurer, Prog. Allergy 8, 1 (1964). s M. Sela, Science 166, 1365 (1969). 9 "'Immunochemistry of Proteins" (M. Z. Atassi, ed.), Voi. 1. Plenum, New York, 1977.

[2]

PROTEINS AND POLYPEPTIDES AS ANTIGENS

51

munochemical studies in the hope that information derived from these systems might be useful in the understanding of the immunochemistry of proteins. Although many data on these synthetic polymer systems have been accumulated in several laboratories, and polymers have contributed to elucidating aspects of the immune mechanism, the information gained from amino acid polymers has not always been helpful in understanding completely the immunochemistry or basis for the immunogenicity of proteins. Knowledge of the antigenic sites of protein antigens may help elucidate further not only the mechanisms of the immune response, but many immunological disorders at the molecular level. Although the last decade has witnessed a great deal of activity investigating the immunochemistry of protein antigens, 5,a so far the antigenic structure of sperm whale myoglobin 1° and of lysozyme have been completed. 1 Factors Influencing the I m m u n e R e s p o n s e

Animal Species The more common animals used for immunization are rabbits, goats, sheep, chickens, horses, guinea pigs, and mice. a2'~3The eventual goals of having antibody against a specific protein or polypeptide may determine the specific species for immunization. Important considerations in choosing a species are the source and availability of the immunogen. As might be expected the larger animals require more antigen for the production of antibody, but when responding can yield more serum than others. Ordinarily one cannot obtain large amounts of serum from repeated bleedings of mice. However, there are techniques available for producing large amounts of ascites fluid rich in antibodies, a4-~9 In addition, recent developments in the technology of the production of "hybridomas" of mye10 M. Z. Atassi, lmmunochemistry 12, 423 (1975). 11 M. Z. Atassi, lmmunochemistry 15, 909 (1978). 12 "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 1. Academic Press, New York, 1967. 13 "Handbook of Experimental Immunology," Vol. 3, Application of Immunological Methods (D. M. Weir, ed.), 3rd ed. Blackweil, Oxford, 1978. 14 j. S. Garvey, N. E. Cremer, and D. H. Sussdorf, "'Methods in Immunology," 3rd ed. Benjamin-Cummings, Reading, Massachusetts, 1977. 15 j. Munoz, Pro¢. Soc. Exp. Biol. Med. 95, 757 (1957). le E. C. Herrmann, Jr. and C. Engle, Proc. Soc. Exp. Biol. Med. 98, 257 (1958). lr E. S. Takasingh, L. Spence, and W. G. Downs, Am. J. Trop. Med. Hyg. 15, 219 (1966). 18 A. C. Sartorelli, D. S. Fischer, and W. C. Downs, J. lmmunol. 96, 676 (1966). 19 A. S. Tung, S. Ju, S. Sato, and A. Nisonoff, J. lmmunol. 116, 676 (1976).

52

PRINCIPLES AND METHODS

[2]

loma cells fused with normal antibody-producing mouse spleen cells has afforded the production of larger amounts of antibody both in vivo and in vitro 2°-~4 (see below). If one wants to increase the likelihood of obtaining a good response against most of the antigenic determinants in a protein, a species as phylogenetically removed as possible from the source (species) of the immunizing material should be injected. However, if a goal is to obtain antisera directed only against a few dissimilar structures or peptide sequences in an immunogen, then the same species should be injected. For instance, antibody against rabbit T-globulin or mouse T-globulin can be produced in rabbits and mice, respectively. However, depending on the genetic background of the host, the antibody produced can be directed against the minor "allotypic" structures. 25 Generally, rabbits, sheep, goats, and horses produce much more antibody per milliliter of serum than do guinea pigs and mice. In addition, most of the antibodies obtained following hyperimmunization of rabbits, sheep, and goats precipitate with the homologous antigens, whereas not all of those produced in guinea pigs and mice precipitate easily, but tend to form soluble antigen-antibody complexes. Genetic Factors

That many "genetic" factors govern immune responses to simple and complex synthetic polymers as well as to proteins such as lysozyme has been recently reviewed. 26 Immune responses of inbred strains of guinea pigs, mice, and rats to many immunogens are controlled by immune response genes present in the major histocompatability complex of the respective species. This has accounted for the unique finding of responders and nonresponders to polypeptides and proteinsY For instance, strain 2, but not strain 13, guinea pigs respond to the random copolymers s0 G. Kohler and C. Milstein, Nature (London) 256, 495 (1975). 2~ G. Kohler and C. Milstein, Fur. J. lmmunol. 6, 511 (1976). z2 G. Galfre, S. C. Howe, C. Milstein, G. W. Butcher, and J. C. Howard, Nature (London) 266, 550, (1977). 23 "'Lymphocyte Hybridomas" (F. Meichers, M. Potter, and N. L. Warner, eds.), in Curr. Top. Microbiol. Immunol. 81 (1978). L. A. Herzenberg, L. A. Herzenberg, and C. Miistein, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd ed., Ch. 25. Vol. 3, Blaekweli, Oxford, 1978. 25 L. A. Herzenberg and L. A. Herzenberg, in "'Handbook of Experimental Immunology'" (D. M. Weir, ed.), 3rd ed., Vol. 3 Ch. 12. Biackweil, Oxford, 1978. 26 "Genetic Control of Immune Responsiveness: Relationship to Disease Susceptibility" (H. O. McDevitt and M. Landy, eds.). Academic Press, New York, 1973. 37 "Immunogenetics and Immunodeficiency" (B. Benacerraf, ed.), Univ. Park Press, Baltimore, Maryland, 1975.

[2]

PROTEINS AND POLYPEPTIDES AS ANTIGENS

53

(Glue°Al#°) n and (Glue°Lys4°)n and the opposite pattern is noted with the polymer (GluS°Tyr5°)~. Similar situations exist with inbred mice, i.e., mice of H-2 haplotypes a, b, d, f, k, s respond to the random polymer (Glua°Al#°)n, but mice of haplotypes p and q do not. In addition a polymer or protein that is immunogenic in one species, need not be immunogenic in another species; i.e., mice do not respond to (Glu6°Lys4°), although guinea pigs and rabbits do. 2s Because of the multigenic control of most immune responses, it is recommended that outbred animals be immunized first. Even with this precaution, it is likely that not all animals will respond similarly. However, the coupling of a poor immunogen either covalently or via electrostatic interactions with an immunogenic protein "carrier" can convert the nonimmunogen to a conjugate that is immunogenic in nonresponders and responders. Responses are then obtained to the complete conjugate, i.e., the carrier as well as the "haptenic" determinants. Some of the limitations to the above procedure are that the conjugation technique may alter the antigenic structure of the determinant and that "antigenic competition" between carrier and coupled macromolecule may occur if the carrier is very immunogenic.

Properties of the Antigen Any consideration of the immunogenicity of a protein must take into account how its physiochemical properties can dictate the outcome of the immune response. We will consider only some of the intrinsic properties of antigens that are important in this respect; other properties will be considered in the section Methods of Immunization. The state of aggregation of a protein has long been recognized as a factor involved in its immunogenic potential. Using bovine y-globulin, Dresser z9 found that mice normally responsive to this protein were nonresponsive to preparations freed of particulate or aggregated material by centrifugation or column chromatography. Others 3° have demonstrated similar results in rabbits with human y-globulin as the immunogen. In both cases the failure to mount an immune response seemed to be due to the induction of a tolerant (paralyzed) state by the deaggregated preparations. Denaturation of protein antigens has been extensively studied over the past 70 years and in general has been shown to decrease immunogenicity relative to that o f the native form. In addition the antigenic specificity of P. Pinchuck and P. H. Maurer, J. Exp. Med. 122, 665 (1965). 2a D. W. Dresser, Immunology 5, 378 (1962). 30 C. Biro and G. Garcia, Immunology 8, 411 (1965).

54.

PRINCIPLES AND METHODS

[2]

many native proteins is lost and new specificities are created. Several reviews 6"31'32 o f this subject are available, and thus an extensive survey o f the literature will not be attempted here, instead a few specific examples will be given that typify the results obtained. Early studies demonstrated that denaturation o f ovalbumin (OA) achieved by any o f several means, e.g., heat, ultraviolet irradiation, sonication, alcohol, urea, or chemical modification resulted in a loss o f reactivity with anti-native OA antibodies. In 1951 Maurer and Heidelberger a3 showed that upon deamination two fractions could be obtained from ovalb u m i n - - o n e lightly deaminated (27-36%), which appeared not to be denatured, and another highly deaminated ( 4 0 - 8 0 ~ ) , which was denatured and insoluble at its isoelectric point. The lightly deaminated preparation reacted completely with anti-native OA serum, and conversely antisera to this preparation reacted completely with native OA. The highly deaminated preparation, however, reacted only weakly with either antiserum. More recently, Jacobsen e t a l . 34 employing a series of chemically modified human serum albumins, have shown that a distinct relationship exists between the antigenicity of these proteins and the Stokes radius values as determined by gel filtration experiments. Using the Stokes radius as an indicator o f unfolding, they have shown that an increase of approximately 0.5 nm results in a 50% loss in activity and an increase of 1.7 nm abolishes activity. Analogous results have been obtained with reduced-carboxymethylated bovine serum albumin, 3~ performic acid-oxidized ribonuclease, 36 reduced-carboxymethylated lysozyme, 37 and several other proteins. The studies described above, as well as numerous others, have led to the conclusion that protein antigens may contain two general classes o f determinant structures, namely, sequential and conformational. Sequential determinants would be those occurring in a linear conformation, as in the unfolded form o f a protein, and conformational determinants would be those that are recognized by their homologous antibodies only when they o c c u r in a particular conformation. The latter class would include determinants formed from amino acids that are located at distant points in the 31 E. A. Kabat, in "Kabat and Mayer's Experimental Immunochemistry,'" 2nd ed. Thomas, Springfield, Illinois, 1961. 32 M. Reichlin, Adv. l m m u n o l , p. 29. Academic Press, New York, 1975. 38 p. H. Maurer and M. Heidelberger, J. A m . Chem. Soc. 73, 2076 (1951). 34 C. Jacobsen, L. Funding, N. P. H. Moller, and J. Steengard, Fur. J. B i o c h e m 30, 392 (1972). 35 E. J. Goetzl and J. H. Peters, J. l m m u n o l . 108, 785 (1972). 86 R. K. Brown, J. Biol. C h e m 237, 1162 (1962). 37j. D. Young and C. Y. Leung, Biochemistry 9, 2755 (1970).

[2]

PROTEINS AND POLYPEPTIDES AS ANTIGENS

55

peptide chain but come into proximity with each other when a particular conformation is achieved. An elegant demonstration of conformational effects has been reported by Arnon and Sela. 38 They were able to isolate a 20-amino acid peptide (Cys°4-Leua3) from peptic digests of egg white lysozyme, which was joined at residues 64 and 80 by a disulfide bridge, thus forming a " l o o p " . This " l o o p " peptide was conjugated to a synthetic branched polypeptide and then used to immunize rabbits. Antibodies immunospecifically isolated from these sera, or from sera of animals immunized with lysozyme, react with the " l o o p " peptide, but only poorly or not at all with the open chain form produced by reduction and carboxymethylation, indicating the requirement for a particular three-dimensional structure in the antigen to bind with antibody. It also seems likely that proteins displaying quaternary structure have unique determinants that can be considered "conformational." Thus Reichlin32 has shown that, in complement fixation tests, antisera against methemoglobin reacted better with oxyhemoglobin than with deoxyhemoglobin. This is most likely due to the known difference in quaternary structure between the two proteins. In addition, the isolated a and /3 chains, which were inactive with the antiserum, could be rendered active by recombination with the appropriate chains from a different species. With myoglobin similar results were obtained, i.e., antisera to myoglobin detected differences between the heme-containing protein and the heinefree protein. It appears at present that most determinants in globular proteins are of the conformational type whereas one finds both types in structural proteins such as collagen, a9 There are numerous properties, other than those mentioned, that are associated with antigens and would affect their ability to induce antibodies or our ability to detect these antibodies. We will consider only four here, since they are more applicable to proteins and polypeptides: accessibility of determinants, complexity of amino acid composition, molecular weight, and effect of ions. It is well documented that groups that function as antigenic determinants are those that are exposed to solvent. This has been elegantly demonstrated by Sela's group s with branched-chain synthetic polypeptides. They found that when tyrosine and glutamic acid residues were attached in a linear fashion to poly(oL-alanine), and the alanine is then linked to poly(L-lysine) as a branch, antibodies were formed against the terminal glutamic acid and tyrosine positions (Fig. 1). However, if alanine was placed in the terminal position and linked to the poly38 R. Arnon and M. Sela, Proc. Natl. Acad. Sci. U . S . A . 62, 163 (1969). 39 M. Crumpton, in "Defence and Recognition" (R. R. Porter, ed.), MTP Int. Rev. Sci. Set. l, Vot. 10. Butterworth, London, 1973.

56

PRINCIPLES A N D M E T H O D S

°ll

[2]

l

Y.

FIG. 1. A multichain copolymer in which L-tyrosine and L-glutamic acid residues are attached to multi-poly(DL-alanyl)-poly(L-lysine). Left: Tyrosine and glutamic acid located in

terminal positions. Right: Tyrosine and glutamic acid positioned internally. Horizontal lines, poly(L-lysine); hatched area, poly(DL-alanine); 0 , L-tyrosine; ©, L-glutamic acid. From M. Sela. s

lysine through the glutamic acid or tyrosine, then most of the antibodies were alanine specific. These data strongly argue that, in order to be antigenic, determinants must be exposed to the environment. This concept is supported by Atassi's studies ~° with myoglobin. Atassi was able to identify five antigenic regions in this protein, and all were located in solventaccessible regions, in this case on the surface of the molecule. It is generally accepted that a relationship exists between the structural complexity of a compound, i.e., the variety of its components, and its ability to induce an immune response. For example, homopolymers of amino acids by themselves are very poor antigens, 4°-42 however, when used in a complex (e.g., with phosphorylated serum albumin), they induce normal levels of antibodies .43 The same effect can be accomplished by the introduction of a second or third different amino acid. 44 Analogous situations exist in some naturally occurring macromolecules. Thus, the low level of antibodies induced by gelatin could be greatly elevated by the introduction of tyrosyl residues. 45"46At the present time, we cannot explain 40 S. B-Efraim, S. Fuchs, and M. Sela, Immunology 12, 573 (1967). 41 p. H. Maurer, Proc. Soc. Exp. Biol. Med. 96, 394 (1957). 42 D. Subrahmanyam and P. H. Maurer, Fed. Proc. 18, 600 (1959). 43 H. Van Vunakis, J. Kaplan, H. Lehrer, and L. Levine, Immunochemistry 3, 393 (1966). 44 p. H. Maurer, Ann. N. Y. Acad. Sci. 103, 549 (1963). 45 M. Sela and R. Arnon, Biochem. J. 75, 91 (1960). 46 M. Sela, B. Schechter, I. Schechter, and F. Borek, Cold Spring Harbor Syrup. Quant. Biol. 32, 537 (1967).

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this phenomenon, although it has been suggested that cooperation between T and B cells in the immune response requires that different specificities exist within the antigen. 47 In considering the relationship of an antigen's molecular size to its capacity to stimulate an immune response, one must examine both humoral and cellular immunity, since their requirements are somewhat different. In general there is a direct relationship between molecular weight and the ability to induce an immune response in high molecular weight compounds. Thus, many polymers, e.g., flagellin, 4s dextran, and pneumococcal polysaccharide, 4a demonstrate increased humoral and cellular responses with increased size, but this is by no means absolute, since several synthetic polypeptide antigens of the same overall composition but widely different molecular weights have induced the same amount of antibody. 5° Attempts to determine the minimum size necessary to evoke a response have been much more definitive. Schlossman e t al. 51 showed, by using a homologous series of a-DNP-oligo(L-lysine) compounds ranging in size from the tetramer to the nonamer, that a chain length of seven units was the smallest size that could induce humoral and cellular immunity in guinea pigs. Smaller oligomers were ineffective. These results were confirmed by Stupp e t al. 52 using ¢-DNP-oligo(L-lysine) compounds, but it was also discovered that the incorporation of oligopeptides into Freund's adjuvant made significant differences in response patterns. Thus, mono-¢DNP-oligo(L-lysine) compounds, containing as few as two lysine residues, when emulsified with mycobacteria in complete Freund's adjuvant, were capable of stimulating anti-DNP antibody production but not delayed hypersensitivity reactions. No antibody was produced when incomplete adjuvant or saline was used. As an explanation it was suggested that the mycobacteria in the adjuvant formed complexes with the positively charged pepfides and thus acted as a carrier. Although it has been known for many years that high concentrations of many salts can inhibit antigen-antibody interaction or dissociate ira-

47 j. Goodman, in " T h e Antigens" (M. Sela, ed.), Voi. 3, pp. 127-183. Academic Press, New York, 1975. 4s M. J. Becker, H. Levin, and M. Sela, Eur. J. lmmunol. 3, 131 (1973). 49 K. Jann and O. Westphal, in "The Antigens" (M. Sela, ed.), Vol. 3, pp. 1-110. Academic Press, New York, 1975. 5o T. J. Gill, III, H. W. Kunz, and D. S. Papermaster, J. Biol. Chem. 242, 3308 (1967). 51 S. F. Schlossman, S. Ben-Efraim, A. Yaron, and H. A. Sober, J. Exp. Med. 123, 1083

(1966). Y. Stupp, W. E. Paul, and B. Benacerraf, Immunology 21, 583 (1971).

58

PRINCIPLES

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[2]

mune complexes, it was only recently discovered that in some systems physiological concentrations of specific ions are necessary to affect interaction. Using a synthetic polypeptide composed of 60% glutamic acid, 30% alanine, and 10% tyrosine (GAT), Maurer e t a l . 53 found that some animals (particularly sheep) respond to immunization with the production of two distinct populations of antibodies--one that reacts with the antigen only if divalent cations are present, and another having no such requirement. It was shown with several sheep and rabbit antisera that addition of a chelating agent (EDTA) with the antigen prevented precipitation of 10-90% of the antibody. These experiments led to the isolation of the antibodies and subsequently to a physicochemical explanation of the cation's role. Liberti e t al. ~ showed that cations, especially calcium, neither affect the antibody itself nor enhance precipitation of preformed antigenantibody complexes. They did show that calcium affects the antigen by decreasing intrinsic viscosity, increasing sw,20 and changing the optical rotatory dispersion pattern. Bivalent cations probably induce conformational changes in the antigens, perhaps by bridging the carboxyl groups of glutamic acids, leading to the creation of " n e w " antigenic determinants. In addition to synthetic polypeptides, this cation effect has been found with polysaccharide and protein antigens. Approximately 10-20% of rabbit antibodies raised against two pneumococcal polysaccharides (type III and type VIII) had a calcium requirement for interaction with the homologous antigen. 55 Not surprisingly, both polysaccharides contain glucuronic acid residues in their determinant structures. Favre and Vollotton 56 found 7 of 14 rabbits immunized with angiotensin II had antibodies that bound the antigen maximally in the presence of calcium. More recently ~7calcium requiring anti-human serum albumin antibodies from both rabbits and sheep have been isolated and characterized. Methods of Immunization Very detailed and helpful techniques dealing with the preparation of immunogens for immunization and the use of various vehicles and methods for immunization are presented in ~'Methods in Immunology and Immunochemistry, ''12 Volume 1, and in the "Handbook of Experimental Immunology."13

53 p. H. Maurer, L. G. Clark, and P. A. Liberti, J. lmmunol. 105, 567 (1970). P. A. Liberti, H. J. Callahan, and P. H. Maurer, Adv. Exp. Med. Biol. 48, 161 (1974). 55 H. J. Callahan and P. H. Maurer, lmmunol. Commun. 4, 537 0975). 66 L. Favre and M. B. Vallotton, Immunochemistry 10, 43 (1973). 5~ M. E. Frankel, H. J. Callahan, and P. A. Liberti, Fed. Proc. 36, 1286 (1977).

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In dealing with limited amounts of valuable protein or polypeptide one should use techniques that might enhance the immune response. Although responses can be obtained against either solutions of soluble antigens or suspensions of particulate antigens, better responses are elicited with the proper use of "adjuvants." In principle, the adjuvants allow use of much less immunogen for some of the following reasons. Not all of an immunogen administered to a host persists long enough to become an effective stimulator for antibody formation. Adjuvants increase the persistence of antigen in the host and can protect the antigen from degradation by the usual proteolytic enzymes. This can allow more antibody-forming cells to be exposed to the limited amount of antigen. Particulate materials are more immunogenic, and therefore it is advisable, if possible, to aggregate the protein artificially providing the procedure does not change the conformation or biological activity. Aggregates of human y-globulin and bovine serum albumin are immunogenic whereas the "monomeric" form of these proteins can be tolerogenic, sa Macromolecules that are highly charged, and even those that are nonimmunogenic, can be made to react with such carriers as methylated bovine serum albumin or phosphorylated bovine serum albumin. The charge interaction leads to an insoluable aggregate, which can be immunogenic. This technique has been successful for producing antibody against charged macromolecules, such as DNA, 59 polynucleotides,6° polyglutamic acid. 42 However, one has to be aware that the reaction might lead to changes in the structure of the immunogen or to masking or creation of new immunogenic determinants. Processing of an antigen by macrophages is an important aspect of the immune response, and therefore any procedure that enhances the uptake by macrophages before presentation to the lymphocytes augments the response. Adjuvants allow concentration of the antigen onto a particulate carrier so that the amount of antigen administered per unit volume is increased, the immunogen can be localized in specific areas for long periods of time, and local destruction and elimination of antigen is retarded. The commonly recognized adjuvants are remarkable for their diversities. Soluble immunogens can be adsorbed onto the following kinds of inorganic suspensions: alumina cream, aluminum phosphate, and aluminum sulfate. Adsorption onto organic carriers such as blood, charcoal, calcium alginate, or polyacrylamide gels has also enhanced responses. A Maalox

58 W. O. Weigle, "Natural and Acquired Immunologic Unresponsiveness." Cleveland World Publ., Cleveland, Ohio, 1967. 59 O. J. Plescia, W. Braun, and N. C. Palczuk, Proc. Natl. Acad. Sci. U.S.A. 52,279 (1964). 6o "Nucleic Acids in Immunology" (O. J. Plescia and W. Braun, eds.). Springer-Verlag, Berlin and New York, 1968.

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PRINCIPLES AND METHODS

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[AI(OH)3] suspension has also been used in conjunction with the addition of bacteria, such as Bordetella pertussis. The most popular and successful adjuvants have been the water in oil emulsions developed by Freund. The basic ingredients of light mineral oil (Bayol) and emulsifying agents mixtures such as Arlacel (A or C) are available commercially. The reagents are emulsified with either solutions or suspensions of the immunogen (incomplete Freund's adjuvant). The addition of mycobacteria (Mycobacterium butyricum, M. tuberculosis) in small amounts to the suspension (complete Freund's adjuvant) leads to a further enhancement of the immune response. This has been attributed to the increased local inflammatory response caused by the mycobacteria, el The well mixed and stable emulsion is injected either intraperitoneally, intradermally, or in the footpads of the host. If complete Freund's adjuvant has been used for the first injection, the secondary injections should not contain the mycobacteria, as further immune responses against the mycobacteria can be detrimental to the host and also lead to enhanced inflammatory responses. There are some additional advantages to using the complete adjuvant. The class of antibody formed is sometimes altered, leading to precipitating antibody, and ascites fluid can be formed following intraperitoneal injections in mice. Although it is not always predictable mice can produce large amounts of this fluid, which has concentrations of antibody almost equal to that found in the serum. On the negative side the investigator should be aware that the emulsion might destroy or mask some of the antigenic determinants of labile antigens. As a matter of convenience the immune response is divided into two stages. The primary response, resulting from an initial interaction with antigen, and the secondary response, resulting from subsequent contact with the same antigen. Quite often it is difficult to clearly delineate between the two; for example, an animal may have seen the antigen previously (particularly microbial antigens) or may have been exposed to cross-reacting antigens; however, for the present these factors will be discounted. The primary immune response is characterized by the appearance, within a few days of 19 S (IgM) antibodies. This is followed by a decline in 19 S levels and a rise in 7 S (IgG) antibody levels, which in general is dose dependent; for example, a good immunogen given in very low doses elicits little 7 S antibody, whereas in high doses quite significant amounts are formed. This phenomenon is nicely illustrated in the work of Uhr and Fine~ R. G. White, in "The Immunologically Competent Cell: Its Nature and Origin" (G. E. Wolstenholme and J. Knight, eds.). Churchill, London, 1963.

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50 OOSEOF ~X

/,,~

1011

l/ iI

L p

°.1f

109 10 e

0.01

0.001

1

5

I 10

I

15

DAYS

FIG. 2. The primary 19 S ( - - ) and 7 S (---) antibody responses to ~bX174 bacteriophage (&X) in the guinea pig. Representative responses to intravenous injections of 1011, 109, or liP phage particles are shown. From Uhr and Finkelstein. 6z

kelstein. °a Using the bacteriophage 4 × 174, a very potent antigen, they showed that injection of 10s of 10a particles led to good IgM response with no IgG production. However, administration of 1011 phage led to high IgM titers followed several days later by a large increase in IgG (Fig. 2). After about 2 weeks the IgM response had fallen off and the antibody was mostly of the IgG class. There has been some question as to whether soluble protein antigens induce the same pattern of primary response as particulate ones, and it now appears that they do, although the length of time between injection of antigen and appearance of antibody (lag phase) may be delayed. The secondary response results from the readministration of antigen at a later time and is characterized by a rapid increase in antibody levels consisting mainly of IgG, but with some transient IgM. The most striking effect observed is the great increase in total serum antibody levels over that obtained in the primary response e3 (Fig. 3). After 2 - 3 weeks there is a e2 j. W. Uhr and M. S. Finkelstein, Prog. Allergy 10, 37 (1967). J. W. Uhr, M. S. Finkelstein, and J. B. Bauman, J. Exp. Med. 115, 655 (1962).

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PRINCIPLES AND METHODS

[2]

10o000

1,000

100

k

10-

0

/

- ~,,

10

/

,,

/ / i -

X

i- 19SANTIBODY ~,X 7SANTIBODY

i/ o 0

I I J

.... 5

t .... I0

I,,,,,J,l,,

....

30

15

I .....

35

0.1 I ....

40

DAYS

FIG. 3. Antibody response to ~bX174 in the guinea pig after two intravenous injections. ---, ~bX174 bacteriophage (~bX); - - , 19 S antibody; response; ---, 7 S antibody response.

rapid decline in the IgG level until antibody concentrations reach a plateau, at which they may persist for weeks or months. The dose of antigen used for immunization, in addition to modulating the classes of immunoglobulin formed, can also have a profound effect on the ability of the animal to produce antibody at all. A refractory state, called tolerance, can be induced with most proteins having a low or moderate molecular weight (e.g., serum proteins) provided that the amount of antigen administered is within a given range. This tolerant state may be defined as the inability or diminished ability of an animal to react to a normally immunogenic material that has been induced by previous administration of the same material. It has shown that tolerance can be achieved within two distinct zones of antigen dosage. High zone tolerance is achieved when quantities of antigen much greater than the optimum immunizing dose are presented. Mitchison, 64 for example, induced tolerance to bovine serum albumin in mice by injection of 10 mg three times per week for 10 weeks. This type of tolerance is readily achieved with weaker immunogens (e.g., soluble proteins), but it is difficult to induce with potent immunogens because the quantities needed tend to be toxic or impractical to use. N. A. Mitchison, in "Immunogenicity" (F. Borth, ed.), p. 87. North-Holland Publ., Amsterdam, 1972.

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Low zone tolerance is induced with subimmunogenic amounts of antigen, approximately 1/zg or less in the mouse. Repeated administration of antigen is usually necessary to induce or maintain the tolerant state, or both. There is still no foolproof method for choosing the route of administration of antigen in order to evoke a humoral response. In general, weaker immunogens are used with Freund's adjuvant and given intramuscularly or subcutaneously. With protein antigens the intravenous route is often used in tolerance induction; the intradermal route, minus adjuvant, is employed to induce delayed hypersensitivity. 65 A final consideration in regard to antigen dosage is that of the amounts, affinity, and specificity of the antibodies produced during the immune response. Siskind e t a l . 6~ have shown that in rabbits immunized with DNP-bovine y-globulin, high doses of antigen (50 mg) resulted in a rapid increase in serum antibody levels followed later in the immune response by a decrease and plateauing at low levels. When a low dose (0.5 mg) was used, the response began slowly but increased with time and eventually exceeded by threefold the high-dose level. These authors also showed a progressive increase in antibody affinity with time after immunization. Although all the doses used induced this effect, the increase was much greater with the lower ones. After a consideration of the cellular events involved in antibody production, it was suggested that these results could be interpreted as follows: the large doses of antigen injected might induce tolerance in "high affinity" cells resulting in a decrease in the amount and affinity of antibody produced. An alternative possibility is that large doses of antigen would favor differentiation of cells for antibody production resulting in the rapid appearance of high antibody titers but with a concomitant depletion in proliferating cells, thus limiting the response. Lower concentrations would give a more sustained and eventually greater response: In brief, the current explanation for an affinity increase is that after initial immunization a number of cell types, of both high and low affinity, are stimulated. Later, as the concentration of available antigen decreases owing to metabolism, only those cells that can bind antigen strongly (high affinity) are stimulated. This theory is also used to explain the generally observed increase in cross-reaction of antibodies that occurs with time during the immune response, by noting that, as affinities increase, determinants that would not have bound well with early (low affinity) antibodies now can be bound well enough to be detected. es j. W. Uhr, Physiol. Rev. 46, 359 (1966). G. W. Siskind, P. Dunn, J. G. Walker, J. Exp. Med. 137, 55 (1968).

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Typical Protocols for Antibody Production

In Vivo Techniques After consideration of the many factors that influence the immune response and the availability of material, most investigators prefer to incorporate the antigen in Freund's complete or incomplete adjuvant. The preparation for immunization need not be absolutely homogeneous, but the antigen to be used in the assay of the immune response should be highly purified. At times the purity of the immunogen may be important, as one might encounter the phenomenon of antigenic competition, wherein the response to the impurities might mask the responses against the putative purified enzyme. For immunization of large animals, such as rabbits, sheep, or goats, small to moderate amounts of antigen should be used (0.1-1.0 mg per kilogram of body weight). A typical protocol would employ 5 or 6 rabbits injected either in the footpads or intramuscularly into multiple sites with about 1-5 mg in complete Freund's adjuvant in a total volume of 0.250.5 ml. With smaller animals, such as mice and guinea pigs, microgram amounts (1-100/~g in 0.02-0.2 ml) are injected in the footpads subcutaneously or intraperitoneally. The animals are bled weekly for 4 - 6 weeks beginning about 3 weeks after the immunization; the sera are separated and tested qualitatively (see below) for the production of antibody. Rabbits and larger animals can be bled from the ear vein or via jugular vein puncture. With mice, sera can be obtained via retroorbital tappings of blood. Several techniques have been developed for producing ascites fluid in mice. ~5-19 In addition to the original adjuvant technique, a modification used in our laboratory involves injecting the antigen in complete Freund's adjuvant intraperitoneally followed by an injection 3 days later of 0.5 ml of pristane. TM Mice are boosted 7-10 days later and again after another 7-10 days. Another effective method is to administer intraperitoneal injections of Sarcoma 180 cells TM subsequent to several intraperitoneal injections of complete Freund's adjuvant. Distension of the abdomen is indicative of ascites development. The amount of ascites fluid that can be obtained at each tapping varies from 0.25 ml to 20 ml. If the level of antibody is rising significantly, or there is no response at all, booster injections need not be given. It is important to have intervals between injections and to be careful not to reinject if there are high levels of antibody. When the antibody level is rising slowly or has decreased, it is beneficial to reinject the animals with incomplete Freund's adjuvant or the antigen solution intraperitoneally. In the presence of high levels of antibody, anaphylactic shock can ensue in mice or rabbits.

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Pooling of sera from different outbred animals is not recommended, as reactions to different determinants might have occurred in different animals. In addition, if the immunizing preparation is not absolutely pure, it is conceivable that some animals might have responded very well to the impurities. Changes do occur in the properties of the antibody produced over a period of time. In general high affinity antibody follows immunization with low doses of antigen, and the best sera in rabbits can be obtained about 3 - 5 months after immunization. Although not always predictable, animals that respond early after immunization usually produce the best antibody, n7 In Vitro Techniques

A unique and revolutionary adaptation of cell hybridization techniques to the construction of myeloma-like cell lines producing monoclonal antibodies with desired reactivities has revolutionized the approach to the production of immunospecific reagents. ~°-~4 Large amounts of specific antibody can be obtained after hybridization of lymphoid cells from an appropriately immunized donor (mouse) with cells from a mouse myeloma that has been adapted to growth in culture. Although the normal in vivo immune response to complex antigens leads to a very heterogeneous population of antibody molecules directed against many determinants, with the hybridoma technique each hybrid clone theoretically produces a single species of antibody specific for a single antigenic determinant. A recent symposium on cell hybridomas ~3and other publications 2°-24 discuss in depth both the technology and the applicability of the procedure. Essentially the protocol (Fig. 4) involves hybridizing spleen cells from a hyperimmunized donor with cells from an in vitro adapted enzyme-deficient myeloma. A neoplastic cell line used for producing fusions is X63AGS, a clonal line of myeloma MOPC21 that has been adapted to growth in vitro, in 8-azaguanine, and lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRTase) required for rapid growth in tissue culture medium containing hypoxanthine, aminopterin, and thymidine (HAT medium). The fusing agent is polyethylene glycol (PEG) of a specific concentration and molecular weight. After fusion (hybridization) the cells are cultured in HAT medium. Before subculturing, the supernatants of the initial hybrid cells can be assayed for antibody production by a number of sensitive techniques that allow one to determine which clones from a complete spleen are involved in antibody production and therefore are worth further subculturing. Once established the clones can sr G. W. Siskind and B. Benacerraf, Adv. lmmunol. 10, 1 (1969).

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PRINCIPLES AND METHODS

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P3/X63-Ag 8 tumor cells grown in HAT medium

\

/

107 Tumor cells

108 Spleen cells

/

\

Mixed, fused in PEG

I

Spleen and fused cells cultured in HAT medium

I Fused celts diluted and cloned for growth

I

Cloned cells screened for antibody iroduction

Antibody-producer cells grown in mass culture

Injected in mice for antibody production (serum/ascites fluid)

FIG. 4. Multistep methods f o r eliciting specific antibody-producing ceils (hybridomas).

HAT, hypoxanthine, aminopterin, and thymidine; PEG, polyethylene glycol.

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be kept growing either in culture or in vivo for many months. In the in vivo technique the culture-grown antibody-forming clones are injected into pristane primed mice. Within a few weeks the ascites-containing fluid that appears has monoclonal antibody. When grown in mass culture, antibody concentrations can reach about 50/zg/ml. Immunoadsorbent techniques can then be used to concentrate the specific antibody. In a number of situations where the hybridized cells have been injected in vivo, concentrations of 5-20 mg per milliliter of antibody have been produced. In the specific area of enzymology, success has been achieved in producing clones against human alkaline phosphatase, hen egg lysozyme, and horseradish peroxidase. °a A major advantage of this technique is that one can obtain monospecific antibodies directed against the immunogenic determinants of the enzyme, some of which might be against the active site of the enzyme. T e c h n i q u e s for Assaying for Antibody Initially a qualitative test, then a semiquantitative and, if deemed useful, a quantitative estimation of antibody can be performed.12-14 The table presents the sensitivity of some of the in vitro serological tests that can be used. (The advantages and disadvantages of the different techniques will be discussed by other authors.) When detecting immune responses in different species, one should reckon with the fact that not all antibody systems lead to a precipitation reaction. Rabbit sera containing precipitating COMPARISON OF MINIMAL CONCENTRATION OF ANTIBODY DETECTABLE BY SPECIFIC TEST

Immunological test 1. Fluid precipitation Interfacial (ring) test 2. Gel precipitation Double diffusion (Ouchterlony) Single diffusion (Oudin) 3. Hemagglutination Passive (indirect) 4. Radioimmunoassay

Antibody detectable (/zg N/ml) 20-30 3-15 10-100 0.001-0.03 0.001-less

See references to hybridomas produced against enzymes in "Lymphocyte Hybridomas" (F. Melchers, M. Potter, and N. L. Warner, eds.), Curt. Top. Microbiol. Immunol. 81, 19-22 (1978).

68

PRINCIPLES AND METHODS

[2]

antibody can be screened by both the agar diffusion techniques and/or reactions in liquid medium. It is best first to test the sera by precipitation in gel techniques. Although this is a secondary reaction, based upon complex interactions of antigen-antibody complexes following the initial interaction, a positive reaction (band formation) is indicative of significant concentrations of antibody. The ring or interfacial test involves carefully overlaying a solution of antibody and its dilutions with antigen so that a sharp interface is formed. Diffusion occurs between the two components until an optimal ratio (equivalence) for precipitation of the complexes is established. The gel diffusion tests involve carrying out the reactions between antigen and antibody in a semisolid medium. There are many modifications of this technique, i.e., Preer, Ouchterlony, and Oudin, all of which lead to " b a n d " formation. 14 In addition to detecting antibody qualitatively and estimating the approximate equivalence ratio needed for optimal precipitation between antigen and antibody, the agar diffusion techniques can indicate the number of antigen-antibody systems that might be present, providing that the diffusion effects due to temperature and concentration are controlled. A disadvantage of the reaction in liquid medium resides in the fact that if the concentration of antigen added is too high, soluble antigen-antibody complexes will form in antigen excess and it may appear as though there is no significant amount of antibody present. Therefore varying concentrations of antigen must be added to both undiluted and varying dilutions of serum in a checkerboard fashion. Although there are many in vitro and in vivo methods for estimating antibody in serum or other fluids, only the quantitative precipitin reaction gives a reliable and accurate measure of antibody in absolute weight units. When a proper assessment of the many factors influencing the reaction is made and rigorous procedures of quantitative analysis are employed in analyzing the washed antigen-antibody specific precipitate, quantitive antibody values are obtained, al In addition to the direct reaction of the antigen with antibody, techniques are available for performing "indirect" reactions. Antigens can be coupled chemically or via a "tannic acid" procedure to red blood cells. The antigen-coated erythrocytes in the presence of specific antibody then agglutinate (clump) as do red blood cells in the presence of antibody to the red blood cells (hemagglutination). Rather than measuring the capacity of an antiserum to combine with antigen, all the above-mentioned antibody tests measure the capacity of an antiserum to produce secondary effects, i.e., precipitation and complement fixation, following the primary antigen-antibody interaction.

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Measuring the antigen binding capacity rather than the absolute amount of antibody in a precipitate becomes important when it is realized that some classes of antibodies, and antibodies from some species, do not precipitate well even in the presence of "optimal" proportions of antigen and antibody. A number of reactions do exist that measure the primary binding between antiserum and low molecular weight haptens employing modifications of equilibrium dialysis techniques, s9 However, the application to large macromolecules has been more difficult to develop.The ammonium sulfate test (Farr) was developed with the antigen bovine serum albumin (BSA), which was labeled with lalI, to fill the need for a primary binding test suitable for nondialyzable large macromolecules. ~° As devised it can measure the capacity of antisera to combine with the soluble macromolecular antigens and can detect both precipitating and nonprecipitating antibody. The principle of the reaction depends upon the fact that the antigen (BSA) is soluble in 50% saturated ammonium sulfate, whereas antigen-antibody complexes, which assume the solubility properties of the antibody, are insoluble under the same conditions. One of the serious limitations of this technique is the need for the antigen to be soluble in 50% saturated ammonium sulfate. However, modifications have been developed employing anti-immunoglobulin serum (rather than ammonium sulfate), which precipitates the soluble complexes. 71 The anti-immunoglobulin serum is directed against the immunoglobulins of the specific species being tested. This reagent must be checked for its ability to precipitate all the immunoglobulin in the serum being assayed. The precipitation of the radioactive antigen in the presence of increasing dilutions of serum is a measure of the amount of the antibody; that is, the greater the dilution of antiserum against a specific immunogen that still combines with and precipitates a constant amount of antigen, the greater is the strength of the serum. This double-antibody or radioimmunoprecipitation test has been used to measure a variety of hormonal, microbial, and tumor antigens. Both the ammonium sulfate and anti-immunoglobulin techniques have been used not only to measure the presence or the concentration of antibody, but to detect very small amounts of antigen in fluids or solutions. (Discussions and applications of the various radioimmunoassay procedures referred to here are given elsewhere in this volume.)

C. W. Parker, "Radioimmunoassay of Biologically Active Compound." Prentice-Hall, New York, 1976. 7o R. S. Farr, J. Infect. D/s. 103, 239 (1958). 74 p. Minden, R. S. Farr, and J. Trembath, lmmunochemistry 12, 477 (1975). s0

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Acknowledgments T h e authors' research was supported by grants from the National Institutes o f Health, Institute for Allergy and Infectious Diseases, A0107825; The American C a n c e r Society, IM5H; the National F o u n d a t i o n - - M a r c h o f Dimes, 1-492.

[3] T h e

Experimental Induction to Nucleic Acids

of Antibodies

By B. DAVID STOLLAR

Antibodies to nucleic acids have found many uses in the specific measurement of naturally occurring or modified nucleic acids both in solution and in situ. To obtain the required antibodies, it has been necessary to link nucleic acids or their components to cartier proteins or synthetic polypeptides to form immunizing complexes because injection of purified nucleic acids alone into normal animals does not stimulate significant antibody production. Once the antibodies are formed, they react with the nucleic acid in the absence of carrier. A variety of immunogens have been developed, with either small fragments, such as nucleotides or oligonucleotides conjugated covalently to proteins, or with high molecular weight polynucleotides in physical complexes with protein carriers. With these immunogens, antibodies specific for each of the normal bases of DNA and RNA, or for modified bases or base sequences, have become available as selective reagents. Antibodies that recognize helical shapes have also become available. The applications of anti-nucleic acid antibodies have included localization of specific modified bases in ribosomes 1 or chromosomes2; identification of denatured DNA in replicating DNA in situ3; studies of the denaturation and renaturation of DNA4; identification of double-stranded RNA intermediates of viral replicationS; gene localization by in situ hybrid detection6; isolation of DNA enriched in specific genes7; measurement of ultraviolet i S. M. Politz and D. G. Glitz, Proc. Natl. Acad. Sci. U.S.A 74, 1468 (1977). 2 R. R. Schreck, V. G. Dev., B. F. Erlanger, and O. J. Miller, Chromosoma 62, 337 (1977). 3 W. J. Klein, S. M. Beiser, and B. F. Erlanger, J. Exp. Med. 125, 61 (1967). 4 L. Levine, J. A. G o r d o n , and W. P. J e n c k s , Biochemistry 2, 168 (1963). 5 V. Stollar, T. E. Shenk, and B. D. Stoilar, Virology 47, 122 (1972). s G. Rudkin and B. D. Stollar, Nature (London) 2,65, 472 (1977). 7 W. E. S t u m p h , J. R. W u , and J. Bonner, Biochemistry 17, 5791 (1978).

METHODS IN ENZYMOLOGY, VOL. 70

CopyrightO 19$0by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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AND METHODS

[3]

Acknowledgments T h e authors' research was supported by grants from the National Institutes o f Health, Institute for Allergy and Infectious Diseases, A0107825; The American C a n c e r Society, IM5H; the National F o u n d a t i o n - - M a r c h o f Dimes, 1-492.

[3] T h e

Experimental Induction to Nucleic Acids

of Antibodies

By B. DAVID STOLLAR

Antibodies to nucleic acids have found many uses in the specific measurement of naturally occurring or modified nucleic acids both in solution and in situ. To obtain the required antibodies, it has been necessary to link nucleic acids or their components to cartier proteins or synthetic polypeptides to form immunizing complexes because injection of purified nucleic acids alone into normal animals does not stimulate significant antibody production. Once the antibodies are formed, they react with the nucleic acid in the absence of carrier. A variety of immunogens have been developed, with either small fragments, such as nucleotides or oligonucleotides conjugated covalently to proteins, or with high molecular weight polynucleotides in physical complexes with protein carriers. With these immunogens, antibodies specific for each of the normal bases of DNA and RNA, or for modified bases or base sequences, have become available as selective reagents. Antibodies that recognize helical shapes have also become available. The applications of anti-nucleic acid antibodies have included localization of specific modified bases in ribosomes 1 or chromosomes2; identification of denatured DNA in replicating DNA in situ3; studies of the denaturation and renaturation of DNA4; identification of double-stranded RNA intermediates of viral replicationS; gene localization by in situ hybrid detection6; isolation of DNA enriched in specific genes7; measurement of ultraviolet i S. M. Politz and D. G. Glitz, Proc. Natl. Acad. Sci. U.S.A 74, 1468 (1977). 2 R. R. Schreck, V. G. Dev., B. F. Erlanger, and O. J. Miller, Chromosoma 62, 337 (1977). 3 W. J. Klein, S. M. Beiser, and B. F. Erlanger, J. Exp. Med. 125, 61 (1967). 4 L. Levine, J. A. G o r d o n , and W. P. J e n c k s , Biochemistry 2, 168 (1963). 5 V. Stollar, T. E. Shenk, and B. D. Stoilar, Virology 47, 122 (1972). s G. Rudkin and B. D. Stollar, Nature (London) 2,65, 472 (1977). 7 W. E. S t u m p h , J. R. W u , and J. Bonner, Biochemistry 17, 5791 (1978).

METHODS IN ENZYMOLOGY, VOL. 70

CopyrightO 19$0by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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irradiation-induced damage to DNAS; and quantitation of circulating components, such as thymidine a or modified bases of tRNA. 1° Immunogens P r e p a r e d by Covalent Linkage of Nucleic Acid Components to Proteins Principles of I m m u n o g e n Formation Nucleic acid components have been used as classical haptens, with immunogens prepared as covalent hapten-protein conjugates in a number of ways. Purine or pyrimidine bases have been conjugated directly to protein for this purpose, 1' but nucleosides, nucleotides, and oligonucleotides have been used much more extensively. Three procedures for preparing such immunogens will be described in detail in this chapter. For ribonucleosides or ribonucleotides, the most convenient linkage involves periodate oxidation of the furanose ring bearing adjacent hydroxyl groups; this is followed by condensation of the dialdehyde product of oxidation with lysine amino groups of protein. A stable covalent bond is then formed on addition of a reducing agent such as sodium borohydride.12 With deoxyribonucleotides, which do not have adjacent hydroxyl groups, water-soluble carbodiimides have been used to form phosphoramidate conjugates through the 5'-phosphate and protein amino groups.13 With cyclic nucleotides, which do not have free adjacent hydroxyl groups or a free phosphate group, the 2'-hydroxyl group can be succinylated with succinic anhydride and this product can be conjugated to protein amino groups with a carbodiimide reagent. 14 Deoxyribonucleosides also lack both the adjacent hydroxyls and free phosphate group; in this case, the 5'-hydroxyl has been oxidized to a carboxyl group, which is then linked to a synthetic polypeptide carrier with a water-soluble carbodiimide. 15 This procedure has been described in this series (see Vol. 12B [175]). Two other procedures have been used less exs E. Seaman, H. Van Vunakis, and L. Levine, J. Biol. Chem. 247, 5709 (1972). 9 W. L. Hughes, M. Christine, and B. D. Stollar, Anal. Biochem. $$, 468 (1973). ~0 L. Levine, T. P. Waalkes, and L. Stolbach, J. Natl. Cancer Inst. 54, 468 (1975). 1~ V. P. Butler, S. M. Beiser, B. F. Edanger, S. W. Tanenbaum, S. A. Cohen, and A. Bendich, Proc. Natl. Acad. Sci. U.S.A. 48, 1597 (1962). 12 B. F. Erlanger and S. M. Beiser, Proc. Natl. Acad. Sci. U.S.A. 52, 68 (1964). ~3 M. J. Halloran and C. W. Parker, J. l m m u n o l . 96, 373 (1966). 14 A. J. Steiner, D. M. Kipnis, R. Utiger, and C. Parker, Proc. Natl. A c a d . Sci. U . S . A . 64, 367 (1967). 15 M. Sela, H. Ungar-Waron, and Y. Schechter, Proc. Natl. Acad. Sci. U.S.A. 52, 285 (1%4).

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PRINCIPLES AND METHODS

[3]

tensively. One involves formation of a p-aminobenzoate conjugate of the 3'-hydroxyl of thymidine, followed by its diazotization and reaction of the diazonium salt with tyrosine in the protein. 16 The second, applicable to guanine-containing oligonucleotides, involves the photooxidation of guanine with methylene blue and visible light, and linkage through the oxidized product to amino groups of protein. 17 Principles of Specificity Antibodies induced by nucleosides or nucleotides show marked specificity for the purine or pyrimidine base component. When cross-reactivity does occur with other bases, it may involve distinct populations of antibodies that can be removed by absorption, or it may involve all the antibody reacting with a lower affinity, so that large concentrations of crossreacting nucleoside are required. Specific sera can be obtained and used, at suitable dilutions, as selective reagents for any of the normal bases of RNA or DNA or for a wide range of modified base analogs. TM Antibodies to nucleosides usually react with nucleosides equally or only slightly better than with nucleotides (Fig. 1). They also react well with polymers in which the bases are accessible, such as denatured DNA. When the periodate oxidation technique (with ring breakage and later reduction) is used for immunogen preparation, the resulting antibodies react slightly better with deoxyribonucleosides than with ribonucleosides. The use of nucleotide-protein immunizing conjugates induces antibodies that recognize the base, sugar, and phosphate group~a; they show marked specificity for the base component, but also react much better with the nucleotide than with the nucleoside or free base (Fig. I). Still, there is not a great differentiation between the ribonucleotide and deoxyribonucleotide. When dinucleotide-protein or trinucleotide-protein" conjugates are used as immunogens, there is some specificity for the base sequence used, 2°-22 though mononucleotides or partial sequences show some cross-reactivity. In such cases, the innermost base (closest to the

is j. p. Coat, S. David, and J. C. Fischer, Bull. Soc. Chim. Fr. 2489 (1965). ~7 E. Seaman, L. Levine, and H. Van Vunakis, Biochemistry 5, 1216 (1966). 18 B. D. Stollar, in '*The Antigens" (M. Sela, ed.), Vol 1, p. 1. Academic Press, New York, 1973. 19 M. Z. Humayun and T. W. Jacob, Biochim. Biophys. Acta 331, 41 (1973). s0 S. S. Wallace, B. F. Erlanger, and S. M. Beiser, Biochemistry 10, 679 (1971). zl B. Bonavida, S. Fuchs, M. Sela, P. W. Roddy, and H. Sober, Fur. J. Biochem. 31, 534 (1972). z z R. M. D'~lisa and B. F. Erlanger, Biochemistry 13, 3575 (1974).

[3]

73

ANTIBODIES TO NUCLEIC ACIDS 100

75

5G

c 2.'

"at- i

0.25

4-

,

,'1~

0.5

1

p6Aoles

I

I

I

0.25

2

|

0.5

t

I

1

2

Inhibitor

FIG. 1. Specificity of antinucleoside and antinucleotide antibodies. (A) Inhibition of the precipitation of antiadenosine antibodies and adenosine-serum albumin by adenosine (O), deoxyadenosine (O), adenosine 5'-monophosphate (A), adenine (A), and ¢ytidine, thymidine, or guanosine (+). (B) Inhibition of the precipitation of anti-GMP antibodies and GMPserum albumin by GMP (11), dGMP ([3), and guanosine (x).

protein in the conjugate) may contribute an unexpectedly large part of the specificity.2° Antibodies to mono-, di-, and trinucleotides of the usual nucleic acid bases react with denatured DNA but not with native DNA, in which the bases are not accessible. It has been difficult in many cases to measure their reactions with ribosomal RNA or single-stranded viral RNA, partly because of the extensive secondary and tertiary folding that may mask many of the bases and partly because of the difficulty in removing all ribonuclease from serum. Some reaction with RNA was measurable with an antiadenosine serum after careful efforts were made to remove ribonuclease. 2a Antibodies to anticodon sequences reacted with tRNA, 22 as did antibodies to modified bases of tRNA 24 and antibodies occurring spontaneously in sera of N Z B / N Z W mice or human patients with systemic lupus erythematosus. 2s,26 ~3 B. J. Rosenberg, B. F. Erlanger, and S. M. Beiser, Biochemistry 12, 2191 (1973). 24 R. Salomon, S. Fuchs, A. Aharonov, D. Giveon, and U. Z. Littauer, Biochemistry 14, 4046 (1975). 25 D. P. Eilat, P. Di Natale, A. D. Steinberg, and A. N. Schechter, J. Immunol. 118, 1016 (1977). ~ D. Eilat, A. D. Steinberg, and A. N. Schecter, J. lmmunol. 120, 550 (1978).

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PRINCIPLES

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Preparations of I m m u n o g e n s

Ribonucleoside-Protein Conjugates The preparation of immunogens by periodate oxidation of ribonucleosides or nucleotides was introduced in 1964 by Erlanger and Beiser ~2 and has been used extensively since. The original procedure has been modified~° and can be simplified to the following steps. About 0.1 mmol of nucleoside or nucleotide (20-40 mg) is dissolved in a solution of 0.1 M sodium metaperiodate in water. The volume of the periodate solution is chosen so as to give about an equivalent amount of this reagent; thus 1.0 ml of a 0.1 M solution is used for 0.1 mmol of nucleoside. The oxidizing mixture is stirred at room temperature for 20 min and then added, dropwise, to 2 ml of hemocyanin or other protein (5-10 mg/ml in 0.1 M bicarbonate-carbonate buffer, pH 9.5); the pH is readjusted to 9.5, if necessary, with 5% K2CO3. The solution is stirred at room temperature for 1 hr. Then 100 mg of sodium borohydride in 5 ml of water are added, and the mixture is placed at 4° for 1 hr or, if more convenient, overnight. The solution is then dialyzed extensively against 0.1 M NaCI, 30 mM NaHCO3 or a neutral buffered saline solution; some conjugates become insoluble if the pH is allowed to drop to less than 6. In this procedure, the extent of substitution is most profoundly affected by the pH of the reaction mixture containing oxidized nucleoside and protein (Fig. 2). Modifications of the basic technique are required in some instances. Guanosine is not soluble until it is oxidized. To avoid formation of unmanageable clumps during oxidation, the 0.1 mmol of guanosine is first dispersed in 1 ml of distilled water. Periodate solution is then added, and the solution is stirred well during oxidation. Nearly all the oxidized guanosine dissolves, usually producing a viscous solution or gel. This state is maintained until the sodium borohydride addition step. A different problem occurs with 7-methylguanosine. The purine base of this nucleoside is hydrolyzed at basic pH, and very significant degradation occurs in an hour at pH 9.5 at room temperature. The mixture of oxidized nucleoside and protein, therefore, is kept at pH 9.1 and at 0-4 °, at which only very limited hydrolysis of the base occurs in 1 hr. 27 A different reducing agent, tert-butylamine borane (Aldrich Chemical Co.), is used; the reduction is done for only 30-60 min at 4 °, and the product is separated from free nucleoside on a Sephadex G-25 column at 4 °. The nucleotide of the 7-methylguanosine is more stable than the nucleoside, and it also has been used to induce antibody to the 7-methylguanine structure. ~8 27 L. Rainen and B. D. Stoilar, Nucl. Acids Res. 5, 4877 (1978). 2s R. D. Meredith and B. F. Erlanger, Fed. Proc. 37, 1503 (1978).

[3]

ANTIBODIES TO NUCLEIC ACIDS .c_

//

20

o o_

O

"(3

75

10

o

z O I

7

I

8 pH

I

9

I

10

FIG. 2. The dependence of nucleoside-protein conjugation on pH. A mixture of periodate-oxidized adenosine and cytidine was added to bovine y-globulin to give final concentrations of 4 mM nucleoside (0.9 mg/ml) and 6.7/aM protein (1 mg/ml) in 0.2 M Veronal buffer titrated to varying pH. These mixtures were incubated at room temperature for 1.5 hr. Then sodium borohydride was added to a final concentration of 0.4 M (15 mg/ml), and samples were incubated for 2.5 hr at 4°. They were then dialyzed extensively against 0.1 M NaCI and analyzed for protein and nucleoside composition.

In this case, the reducing agent was cyanoborohydride.2s To verify that the conjugate contains intact purine base, a difference spectrum of the hapten-protein conjugate minus protein should be obtained; it should be very close to the spectrum of the intact nucleoside or nucleotide alone. Further, the antibodies should be specific for the intact base in comparison with the degradation productY Modifications are made also in order to use this reaction to conjugate nucleosides to erythrocyte surfaces, allowing use of the coated cells as targets for assays of antibody-forming splenic lymphocytes.29 Nucleoside, 10-20 mg, is oxidized in 1.5 ml of 0.1 M sodium periodate in 0.15 M NaHCO3 for 20 min at room temperature; the reaction is stopped by the addition of 15/zl of ethylene glycol. Sheep erythrocytes are washed twice with 0.15 M NaHCOa, and 0.5 ml of packed cells is then suspended in 2.0 ml of the bicarbonate solution in a 40-ml centrifuge tube. The oxidized nucleoside is added dropwise to the cell suspension and the mixture is kept at room temperature for 15 min. tert-Butylamine borane (Aldrich Chemical Co.), 100 mg in 5 ml of 0.15 M NaHCOa, is added. The suspension is kept at room temperature for 3 min, and the tube is then quickly filled with bicarbonate solution and centrifuged at 1500 rpm for 10 min. 29 B. D. Stollar and Y. Borel, Nature (London) 267, 158 (1977). This procedure is quoted with permission of Macmillan Journals Ltd., publishers of Nature.

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PRINCIPLES AND M E T H O D S

[3]

The modified cells are washed twice more with the bicarbonate solution and are ready for use as target cells in the hemolytic plaque assay.

Carbodiimide-Linked Nucleotide-Protein Conjugates This procedure was introduced by Halloran and Parker for use with mono- and oligonucleotides. 13 Humayun and Jacob modified it to reduce the amount of insoluble aggregate they obtained with the original method, especially with purine nucleotides. 19 In a reaction following the modified procedure, about 0.25 mmol of solid 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to 0.1 mmol of nucleotide in 0.5 ml of water. The pH is adjusted to 7 with dilute NaOH if necessary, and the solution is incubated at 60° for 10 min. The mixture is then added dropwise to a protein solution of 10-20 mg in 0.2-0.4 ml of 0.15 M NaCl, and this solution is kept in the dark overnight at room temperature. The product is then separated from the free nucleotide and the hydrolyzed carbodiimide reagent by dialysis or gel filtration. For some purposes, it may be important to note that the carbodiimide can modify the protein carboxyl groups, especially at low pH. 3° The conjugate, therefore, is often very positively charged and will form a precipitate with DNA. The carboxyl group modification can be reversed to a large extent by dialysis of the conjugated protein at pH 10, with no loss of nucleotide substitution. Exposure of protein to carbodiimide at high pH (9.5) produces a stable modification of lysine amino groups. 3° It should be noted also that carbodiimide can modify guanine, thymine, and uracil if the pH is above 8. 31

Succinylated Nucleotides Steiner et al. prepared an immunogen by 2'-O-succinylation of cyclic AMP and linkage of the product to protein with a water-soluble carbodiimide. 14 Although amino functions, such as the 6-amino group of adenine, can be succinylated also, the reaction of the 2-hydroxyl group is much more rapid and can be achieved selectively. 3~ This procedure may be of more general use, as with oligonucleotides having a free 3'- or 5'-hydroxyl group. In the use of this procedure with 3',5'-cyclic AMP (cAMP), 33 morphoa0 j. p. Riehm and H. A. Scheraga, Biochemistry 5, 99 (1966). This detailed study o f this modification used p H 4.5. We have found it to occur to some extent at pH 6 as well. a~ N. W. Y. Ho and P. T. Gilham, Biochemistry 6, 3632 (1967). as j. G. Falbriard, T. H. Pasternak, and E. W. Sutherland, Biochim. Biophys. Acta 148, 99 (1967). a3 A. L. Steiner, C. W. Parker, and D. M. Kipnis, J. Biol. Chem. 247~ 1106 (1972). This procedure is quoted with permission o f the author and the Journal of Biological ChemistO'. We have also applied it to noncyclic deoxyribonucleotides.

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ANTIBODIES TO NUCLEIC ACIDS

77

line N,N'-dicyclohexylcarboxamidine (0.76 mmol) is dissolved in 7.5 ml of hot anhydrous pyridine and 0.7 mmol of cAMP (free acid) is added slowly over a period of 30-60 min. After this mixture has cooled, 10 mmol of succinic anhydride are added and the suspension is stirred at room temperature for 18 hr. Unreacted succinic anhydride is hydrolyzed by the addition of 3.75 ml of water, and the reaction mixture is allowed to stand for an additional 2 hr at 4 °. Thin-layer chromatography of the reaction mixture on cellulose with butanol-glacial acetic acid-water (12:3:5 v/v) shows 2'-O-succinyl-cAMP which runs ahead (Re 0.42) of cAMP (Re 0.30). Pyridine is removed by repeated rotary evaporation at 40 ° under reduced pressure, and the residue is dissolved in 2-3 ml of water after the pH is adjusted to 4.5. The succinyl-cAMP is purified by chromatography on a column (1.5 × 44 cm) of Dowex 50 (H ÷ form) with distilled water as the eluent, at a flow rate of 30 ml/hr. Succinic acid appears in the first 50 ml, succinylated cAMP elutes in tubes 30 to 45, and cAMP in tubes 50 to 65. The yield of succinylated cAMP varies from 45 to 60%. The product is conjugated to protein by incubation of 20 mg of protein in 2 ml of water with l0 mg of succinylated cAMP and 10 mg of l-ethyl-3(3-dimethylaminopropyl)carbodiimide, at pH 5.5 for 16 hr at room temperature in the dark. The conjugated protein is separated from free reagents by dialysis.

Measurement of Hapten Substitution The extent of substitution is determined from the ultraviolet (UV) absorbance spectrum and the protein concentration. The latter is determined by a standard chemical assay. The protein contribution to the A~e0 of the conjugate is determined from the known spectrum of unmodified protein for the measured concentration. The difference between the total A260of the conjugate and the A~eodue to protein is contributed by the nucleoside. The molar concentration of nucleoside is calculated from this difference, using published extinction coefficients.34 Immunization A wide range of doses and immunization schedules has been used successfully. A convenient schedule for immunization of rabbits is to inject 200-500 ~g of conjugated protein, emulsified in complete Freund's adjuvant, intradermally and subcutaneously on the first day, and to inject the same dose in incomplete adjuvant on days 14 and 21. Serum can be obtained 5-7 days later. Further bleedings may be done weekly, with intradermal booster injections given if the serum antibody levels fall. After an 34"Handbookof Biochemistry,MolecularBiology"(G. Fasman, ed.), 3rd ed., Section B, Vol I. Chem. Rubber Publ. Co., Cleveland,Ohio, 1976.

78

PRINCIPLES AND METHODS

[3]

intravenous booster injection, the serum antibody level may peak and fall quickly, especially in early courses of immunization. In a recent study, involving immunization with 7-methylguanosine-bovine serum albumin, a schedule of small doses and intradermal and subcutaneous injections induced more antibody (and antibody that was less cross-reactive with guanosine) than did intravenous immunization. ~7 In mice, the response varies in different strains, but in both moderateand high-responding strains, a dose of 200/~g of nucleoside-hemocyanin induces higher antibody production than l0/zg or 50/~g.a5 Injections of antigen in complete Freund's adjuvant are given intraperitoneally on days 1 and 21, and high levels of antibody are present on day 26; a further injection on day 31 gives still higher levels on day 36; further immunization does not usually increase the antibody levels beyond that.

Assays for Antibody Two-dimensional immunodiffusion assays indicate qualitatively whether specificantihaptcn antibodies have been formed; ifthey arc present in thc serum, the precipitation line sccn with conjugated protein will spur ovcr that seen with the carrier protein alone, and precipitation will occur with haptcn linked to an unrelated carrier molecule. Quantitative precipitation tests indicate both h o w much haptcn-spccific antibody is present and how much unconjugatcd protein would bc required to absorb out all the antibodies directed against the protein alone. This absorption can then bc done by incubating equivalence conccntrations of antigen and serum and removing the precipitate.Absorption can also bc done with protein-agarosc affinitycolumns. In addition, the antibody can be purifiedwith hapten-bearing affinitycolumns, from which hapten-spccific antibody can be elutcd with 2 M acetic acid or with excess hapten. Antinucleotidc scra have been tested also for binding of radiolabcled hapten-protein conjugates or labeled D N A and with quantitative complement fixation assays. Once characterized, specific serum or purified antibodies may bc uscd also in immunohistochcmical tests at the level of light or electron microscopy. Single-Stranded Polynucleotides as H a p t e n s Principles of Immunization and Specificity Plescia e t al.36 contributed a major advance in the induction of anti-nucleic acid antibodies when they demonstrated that insoluble complexes of 35y. Borel and B. D. Stollar,Eur. J. Immunol. 9, 166 (1979). ~eO. J. Plescia, W. Braun, and N. C. Palczuk,Proc. Natl. Acad. Sci. U.S.A. 52, 279 (1964).

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ANTIBODIES TO NUCLEIC ACIDS

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denatured DNA and the positively charged methylated bovine serum albumin (MBSA) could elicit antibodies to the nucleic acid portion of the complex. This procedure has been applied since to synthetic homopolymers, 37 to modified DNA such as UV-irradiated 8 or photooxidized (visible light and dye) DNA, 17 to poly(adenosine-diphosphoribose), as and to a variety of helical nucleic acids as well. Native DNA, native tRNA, and single-stranded viral RNA have not been rendered immunogenic in this way.

Immunization with denatured D N A - M B S A gives rise to antibodies, often mainly of the IgM class, 3a that react with single-stranded DNA but not with native DNA. The largest antigenic determinant for such antibodies is about the size of a pentanucleotide. 4° When DNA with an unusual base, such as the glucosylated hydroxymethylcytosine of T-even phage, is used, the specificity is directed largely to the modified base and much more IgG may be produced. 41"42 Similarly, when UV-irradiated DNA or photooxidized DNA is used, the modified bases of the lesions provide the major specificity determinants, a'l~'4a Immunization with homopolynucleotide-MBSA complexes gives rise to antibodies that are specific for an oligonucleotide segment of the polymer. They probably recognize a number of bases in sequence, perhaps in a stacked array. Poly(I), poly(C), and poly(A) each induce specific antibodies that show little cross-reaction with the other homopolynucleotides, and slight cross-reaction with denatured DNA. 37 Procedures for preparation of the immunizing antigen, injection schedules, and assays for antibody formation are similar to those used for helical polynucleotide-MBSA antigens and are discussed in detail in the following section and in Vol. 12B [174] of this series. Helical Nucleic Acids as Antigens Principles of Specificity Antibodies can distinguish fine structural differences among helical polynucleotides. 44,45In doing so, they probably recognize conformational 37 E. Seaman, H. Van Vunakis, and L. Levine, Biochemistry 4, 1312 (1965). 3s y . Kanai, M. Miwa, T. Matsushima, and T. Sugimura, Biochem. Biophys. Res. Commun. $9, 300 (1974). 39 A. L. Sandberg and B. D. Stoilar, Immunology 11, 547 (1966). 40 A. Wakizaka and E. Okuhara, lmmunochemistry 12, 843 (1975). 4~ E. Seaman, L. Levine, and H, Van Vunakis, Biochemistry 4, 2091 (1965). 42 R. Gruenewald and B. D. Stollar, J. lmmunol. 111, 106 (1973). 43 L. A. Zamchuk, N. A. Braude, and D. M. Goldfarb, Immunochemistry 13, 81 (1976). 44 F. Lacour, E. Nahon-Merlin, and W. Michelson, Curt. Top. Microbiol. lmmunol. 62, 1 (1973). 45 B. D. Stollar, Crit. Rev. Biochem. 3, 45 (1975).

80

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variations that alter the steric relationship of the backbone phosphate and furanose groups. This allows the development of a particularly useful type of antibody reagent that reacts with a given class of helical structure. For example, one can obtain antibodies that react with double-stranded RNA (dsRNA) of any origin, but do not react with helical native DNA and react only very weakly with R N A - D N A hybrid helices. Similarly, one can induce antibodies that react with R N A - D N A hybrids as a class of structure but not with dsRNA or dsDNA. A third reagent, present in sera of some patients with systemic lupus erythematosus (SLE), but not yet inducible in normal experimental animals, reacts with native dsDNA but only weakly with R N A - D N A hybrids and not at all with dsRNA. Used in suitable assays, these reagents allow the selective measurement of dsRNA, dsDNA, or R N A - D N A hybrid in the presence of large amounts of other nucleic acids. Consistent with the suggestion that specificity is determined by helical conformation of the antigen (and important for the wide application of the antibodies) is the finding that reactions of these antibodies do not depend on specific base sequences or even base composition in the polynucleotide. Antibodies induced by poly(A).poly(U) react very well with poly(I).poly(C) or viral dsRNA of mixed base composition. Quantitative distinctions do occur within this class of structures, 46 and there may be some advantage to using a viral dsRNA as immunogen, for example, to obtain the strongest reactions with natural dsRNA of mixed base composition47; however, antibodies induced by the synthetic forms have been effective reagents for viral dsRNA in several studies. Similarly, antibodies to the hybrid poly(A).poly(dT) react well with poly(I).poly(dC) or hybrids of natural RNA and DNA. The SLE anti-native DNA antibodies react with synthetic poly(dAT) or native DNA of any viral, plant, or animal origin. Narrower specificities have been obtained with antibodies to some unusual helical structures. Poly(dG).poly(dC) induces antibodies specific for the immunogen and unreactive with other deoxyribonucleotide polymers, such as poly(dAT) or native DNA. Double-helical polyribonucleotides with modified furanoses, such as poly(A).poly(2'-O-methylU), induce antibodies that react with a number of polymers bearing 2'-furanose substitutions (such as methyl or ethyl groups on either the purine or pyrimidine-containing strand). 46 Poly(G).poly(C) induced antibodies of narrow specificity in our studies, but Lacour and co-workers obtained antipoly(G).poly(C) that cross-reacted with several forms of viral RNA. 48 Antibodies specific for triple-helical polynucleotides clearly distin4e M. I. J o h n s t o n and B. D. Stoilar, Biochemistry 17, 1959 (1978). 47 R. I. B. Francki and A. O. Jackson, Virology 48, 275 (1972). 48 E. Nahon-Merlin, A. M. Michelson, C. Verger, and F. L a c o u r , J. lmmunol. 107, 222 (1971).

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ANTIBODIES TO NUCLEIC ACIDS

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guish between three-stranded and two-stranded structures, and among different three-stranded structures. Examples of polymers that are qualitatively distinct are poly(U).poly(A).poly(U), poly(U).poly(dA).poly(U), and poly(U).poly(A).poly(I).49,5° Preparation of I m m u n o g e n s Synthetic polynucleotides are convenient immunogens for obtaining antibodies to helical nucleic acids; naturally occurring helical forms may be preferable if available in adequate supply (100/zg or more). The synthetic forms may be purchased as helical polymers, such as poly(A).poly(U) for dsRNA or poly(A).poly(dT) for hybrids. Alternatively, a wide variety of double- or triple-helical forms can be prepared from mixtures of homopolymers; because they have perfect base pairing homology, most complementary homopolymers anneal readily. Their interaction can be verified by measurement of the UV absorbance spectra of the separate homopolymers and of the annealed mixture; hypochromicity will be observed in the mixture (though not always at 260 nm). To establish whether a double- or triple-stranded structure is produced, one can mix the homopolymers in varying proportions and determine whether the greatest hypochromicity occurs with a 1 • 1 mixture or a 1 : 2 mixture of homopolymers. The assay of stoichiometry by measurements of hypochromicity with varying proportions can help to minimize the presence of an excess of free single-stranded homopolymer and the formation of corresponding antibody. If some antibody to one of the homopolymers is formed, it should be removed from the serum by absorption. To form double-stranded poly(A).poly(U), equimolar amounts of the two homopolymers are mixed at a concentration of 0.6-1.5 p,M nucleotide (about 200-500/zg/ml) in 0.1 M NaC1, 0.01 M phosphate pH 7 at room temperature and allowed to anneal for a few hours. Hypochromicity occurs at 260 nm when a double helix is formed and at both 260 and 280 nm when a triple helix is formed. 51 At room temperature and 0.1 M NaC1, only the stoichiometry determines which helix will form. At high ionic strength (0.7 M NaCl), and at high temperature, even a 1 : 1 mixture will form a triple helix with time; triple-strand formation is also favored by Mg2+.51 Poly(I) and poly(C) form only a double helix, and hypochromicity is greatest at 250 nm. Since poly(I) can form helical structure by itself, this mixture of homopolymers is heated to 100° in a boiling water bath to melt the poly(I) structure, and the mixture is allowed to cool slowly to room 49B. D. Stollarand V. Raso, Nature (London) 250, 231 (1974). 50L. Rainen and B. D. Stollar,Biochemistry 16, 2003 (1977). 51M. Riley, B. Maling,and W. Chambedin,J. Mol. Biol. 20, 359 (1966).

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[3]

temperature. Mixtures with poly(G) or poly(dG) are also heated to disrupt homopolymer secondary structure. Poly(A).poly(dT) shows hypochromicity at 260 nm; a triple-helix can form with excess poly(dT) but only in solutions of high ionic strength. 5~ Poly(dA) and poly(U) form only a triple helix, with hypochromicity at all wavelengths from 220 to 285 nm. 5' For preparation of the aggregated polynucleotide-MBSA complex, it is convenient to use the polynucleotide at a concentration of about 200500/xg/ml. A stock solution of 10 mg of MBSA per milliliter in water is prepared; the powdered or lyophilized MBSA does not dissolve readily in saline. An amount of MBSA equal to the total weight of the polynucleotide is added to the annealed polymer. A white precipitate should be visible after the MBSA is added. It is an easily handled fine suspension with most polymers, but fibrous strands may be formed with samples of very high molecular weight. Preliminary mechanical homogenization or sonication usually reduces such polymers to a size that will form a manageable suspension with the MBSA. The polynucleotide-MBSA suspension is then emulsified with an equal volume of Freund's adjuvant until a stable thick white emulsion is formed. Complete adjuvant is used for the first immunization, and incomplete adjuvant for subsequent injections. Immunization Schedule A primary dose of 50-200/zg of polynucleotide, in the MBSA complex and emulsified with complete adjuvant, is injected into each rabbit at several intradermal sites along the back and subcutaneously; a total volume of 1 ml per rabbit is convenient. Similar doses, but with incomplete adjuvant, are given intradermally and subcutaneously on days 14 and 21, and the animals are bled 5 - 7 days later. They can be bled at weekly intervals, and additional booster immunizations can be given if antibody levels should fall. During early courses of immunization, they may peak and fall rapidly .39 Assay of Antibodies Sera can be tested by immunodiffusion or, with greater sensitivity and speed, by counterimmunoelectrophoresis (Fig. 3). In this assay, the negatively charged polynucleotide antigens move toward the anode and antibodies move toward the cathode during electrophoresis, and they meet and precipitate in less than an hour. About 7 ml of melted 0.8% agar in 50 mM Tris-HC1, pH 8, is placed on a 5 x 7.5 cm microscope slide. It is important to use agar, not agarose, since the mobility of the antibody depends on endosmotic flow. A trough for 150/zl of antiserum is placed opposite several wells; each well receives 50/.tl of antigen at a concentration of 5-10 ~g/ml. Electrophoresis, in 50 mM Tris-HC1, pH 8, buffer is

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FIG. 3. Counterimmunoelectrophoresis assay of anti-poly(A).poly(U) antiserum. Serum (150/zl) was placed in the trough, and 50 V.1containing 0.25 p.g of antigen was placed in each well. The antigen preparations were, from left to right, poly(A); poly(A) + poly(U), 85 : 15; poly(A) + poly(U), 67 : 33; poly(A) + poly(U), 50 : 50; poly(A) + poly(U) 33:67; poly(A) + poly(U), 10:90; and poly(U). Electrophoresis was run at 300 V, giving 10 mA per slide, for 45 min. The gel was 0.8% agar in 50 mM Tris-HC1 pH 8. run at 10 m A per slide for 3 0 - 4 5 min. With this assay one can quickly test reactivity with the annealed helical antigen, separate h o m o p o l y m e r s , and potentially cross-reacting p o l y m e r s . F o r m o r e quantitative assay, sera can be tested for binding of radiolabeled antigen; a double-antibody assay, using the y-globulin fraction of an anti-rabbit I g G serum, is preferred. Quantitative precipitation and quantitative m i c r o - c o m p l e m e n t fixation are also valuable assays for these antibodies. I n c r e a s i n g Specificity b y A b s o r p t i o n When antibodies to single-stranded p o l y m e r s or to cross-reacting helices are present, they m a y be r e m o v e d by absorption. A preliminary small-scale quantitative precipitation c u r v e (with 50 or 100 kd o f serum and 2 - 5 0 ~g of antigen per tube) will indicate the amount of p o l y m e r required for equivalence for a given volume of serum. A corresponding mixture is then p r e p a r e d on a larger scale and incubated for 1 hr at 37 ° and overnight at 4 °. The precipitate is r e m o v e d by centrifugation. Since excess free p o l y m e r m a y remain in the supernatant, the a b s o r b e d serum is

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passed through a small (2-5 ml) column of DEAE-cellulose equilibrated with 15 mM potassium phosphate, pH 7; antibody passes through directly, whereas polynucleotide binds to the column. Sera may be absorbed also by affinity columns, such as one on which polynucleotide is linked to CNBr-treated agarose. 52 Immunospecific Purification of Antibodies from Specific Precipitates Antibodies specific for helical structure can be purified by a procedure that does not require acid, alkali, or denaturants such as urea or guanidine. 53 Equivalence proportions of polynucleotide and serum (determined from a preliminary quantitative precipitin curve) are mixed and incubated at 37° for 1 hr and at 4° overnight. The precipit~ite, obtained by centrifugation, is washed with cold phosphate-buffered saline (0.14 M NaCl, l0 mM phosphate, pH 7) three times to remove extraneous serum proteins. It is then drained thoroughly, and excess buffer is wiped off the wall of the tube. The precipitate is resuspended in distilled water (in a volume close to that of the starting serum sample) and heated to 45-50 °. This denature s the helix, freeing the antibody. Pancreatic ribonuclease (or a mixture of pancreatic and T1 ribonucleases) is added to digest one or both of the homopolymer strands, and the mixture is incubated at 50° for 1 hr. It usually becomes turbid because the free immunoglobulin is poorly soluble in distilled water. One-tenth volume of 1.5 M NaCl is then added to dissolve the free antibody. Residual precipitate is removed, and the soluble material is applied to a Sephadex G-200 column. This separates purified IgG and IgM antibody populations from each other and from oligonucleotide fragments and residual ribonuclease. Immunospecific Purification with Affinity Adsorbants Guigues and Leng have described the preparation of polynucleotideSepharose columns for this purpose, with linkage of oligo- and polyribonucleotides to the gel through a 6-aminohexanoic acid spacer. ~4 For this linkage, Sepharose-aminohexanoic acid is added to 50 mg of nucleic acid in 50 ml of 0.1 M NaCl. Then 100 mg of a water-soluble carbodiimide is added, and the mixture is incubated at pH 5 for 2-5 hr. The substituted Sepharose is washed sequentially with high-salt solution, 2 M acetic acid, and then neutral buffer. (With purine-containing polydeoxyribonucleotides, the acid step should be avoided at this stage, since such polynucleotides are subject to depurination below pH 5.) Serum or its ~/-globulin 52M. S. Poonian,A. J. Schlabach,and A. Weissbach,Biochemistry 10, 424 (1971). Methods for preparing affinitycolumnswith nucleic acids have been reviewed by H. Potuzak and P. D. G. Dean, FEBS Lett. 88, 161 (1978). 53B. D. Stollarand V. Stollar, Virology 42, 276 (1970). M. Guiguesand M. Leng,Eur. J. Biochem. 69, 615 (1976).

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fraction is applied to the column, and the column is washed with phosphate-buffered saline. Additional washing with a solution of 0.125 M borate, pH 8.5, 1.0 M NaCl, 0.1% Tween 20 removes nonspecifically bound protein. 55The antibodies are eluted with 2 M acetic acid in the cold and neutralized immediately. The immunospecifically purified antibodies have been particularly useful in physicochemical studies of the antibody-nucleic acid interaction; for some purposes, the monovalent Fab fragments of the purified antibodies have been used. 54,56 The use of absorbed and purified antibodies also ensures the specificity of immunofluorescent and serological measurements of helical nucleic acids of a given class in the presence of other nucleic acid forms. Acknowledgments Research in the author's laboratory has been supported by grants (currently grant PCM-79-04057) from the National Science Foundation and grant AI14534 from the National Institutes of Health. 5s j. A. Smith, J. G. R. Hurrell and S. J. Leach, Anal. Biochem. 87, 299 (1978). M. Leng, M. Guigues, and D. Genest, Biochemistry 17, 3215 (1978).

[4] T h e P r e p a r a t i o n o f A n t i g e n i c H a p t e n - C a r r i e r Conjugates: A Survey By BERNARD F . ERLANGER

Substances of molecular weight less than 1000 are not ordinarily antigenic. However, antibodies can be raised to small molecules by immunization with conjugates made up of low molecular weight substances (haptens) covalently linked to proteins or synthetic polypeptides. The ability to couple many different structures to macromolecules, the high degree of antigenicity of many of the conjugates, the development of sensitive methods of detecting and quantitating reactions between antibody and hapten, and the perfection of techniques for obtaining highly purified preparations of antihapten antibodies have contributed to the development of many of our modern immunological concepts. Much of our current knowledge of the requirements for immunogenicity, the structure of antigenic determinants, and the nature of antibody--its purification, hetMETHODS IN ENZYMOLOGY,VOL. 70

Copyright© 19~0by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181970-1

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fraction is applied to the column, and the column is washed with phosphate-buffered saline. Additional washing with a solution of 0.125 M borate, pH 8.5, 1.0 M NaCl, 0.1% Tween 20 removes nonspecifically bound protein. 55The antibodies are eluted with 2 M acetic acid in the cold and neutralized immediately. The immunospecifically purified antibodies have been particularly useful in physicochemical studies of the antibody-nucleic acid interaction; for some purposes, the monovalent Fab fragments of the purified antibodies have been used. 54,56 The use of absorbed and purified antibodies also ensures the specificity of immunofluorescent and serological measurements of helical nucleic acids of a given class in the presence of other nucleic acid forms. Acknowledgments Research in the author's laboratory has been supported by grants (currently grant PCM-79-04057) from the National Science Foundation and grant AI14534 from the National Institutes of Health. 5s j. A. Smith, J. G. R. Hurrell and S. J. Leach, Anal. Biochem. 87, 299 (1978). M. Leng, M. Guigues, and D. Genest, Biochemistry 17, 3215 (1978).

[4] T h e P r e p a r a t i o n o f A n t i g e n i c H a p t e n - C a r r i e r Conjugates: A Survey By BERNARD F . ERLANGER

Substances of molecular weight less than 1000 are not ordinarily antigenic. However, antibodies can be raised to small molecules by immunization with conjugates made up of low molecular weight substances (haptens) covalently linked to proteins or synthetic polypeptides. The ability to couple many different structures to macromolecules, the high degree of antigenicity of many of the conjugates, the development of sensitive methods of detecting and quantitating reactions between antibody and hapten, and the perfection of techniques for obtaining highly purified preparations of antihapten antibodies have contributed to the development of many of our modern immunological concepts. Much of our current knowledge of the requirements for immunogenicity, the structure of antigenic determinants, and the nature of antibody--its purification, hetMETHODS IN ENZYMOLOGY,VOL. 70

Copyright© 19~0by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181970-1

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PRINCIPLES AND METHODS

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erogeneity, valence, size of combining site, and biological p r o p e r t i e s - has resulted from the use of such conjugates. The immunochemistry o f low molecular weight molecules had its beginning in the pioneering work of Landsteiner. In 1917, when Landsteiner set out to prepare what he called "artificial conjugated antigens," it was " t o investigate an almost dogmatic belief . . . that a special chemical constitution, peculiar to proteins, was required for the production of antibodies. ''1 We know better now, of course, but it is to these studies by Landsteiner that we owe much of what appears in this volume. The earliest of his conjugated proteins were prepared by the acylation of the amino groups o f serum albumin with chlorides or anhydrides of butyric, isobutyric, mono-, di-, and trichloroacetic, anisic, and cinnamic acids. This was followed by his better-known studies in which diazonium compounds were allowed to react with histidine, tyrosine, and tryptophan residues o f a protein. With these conjugates, he established that the original specificity of the protein carrier was changed by the newly introduced groups which, by themselves, were not antigenic, and that cross-reactions among sera depended now upon the structural relationships among the acyl or azo groups that were covalently linked to the protein. He also noted that, in most cases, antibody was produced to the protein carrier as well and, to be certain of antibodies to the new determinant group, one had to test the sera with conjugates made with an unrelated or homologous (to the immunized animal) protein. It was his practice to r e m o v e any of the anticarrier antibody by absorption of the serum with the free protein. This is still done, although it is not necessary for radioimmunoassays. Landsteiner also sought to determine the optimal n u m b e r of haptenic groups that gave the best antibody response, and he concluded that too much or too little hapten led to a poor response. With serum albumin as the carrier, 10 haptenic groups seemed to be optimal. The major finding by Landsteiner, however, related to the exquisite specificity of the antisera, as was so beautifully demonstrated by his classical studies with L-, O-, and meso-tartaric acids. Thus, Landsteiner's work established many of the ground rules by which we operate today. Our contributions since his time have been mainly refinement o f techniques and procedures and the expansion o f his ideas. The major exception to this statement, and a crucial one indeed, is the development by Berson and Yalow 2 of the technique o f radioimmunoi K. Landsteiner, "The Specificityof Serological Reactions" Harvard Univ. Press, Cambridge, Massachusetts, 1945. z S. A. Berson and R. S. Yalow, Radioimmunoassay: A status report, in "Immunobiology: Current Knowledge of Basic Concepts in Immunology and Their Clinical Application" (R. A. Good and D. W. Fisher, eds.), pp. 287-293. Sinauer, Stamford, Connecticut, 1971.

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- A M I N O G R O U P S O F LYSINE RESIDUES (59)

×O CH2--CI-12--CH2--CH2--CH--C ~" N'H--

I

I

NH 2

NH

I

a-AMINO G R O U P S (1) /O -- CH--CI---NH

-

I NH 2

PHENOLIC HYDROXYL

G R O U P S O F TYROSINE RESIDUES (19) zO NH

I G R O U P S O F C Y S T E I N E RESIDUES (I)

SULFHYDRYL

×O CI-12--¢H--C"/ NH--

i

I

SH

NH

I I M I D A Z O L E G R O U P S O F HISTIDINE RESIDUES (/79 zO HC

C --CHz---

I

I

N.~c/NH H

CH--C'/---NH

-

l NH I

FIG. 1. Available functionalgroups in bovine serum albumin.

assay. It is this procedure that has led to the dramatic expansion of immunological techniques into the fields of biochemistry and pharmacology. In 1956 our laboratory, in collaboration with Beiser and Lieberman, became interested in preparing steroid-protein conjugates that were to be used to elicit antisteroid antibodies. An examination of the literature at that time showed that the azo coupling techniques of Landsteiner were still dominant. Like him, we chose to use the serum albumins because they were inexpensive and likely to yield soluble conjugates. However, an examination of the amino acid content of bovine serum albumin (BSA) (Fig. 1) convinced us that substitution by such relatively complex haptens as steriods should be attempted by reaction with the more plentiful Eamino groups of the lysine residues rather than by an azo coupling reaction with tyrosine, tryptophan, and imidazole residues. This meant forma-

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tion of amide bonds, for which a number of convenient new methods had been developed for the synthesis of peptides.Z A systematic approach was developed in which carboxylic acid groups were introduced into the haptens in various ways so that reaction with the amino groups of the protein carrier could be effected. Rather than deal with the steroid work separately, we will incorporate it into a general survey of the methods of preparing immunogenic haptenprotein conjugates in which the hapten is a pharmacologically interesting compound. The arrangement will be governed by the nature of the reactive functional groups of the hapten. In this way, the information can be applied most easily to new compounds being considered for use as determinant groups. No attempt will be made to present an exhaustive review of the literature. Instead, the various procedures described will be illustrated by specific examples to which the reader can refer for practical aspects of the experimental methods. Choice of Carrier The protein carriers used in various laboratories include globulin fractions, the serum albumins of various species, hemocyanin, ovalbumin, thyroglobulin, and fibrinogen. Hapten-protein conjugates of serum albumin are, in general, more soluble than conjugates of y-globulin or of ovalbumin. Thus, for example, steroid-protein conjugates of bovine, rabbit, and human serum albumin were soluble above pH 5.54"5; similar conjugates made with y-globulin and egg albumin frequently precipitated out of solution during preparation and could not be redissolved. Insoluble conjugates can be used for immunization, but subsequent characterization of the antibody then becomes a more difficult problem. Under certain circumstances, it may be advantageous to have both soluble and insoluble conjugates containing the same determinant group. The latter can be used for the isolation and purification of hapten-specific antibody. 6 For a review of insoluble hapten-carrier conjugates, we refer the reader to Jakoby and Wilchek r and Williams and Chase. 8 a j. p. Greenstein and M. Winitz, "'Chemistry of the Amino Acids," Vol.2. Wiley, New York, 1961. 4 B. F. Erlanger, F. Borek, S. M. Beiser, and S. Lieberman, J. Biol. Chem. 228, 713 (1957). 5 B. F. Erlanger, F. Borek, S. M. Beiser, and S. Lieberman, J. Biol. Chem. 234, 1090 (1959). 6 H. Szafran, S. M. Beiser, and B. F. Erlanger, J. lmmunol. 103, 1157 (1969). 7 W. B. Jakoby and M. Wilchek, eds., This series, Vol. 34. s C. A. Williams and M. W. Chase, eds., "Methods in Immunology and Immunochemistry," p. 335 et seq. Academic Press, New York, 1967.

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W h e t h e r the choice o f c a r d e r significantly influences the antihapten response is a controversial subject. In the author's opinion, no really definitive study has been c a r d e d out. On the other hand, there are some who believe that K L H is a superior carrier2 Thyroglobulin is a choice of others. ~° As noted above, we chose the serum albumins, as have the great majority of the laboratories engaged in developing radioimmunoassays. With respect to the advantage of using a protein rather than a synthetic polypeptide as a carrier, we can refer to Jaffe e t a l . , 11 who showed that an active fragment ofgastrin, its C-terminal tetrapeptide amide, was antigenic when covalently attached to serum protein carriers but not when linked to poly(L-lysine) or to poly(L-glutamic acid). Walker e t al.lZ in 1973 made similar comparisons with steroid conjugates and obtained the same result, i.e., bovine serum albumin was a better carrier than poly(L-lysine). (But also see below. 42-44) Optimal E p i t o p e D e n s i t y Another important question concerns the optimal number o f haptens bound to the carrier protein (i.e., optimal epitope density). N i s w e n d e r and Midgley, 13 using steroid protein conjugates, suggested that at least 20 molecules of hapten should be covalently attached to a BSA carrier. Less than that results in an inferior antigen. Klause and Cross ~4 in studies on (DNP)nBSA conjugates obtained good responses with as few as five D N P groups, with excellent booster responses. Comparable responses were obtained with (DNP)19BSA. On the other hand, (DNP)50BSA and DNPh0BSA elicited an IgM response only; no change (i.e., boost) in titer occurred after 21 days, even with repeated immunization. It has been our experience that the nature o f the hapten exerts an influence, but that good antibody titers can usually be obtained with epitope densities anywhere between 8 and 25. On the other hand, we have not hesitated to immunize with conjugates with fewer haptenic groups (as few as two) if we were unable to prepare " b e t t e r " conjugates (for example, with expensive olig o n u c l e o t i d e - p r o t e i n conjugates). We have n e v e r failed to obtain a response, although on occasion we have had to wait longer for a suitable titer. 9 M. B. Rittenberg and A. A. Amkraut, J. lmmunol. 97, 421 (1966). 10F. Bartos, G. D. Olsen, R. N. Leger, and D. Bartos, Res. Commun. Chem. Pathol. Pharmacol. 16, 131 (1977). It B. M. Jaffe, W. T. Newton, and J. E. McGuigan, Immunochemistry. 7, 715 (1970). 12C. S. Walker, S. J. Clark, and H. H. Wotiz, Steroids 21,259 (1973). 13G. D. Niswender and A. R. Midgley,Jr., in "Immunological Methods in Steroid Determination" (F. G. Peron and B. F. Caldwell, eds.), Ch. 8. Appleton, New York, 1970. 1~G. G. B. Klause and H. M. Cross, Cell. lmmunol. 14, 226 (1974).

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PRINCIPLES A N D M E T H O D S

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Location of the Linkage Site Before we go on to describe the various chemical reactions used to prepare conjugates, another important consideration must be recognized: the point of attachment on the hapten. Landsteiner established in his early studies that antibody specificity is directed primarily at the part of the hapten molecule farthest removed from the functional group that is linked to the protein carrier. 1 Our experiences have been similar. For example, many steroids share a common ring A structure. In agreement with Landsteiner, it was found that anti-testosterone-3-BSA was better able to distinguish among closely related steroids than was anti-testosterone-17BSA. 15-17 Other similar studies exist in the literature, la'~s Even better specificity has been obtained with conjugates in which the attachment to the steroid is via a spacer joining a protein to a position on the haptenic molecule that is not important for its biological specificity, e.g., the C-6 position of an estrogen, ~a-z~ on the C-6 or C-11 of progesterone. TM An excellent investigation on the effect of the site of conjugation of corticosteroids is that of Nishina e t a l . 22 The antibody raised to these conjugates is thus specific for all the important structural features of the hapten. Preparation of the Conjugates Regardless of the protein carrier used, the same functional groups are available for attachment to the hapten: the carboxyl groups of the C terminal and of the aspartic and glutamic acid residues, the amino groups of the N terminal and the lysine residues, the imidazo and phenolic functions of the histidine and tyrosine residues, respectively, and the sulfhydryl group of cysteine residues (Fig. 1). All have been used for the preparation of immunogenic hapten-protein conjugates. Theoretically, the guanidino group of arginine is also available, but, to our knowledge, it has not been utilized in the preparation of conjugates. The functional groups of the hapten govern the selection of the method to be used to conjugate the hapten to the functional groups of the carder. The procedures described below, therefore, have been classified accordis S. M. Beiser and B. F. Erlanger, Nature (London) 214, 1044 (1967). 16 S. M. Beiser, B. F. Erlanger, F. J. Agate, and S. Lieberman, Science 129, 564 (1959). 17 S. Lieberman, B. F. Erlanger, S. M. Beiser, and F. J. Agate, Recent Prog. Horm. Res. 15, 165 (1959). 18 j. E. Buster and G. E. Abraham, Anal. Lett. 6, 147 (1973). 19 H. R. Lindner, E. Peril, A. Friedlander, and A. Zeitlin, Steroids 19, 357 (1972). zo D. Exley, M. W. Johnson, and P. D. G. Dean, Steroids 19, 605 (1971) 21 S. L. Jeffeoate and J. E. Searle, Steroids 19, 181 (1972). 23 T. Nishina, A. Tsuji, and D. Fukushima, Steroids 24, 861 (1974).

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ing to the functional group of the hapten utilized for conjugation. In this way, it is hoped that the information may be applied most easily to compounds being considered for use as determinant groups. H a p t e n s with Carboxyl Groups This class of haptens includes those that have a carboxyl group, such as acetylsalicylic acid (aspirin) or the peptides angiotensin and bradykinin. In addition, many haptens, such as some steroids, may have reactive groups to which a carboxyl group can be attached as, for example, by reaction with succinic anhydride (see below). For conjugation to proteins, the same procedures may be used regardless of whether the carboxyl group is present as an inherent part of the hapten or as an added moiety.

Mixed Anhydride Procedure This is a simple, direct procedure 3'2a that does not require the preparation and isolation of an active derivative. The coupling procedure is carfled out directly with the hapten, and the product usually contains 1~-25 hapten groups per molecule of albumin. As an example of this method, the coupling of cortisone-21-hemisuccinate to protein 4 is illustrated (Fig. 2). The haptenic group was converted in situ to an acid anhydride, which could then react in an aqueous-acetone solution with the amino groups of serum albumin. Uridine 5'-carboxylic acid, ~4 testosterone-17-hemisuccinate,4 monosuccinyl ecdysterone,~5 3-O-succinyldigitoxigenin,26 cholic acid) 7 thyroxine, 2a prostaglandins) a synthetic estrogens, 3° clonazepam-3-hemisuccinate, al and reserpine 32 are among the compounds linked in this way.

Carbodiimides This is another direct method that has been used extensively in preparing conjugated antigens. Uridine 5'-carboxylic acid was coupled to a mulza j. R. Vaughan, Jr. and R. L. Osato, J. Am. Chem. Soc. 74, 676 (1952). 24 M. H. Karol and S. W. Tanenbaum, Proc. Natl. Acad. Sci. U.S.A. 57, 713 (1967). 25 M. L. deRiggi, M. H. Him, and M. A. Delaage, Biochem. Biophys. Res. Commun. 66, 1307 (1975). 2e G. C. Oliver, Jr., B. M. Parker, D. L. Brasfield, and C. W. Parker, J. Clin. Invest. 47, 1035 (1968). 27 G. J. Beckett, N. M. Hunter, and W. P.-R. Iain, Clin. Chim. Acta 88, 257 (1978). 2s W. H. Churchill and D. F. Tapley, Nature (London) 202, 29 (1964). 39 B. M. Jaffe, J. W. Smith, W. T. Newton, and C. W. Parker, Science 171, 494 (1971). 30 R. J. Warren and K. Fotherby, J. Endocrinol. 62, 605 (1974). 31 W. R. Dixon, R. L. Young, R. Ning, and A. J. Liebman, J. Pharm. Sci. 66, 235 (1977). 33 A. Levy, K. Kawashima, and S. Spector, Life Sci. 19, 1421 (1976).

92

PRINCIPLES AND METHODS 0

0

H

CHz" 0 "C'(CHz)2"COOH

t

C=O • OH

0///,,,~

[4]

It

CHz .OC. (CH2}2,,CO-O.CO.OR

I

o

II

RO.C.Cl,

C=O OH

pH9-9.5 / PROTEIN.NHz ~ Hz'OC'ICHz)z* CO, NH ) C" 0

"-~'OH /

PROTEIN

+ CO2 -h ROH

FIG. 2. Preparation of cortisone-21-hemisuccinate conjugate by mixed anhydride procedures.

tichain polypeptide, poly(DL-alanyl)-poly(L-lysine) with dicyclohexylcarbodiimide in a 95% dimethylformamide medium as solvent, a3 Coupling reactions can be carried out in aqueous solution by use of the water-soluble carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide • HCI or 1-cyclohexyl-3-[2-morpholinyl-(4)-ethyl]carbodiimide methop-toluenesulfonate,a4,a5 both commercially available reagents. Angiotensin and bradykinin, both small polypeptides (MW approximately 1000), were coupled to proteins in the first utilization of this procedure, a" The authors believed that the reaction was between the N-terminal amino group of the peptide and the protein, but provided no evidence for this. On the other hand, they later used similar techniques to couple angiotensin to polylysine with the water-soluble reagent N-ethylbenzisoxazole,37 a reaction that is possible only if the carboxyl group of angiotensin participates. One case in which it was definitely established that carbodiimides activate the carboxyl group of the peptide relates to the production of antibody to gastrin tetrapeptide. 11 In this case, the amino end of the peptide was blocked by a t e r t - b u t y l o x y c a r b o n y l (t-BOC) group. The t-BOC group was subsequently removed with trifluoroacetic acid. Carbodiimides were also used by Dietrich 3a and by Haber et al. a9 Additional components of a3 M. Sela, H. Ungar-Waron, and Y. Schechter, Proc. Natl. Acad. Sci. U.S.A. 52, 285 (1%4). J, C. Sheehan, P. A. Cruickshank, and G. L. Boshart, J. Org. Chem. 26, 2525 (1%1). a5 j. C. Sheehan and J. S. Hlavka, J. Org. Chem. 21, 439 (1956). ae T. L. Goodfriend, L. Levine, and G. Fasman, Science 143, 1344 (1964). a7 T. L. Goodfriend, G. Fasman, D. Kemp, and L. Levine, lmmunochemistry 3, 223 (1966). as F. M. Dietrich, Immunochemistry 4, 65 (1%7). aa E. Haber, L. B. Page, and G. A. Jacoby, Biochemistry 4, 693 (1965).

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biological interest that were coupled to carriers with water-soluble carbodiimides include gastrin, 4° adenosine Y,5'-cyclic phosphate, 41 morphine, 42 lysergic acid diethylamide, 43 and prostaglandin. 44,45 In three cases, 4z-44 the carrier used was polylysine and immunization was done with a complex of the conjugate and succinylated hemocyanin. This improved technique minimized the extent of the immunological response to the carrier portion of the immunogen. Tobramycin, 46 1-/3-E-arabinofuranosylcytosine,47 cocaine metabolites, 4a prednisone-21-hemisuccinate, 4a synthetic narcotic analgesic drugs, 50 DL-methadol-hemisuccinate, 1° ochratoxin A, 51 digitoxigenin, 26 and gentamicin 52 are among additional compounds linked to proteins by means of carbodiimides. Digitoxigenin 26 was linked to a carrier protein, by both the carbodiimide and the mixed anhydride procedures, in the same laboratory. The mixed anhydride yielded a conjugate with 13 haptenic groups compared with 5 groups via the carbodiimide procedure. Specific antibodies to a-melanotropin were obtained with carbodiimide using the peptide with its internal lysine blocked by an ~-methylsulfonylethoxycarbonyl group. Linkage to the amino groups of a protein then occured via the single glutamic acid residue remaining in the peptide, after which the protecting group was removed by 2 N Na2CO3. 53 If the haptenic molecule is not soluble in water, it can be dissolved in water-miscible solvents, such as dimethylformamide, and added to the aqueous protein solution. 45 40 j. D. Young, D. J. Byrnes, D. J. Chisholm, F. B. Griffiths, and L. Lazarus, J. Nucl. Med. 10, 746 (1969). 41 A. L. Steiner, D. M. Kipnis, R. Utiger, and C. Parker, Proc. Natl. Acad. Sci. U.S.A. 64, 367 (1969). 42 H. Van Vunakis, E. Wasserman, and L. Levine, J. Phmmacol. Exp. Ther. 180, 514 (1972). 43 H. Van Vunakis, J. T. Farrow, H. B. Gjika, and L. Levine, Proc. Natl. Acad. Sci. U.S.A. 68, 1483 (1971). 44 L. Levine and H. Van Vunakis, Biochem. Biophys. Res. Commun. 41, 1171 (1970). 45 F. A. Fitzpatrick and G. L. Bundy, Proc. Natl. Acad. Sci. U.S.A. 75, 2689 (1978). A. Broughton, J. E. Strong, L. K. Picketing, and G. P. Brodey, Antimicrob. Agents Chemother. 10, 652 (1976). 47 T. Okabayashi, S. Mihara, D. B. Repke, and J. G. Moffat, Cancer. Res. 37, 619 (1977). 4a B. Kaul, S. J. Millian, and B. J. Davidow, Pharmacol. Exp. Ther. 199, 171 (1976). 49 A. W. Meikle, J. A. Weed, and F. H. J. Tyler, J. Clin. Endocrinol. Metab. 41,717 (1975). 5o H. Van Vunakis, D. S. Freeman, and H. B. Gjika, Res. Commun. Chem. Pathol. Pharmacol. 2, 379 (1975). 51 O. Aalund, K. Brunfeldt, B. Hald, P. Krogh, and K. Poulsen, Acta Pathol. Microbiol. Scand. C 83, 390 (1975). 52 j. E. Lewis, J. C. Nelson, and H. A. Elder, Nature (London) 94, 214 (1972). H. G. Kopp, A. Ebede, P. Vitius, W. Lichensteiger, and R. Schwyzer, Fur. J. Biochem. 75, 417 (1977).

94

PRINCIPLES AND METHODS + 0 NR' 0 NR' O NHR' II II II II H+ II II RC-OH + C.--~RC-O-C ~ RC-O-C INIR' INHR' /HR, O ,, R~-NR'

I C:O

[4]

R"NHz ropid

>

R"NHz very slow

0II H L RC--NR" O+

R' H II __ N HR' N--C

NIl.iR' Fio. 3. Mechanism of carbodiimide-mediated preparation of amides.

The conditions of the reaction are very simple. The carder, an excess of hapten and the reagent are simply stirred together in an aqueous solution for 30 min to several days depending upon the procedure. The reaction is followed by dialysis, and the product is isolated by lyophilization. The reaction mechanism is as shown in Fig. 3. There are two possible pathways, the desired one being catalyzed by H ÷. The protein carrier, however, is most reactive at higher pH, where dissociation of the lysine ammonium groups occurs. A compromise is therefore necessary to provide the most favorable conditions; a pH near 6 is usually chosen. In our experience, the use of water-soluble carbodiimides has not always been successful. On occasion, extensive alteration of the carder has occurred with little if any substitution by haptenic groups. It is possible to be led astray because antibody is produced to the altered protein, and this antibody does not react with the protein in its original state. Nevertheless, it is a generally efficacious method of preparing conjugates. It is of interest that water-soluble carbodiimides have been used to couple nucleotides directly to proteins, presumably by formation of a P-N bond.54-s8

Miscellaneous Carboxyl Methods An aspirin-protein conjugate was prepared by first converting aspirin (acetylsalicylic acid) to the acetylsalicylazide.59 The azide was coupled to rabbit serum globulin in a 1:1 dioxane-water solution maintained alkaline to phenolphthalein by the addition of base. About 25 to 35 haptenic M. J. Halloran and C. W. Parker, J. lmmunol. 96, 373 (1966). 55 M. J. Halloran and C. W. Parker, J. lmrnunol. 96, 379 (1966). se M. Z. Humayun and T. M. Jacob, Biochim. Biophys. Acta 331, 41 (1973). 5T S. A. Khan, M. Z. Humayun, and T. M. Jacob, Nucl. Acids Res. 4, 2997 (1977). 5s S. A. Khan and T. M. Jacob, Nucl. Acids Res. 4, 3007 (1977). 5a G. C. Butler, C. R. Harington, and M. E. Yuill, Biochem. J. 34, 838 (1940).

[4]

ANTIGENIC HAPTEN--CARRIER CONJUGATES

95

o

O RC" OH+HO o

II H

DCC~ R 'O

R'NH2 RC-NR'

o

o

\\

FIG. 4. Preparation of amides using N-hydroxysuccinimide. DCC, dicyclohexyicarbodiimide. groups were conjugated per molecule of globulin. A similar procedure was used for thyroxine.e° Antibodies specific for thyroxine have also been obtained by using, as antigen, tetraiodothyropropionic acid coupled to protein by the mixed anhydride method. 28 The conversion of aspirin to an acid chloride that can react directly with protein was also reported. 61 The insect juvenile hormone DL-10,11-epoxyfarnesoic acid was coupled to protein by a procedure that should find extensive use. 6~,6s We had been unable to effect the reaction with water-soluble carbodiimides, obtaining only unsubstituted, altered protein (see above). The N-hydroxysuccinimide ester was prepared by reaction o f the juvenile h o r m o n e with N-hydroxysuccinimide (commercially available) in the presence of dicyclohexylcarbodiimide (Fig 4). N - H y d r o x y s u c c i n i m i d e esters are used in peptide synthesis. 64 They are quite stable if kept dry but react quickly and in good yield with amino groups to form amide or peptide bonds. Conjugates containing 20 juvenile hormone groups were used to raise specific antibodies in rabbits. Antibodies to e c d y s o n e were made similarly. 6~ Carbonyldiimidazole is another commercially available reagent that has been used to link haptens to proteins by means o f amide bonds, for example in the preparation o f a BSA conjugate of fluoxymesterone. 66 R. F. Clutton, C. R. Harrington, and M. E. Yuill, Biochem. J. 32, 1119 (1938). el L. M. Weiner, M. Rosenblatt, and H. Howes, J. lmmunol. 90, 788 (1963). 62 R. C. Lauer, P. Soloman, K. Nakanishi, and B. F. Erlanger, Fed. Proc. 32, 500 (1972). sa R. C. Lauer, P. H. Soloman, K. Nakanishi, and B. F. Erlanger, Experientia 30, 558 (1974). 84G. W. Anderson, J. E. Zimmerman, and F. M. Callahan, J. Am. Chem. Soc. 86, 1839 (1964). e5 R. C. Lauer, P. H. Soioman, K. Nakanishi, and B.F. Erlanger, Experientia 30, 560 (1974). 66W. A. Colburn, Steroids 25, 43 (1975). so

96

PRINCIPLES AND METHODS

[4]

H a p t e n s with Amino Groups Two classes of haptens with available amino groups must be conside r e d - t h e aromatic amines and the aliphatic amines.

Aromatic Amines Much of Landsteiner's pioneer work 1 was carried out with haptens that were aromatic amines. The compounds were converted to diazonium salts with nitrous acid and allowed to react with proteins at alkaline pH (approximately 9). Reaction occurred primarily with histidine, tyrosine, and tryptophan residues of the protein carrier. For a representative procedure, see Kabat 67 (p. 799 et seq.). An interesting application of this procedure was the preparation of a chloramphenicol-protein conjugate which was used to elicit antibodies specific for chloramphenicol. °s In this case, a prior reduction of the nitro group of chloramphenicol to an amino group was required. As early as 1937, carcinogenic compounds were conjugated to protein carriers by means of their isocyanate derivatives which were prepared from amines. 69 Immune sera were raised, and their properties were studied. 69,7°

Aliphatic Amines Aliphatic amines can be caused to react with proteins by using watersoluble carbodiimides. Examples include bradykinin and angiotensin, 36 tobramycin, 46 gentamicin, 53 adriamycin, 71 5-hydroxytryptamine (serotonin), 72 cortisol-21-amine, 7z and spermidine, r4 Aliphatic amines can be converted to a p-nitrobenzoylamide by reaction with p-nitrobenzoyl chloride. The amide derivative can then be re-, duced to a p-aminobenzoyl derivative which can be coupled to proteins by diazotization, as described above. Among the haptens conjugated this 67 E. A. Kabat, "Kabat and Mayer's Experimental Immunochemistry," 2nd ed. Thomas, Springfield, Illinois, 1961. 68 R. N. Hamburger, Science 152, 203 (1966). ~ H. J. Creech, Cancer Res. 12, 557 (1952). 70 H. J. Creech and W. R. Franks, Am. J. Cancer 30, 555 (1937). 71 y . -H. Chien and L. Levine, lmmunochemistry 12, 291 (1975). 72 B. Peskar and S. Spector, Science 179, 1340 (1973). 73 y. Kobayashi, T. Ogihara, K. Amitani, F. Watanabe, T. Kigushi, I. Ninomiya, and Y. Kumahara, Steroids 32, 137 (1978). 74 F. Bartos, D. Bartos, A. M. Dolney, D. P. Grettie, and R. A. Campbell, Res. Commun. Chem. Pathol. Pharmacol. 19, 295 (1978).

[4]

ANTIGENIC HAPTEN--CARRIER CONJUGATES

97

way are a series of C-terminal peptide sequences of the tobacco mosaic virus protein 75-77 and angiotensin, r8"79 Angiotensin has also been attached by its N-terminal amino group to the amino groups of a carrier by means of the bifunctional reagent m-xylylene diisocyanate,aa Tolylene 2,4-diisocyanate has been used in a similar manner to prepare bradykinin conjugates, s° Haptens containing amino groups have also been covalently linked to amino groups of protein carriers with glutaraldehyde. Among the haptenic groups conjugated in this manner are adrenocorticotropic hormone (ACTH), sl glucagon, 82 and normetanephrine.83 A novel procedure has been used to link nortryptyline to BSA. 84 Its aliphatic secondary amine was converted to a succinamic acid derivative that was caused to react with the protein by using a water-soluble carbodiimide. The antibody was used to assay for various tricyclic antidepressants, including imipramine. In another novel procedure involving nortryptyline, the secondary amine was allowed to react with N-(4-bromobutyl)phthalimide. After removal of the phthalimido group, the resulting primary aliphatic amine was caused to react with carboxyl groups on BSA by using a water-soluble carbodiimide. 85"86 H a p t e n s with Available Hydroxyl Groups This class of haptens includes alcohols, phenols, sugars, polysaccharides, and nucleosides. In most cases, derivatives of this class of compounds must be made in order to introduce functional groups capable of reacting with proteins. Hemisuccinates

A simple procedure, first introduced in our work with steroid-protein conjugates, is the conversion of the alcohol to the half ester of succinic 75 F. A. Anderer, Biochim. Biophys. Acta 71, 246 (1963). 76 F. A. Anderer and H. D. Schlumberger, Biochim. Biophys. Acta 115, 222 (1966). 77 F. A. Anderer and H. D. Schlumberger, Biochim. Biophys. Acta 97, 503 (1965). 78 S. D. Deodhar, J. Exp. Med. 111,419 (1960). 79 S. D. Deodhar, J. Exp. Med. 111, 429 (1960). s o j. Spragg, K. F. Austen, and E. Haber, J. Immunol. 96, 865 (1966). s~ M. Reichlin, J. J. Schnure, and V. K. Vance, Proc. Soc. Exp. Biol. Med. 128, 347 (1968). s2 L. A. Frohman, H. Reichlin, and J. E. Sokal, Endocrinology 87, 1055 (1970). 83 B. A. Peskar, B. M. Peskar, and L. Levine, Eur. J. Biochem. 26, 191 (1972). 84 D. J. Brunswick, B. Needleman, and J. Mendels, Life Sci. 22, 137 (1978). s5 G. W. Aherne, E. M. Piall, and V. Marks, Br. J. Clin. Pharmacol. 3, 561 (1976). s 6 K. P. Maguire, G. D. Burrows, T. R. Norman, and B. A. Scoggins, Clin. Chem. 24, 549 (1978).

98

PRINCIPLES AND METHODS

[4]

acid (i.e., the hemisuccinate). The hemisuccinate has an available carboxyl group that can be made to react by any of the procedures described above. Conversion to the hemisuccinate requires a reaction with succinic anhydride in pyridine. A representative procedure can be found in the papers on steroid-protein conjugates. 4~ Another example is the preparation of the hemisuccinate of cyclic adenosine monophosphate (cAMP). 41 More recently, in an excellent study of the specificity of antiestrogen antibodies, s7 1la-hydroxyhemisuccinates of estrone and estradiol were prepared and linked to BSA by the mixed anhydride technique. Other examples include hemisuccinates of fl-dl-methadol, ~° 3-hydroxyclonazepam,31 ecdysterone, 25 propanolol, 88 Aa-tetrahydrocannabinol,sa and 1-fl-D-arabinofuranosylcytosine.47

Chlorocarbonates Another alternative is the reaction of the determinant group with an equimolar quantity of phosgene to yield the highly reactive chlorocarbonate, which reacts directly with the amino groups of the protein in the presence of bicarbonate. An example is the conversion of testosterone to testosterone- 17-chlorocarbonate.4

Aminophenyl Derivatives Phenols can be converted to active reagents by reaction with diazotized p-aminobenzoic acid. In this way, a carboxyl group is introduced into the molecule. This type of reaction was carried out successfully with 17fl-estradiol. 9° More recently, a similar procedure was used to make conjugates of AT-tetrahydrocannabino189 and reserpine, zz The classical procedure for the coupling of sugars to proteins involves the formation ofp-nitrophenylglycosides, the conversion of the latter by hydrogenation to p-aminophenylglycoside, and then attachment to the protein by diazotization. This method was used by Landsteiner 1 for a number of preparations. A variant of this method, used by Goebel 9L9zand Goebel and Hotchkiss, 93 was conversion to the aminobenzyl ether followed by diazotization. F. C. den Hollander, B. K. van Weemen, and G. F. Woods, Steroids 23, 549 (1974). K. Kawashima, A. Levy, and S. Spector, J. Pharmacol. Exp. Ther. 196, 517 (1976). s a p . T. Tsui, K. A. Kelly, M. M. Ponpipon, and A. H. Sehon, Can. J. Biochem. 52, 252 (1974). 9o N. Weliky and H. H. Weetall, Immunochemistry 2, 293 (1965). 91 W. F. Goebel, J. Exp. Med. 64, 29 (1936). 92 W. F. Goebel, J. Exp. Med. 68, 469 (1938). 93 W. F. Goebel and R. D. Hotchkiss, J. Exp. Med. 66, 191 (1937). s7 ss

[4]

ANTIGENIC HAPTEN--CARRIER CONJUGATES

p

IOZ U

II

OH OH

0

pH8.5-9.2

~'or BHaCN-

\N/

99

0

I ProtoNHz

HO-'CH HC-OH

I

\N/ I

Prot

Prot P= Purine or Pyrimidine R = H ,- PO3H2 or 5'- nucleotide

Prot*NH2= Carrier protein, NHz groups

FIG. 5. Preparation of conjugates using periodate procedure.

Oxidation to Dialdehydes

A relatively simple procedure developed for the preparation of nucleoside- and nucleotide-protein conjugate ~-98 makes use of the reaction of vicinal hydroxyl groups with periodate to yield dialdehydes (Fig. 5). The dialdehydes, without isolation, are caused to react with the amino groups of protein at pH 9.5 in aqueous solution to yield aldimines, which are stabilized by reduction with sodium borohydride. Only the final conjugate is isolated in this procedure, which is simple to run and yields conjugates with as many as 30 determinant groups per molecule of albumin. It should be applicable to all compounds with vicinal hydroxyl groups, such as glycols, glycerol derivatives, and glycosides, and has been used successfully for the preparation of digoxin-protein conjugates 99 and ouabain.1°° Modi-

~" B. F. Erlanger and S. M. Beiser, Proc. Natl. Acad. Sci. U.S.A. 52, 68 (1964). 95 B. F. Erlanger, D. Senitzer, O. J. Miller, and S. M. Beiser, Acta Endocrinol. (Copenhagen) Suppl. 168, 206 (1972). R. M. D'Alisa and B. F. Erlanger, Biochemistry 13, 3575 (1974). 97 R. M. D'Alisa and B. F. Erlanger, J. Immunol. 116, 1629 (1976). S. M. Beiser, S. W. Tanenbaum, and B. F. Erlanger, this series, Vol. 12B, p. 889. V. P. Butler and J. P. Chen, Proc. Natl. Acad. Sci. U.S.A. 57, 71 (1967). 1oo T. W. Smith, J. Clin. Invest. 51, 1583 (1972).

100

PRINCIPLES AND M E T H O D S

[4]

fications have been made to prepare conjugates with alkaline-sensitive nucleosides and nucleotides. 1°"°3 In a recent procedure to detect carcinogen-DNA adducts by radioimmunoassay, 1°4 N-(guanosin-8-yl)acetylaminofluorine was linked to BSA by the periodate method, and the conjugate was used to elicit specific antibodies to this product, which is formed when N-acetoxy-2-acetylaminofluorene reacts with DNA. Oxidation to Carboxyl Oxidation of the 5'-hydroxyl groups of uridine, z4"1°5 pseudouridine, 24 and other nucleosides 33 has made it possible to conjugate these compounds to proteins by methods amenable for the reaction of carboxylic acid derivatives with proteins. Miscellaneous Hydroxyl Methods Another method of seemingly general applicability to carbohydrates was used by Coat et al. 106to conjugate uridine to proteins. The isopropylidine derivative was allowed to react with p-nitrobenzoyl chloride to yield the 5' ester. Removal of the isopropylidine protecting group and hydrogenation of the nitro group made it possible to link the uridine derivative to the protein by a diazotization reaction. Some rather novel chemistry was described in two recent very interesting papers describing a radioimmunoassay procedure for ADP-ribose. 1°7 Adenine-N6-carboxymethylated NAD was prepared and converted to N6-carboxymethylated ADP-ribose by NAD glycohydrolase. N6-Carboxymethylated ADP-ribose was then linked to BSA using watersoluble carbodiimide. The bifunctional reagent sebacoyl dichloride has been used to convert alcohols to acid chlorides, which, at pH 8.5, react readily with proteins. This procedure was used by Bailey and Butler l°s to prepare a cholesterolprotein conjugate. ~o~ L. Rainen and B. D. Stollar, Nucl. Acids Res. 4, 4877 (1978). lo2 R. D. Meredith and E. F. Erlanger, Fed. Proc. 37 (6), 1503 (1978). 1o3 j. Wollack, Fed. Proc. 37 (6), 1632 (1978). ~o4 M. C. Poirer, S. H. Yuspa, I. B. Weinstein, and S. Blobstein, Nature (London) 270, 186 (1977). ~05 M. Sela and H. Ungar-Waron, Fed. Proc. 24, 1438 (1965). ~0~ j. p. Coat, S. David, and J. C. Fischer, Bull. Soc. Chim. Ft. 21, 2489 (1965). ~07 R. Bredehorst, A. M. Ferro, and H. Hilz, Fur. J. Biochem. 82, 105 (1978). los j. M. Bailey and J. Butler, in "'The Reticuloendothelial System and Atherosclerosis" (N. R. DiLuzio and R. Paoletti, eds.), pp. 433-441. Plenum, New York, 1967.

[4]

ANTIGENIC HAPTEN--CARRIER CONJUGATES

101

H a p t e n s with C a r b o n y l G r o u p s K e t o n e s and aldehydes can be used as haptenic determinant groups by converting them to O - ( c a r b o x y m e t h y l ) oximes. This is done by reacting t h e m with O - ( c a r b o x y m e t h y l ) h y d r o x y l a m i n e ( N H 2 O C H 2 C O O H , sold c o m m e r c i a l l y as c a r b o x y l m e t h o x y l a m i n e or a m i n o o x y a c e t i c acid). This serves to introduce a carboxyl group, which is exploited as described above. E x a m p l e s o f this m e t h o d o l o g y can be found in the coupling of testosterone-3-(O-carboxymethyl) oxime, estrone-17-(O-carboxymethyl) oxime, and progesterone-20-(carboxymethyl) oxime to bovine serum albumin with the mixed anhydride technique. 4,5 Prepared in a similar manner were the 3-(O-carboxymethyl) oxime derivative of m e d r o x y p r o g e s terone acetate, l°a the 3 - c a r b o x y m e t h y l oxime of aldosterone-18-21-diacetate. 11° Similarly, O - c a r b o x y m e t h y l derivatives were prepared from the synthetic progestogens norethisterone and norgestrel. H1 5 a - D i h y d r o t e s t o s t e r o n e - l l - ( O - c a r b o x y m e t h y l ) oxime was synthesized H2 in an elegant multistep p r o c e d u r e that included a microbiological reduction and a selective hydrolysis of a dioxime. The final p r o d u c t was p r e p a r e d by reaction of 17fl-hydroxy-5a-androstane-3, 11-dione-11-oxime with sodium chloroacetate to give the O - c a r b o x y m e t h y l oxime. The ketone groups of aldosterone, corticosterone, and cortisol were derivatized with p - h y d r a z i n o b e n z o i c acid. ~13 The resulting carboxylic acid derivatives could be linked to BSA with water-soluble carbodiimide. Aldehydes can be conjugated to proteins directly by Schiff base formation followed by stabilization of the bond by reduction with sodium borohydride. Pyridoxal and pyridoxal p h o s p h a t e are examples of haptens conjugated in this manner. ~4'H5 Other Reactions Penicillenic acid was conjugated to protein by an interesting p r o c e d u r e that included modification o f the protein carrier, u6 Penicillenic acid has a reactive sulfhydryl group capable of forming disulfide bonds with other lo9 M. Hiroi, F. Z. Stanczyk, U. Goebelsmann, P. F. Brenner, M. E. Lumkin, and D. R. Mishele, Jr., Steroids 26, 373 (1975). 110C. A. Bizoleon, J. -F. Riviere, P. Franchimont, A. Faure, and B. Claustrat, Steroids 23, 809 (1974). 111R. J. Warren and K. Fotherby, J. Endocrinol. 62, 605 (1974). 112T. S. Baker and D. Exley, Steroids 29, 429 (1977). 113B. Africa and E. Haber, lmmunochemistry 8, 479 (1971). 114F. Cordoba, C. Gonzalez, and P. Rivera, Biochim. Biophys. Acta 127, 151 (1966). 115R. Ungar-Waron and M. Sela, Biochim. Biophys. Acta 124, 147 (1966). ,e A. L. deWeck and H. N. Eisen, J. Exp. Med. 112, 1227 (1960).

102

PRINCIPLES AND METHODS

[4]

sulfhydryl groups. The carrier proteins (e.g., human y-globulin or bovine y-globulin) were artificially enriched in sulfhydryl groups by reaction with N-acetylhomocysteine thiolactone. 117 The coupling reaction with an excess of penicillenic acid was then carried out in acetate buffer at pH 4 in the presence of H202. Antibody to progesterone was also obtained by immunization with conjugates prepared from thiolated proteins.H.8 Bovine serum albumin was thiolated by reaction with S-acetylmercaptosuccinic anhydride. After deacetylation, coupling was achieved with 6fl-bromoprogesterone. Bis-diazotized benzidine can be used as a bridging reagent between proteins and haptens containing aromatic groups that react with diazonium compounds. A conjugate of thyrotropin-releasing hormone (which contains a reactive histidine residue) was obtained in this way.~9 A novel approach has been to allow serotonin to react with protein via the Mannich reaction? 2° This is a simple reaction that enables one to use formaldehyde as a bridge between the amino groups of a protein and compounds containing one or more reactive hydrogens. The Mannich reaction has also been used to prepare reserpine conjugates, s2 The antibody titers were not as satisfactory as those elicited by conjugates prepared by a pcarboxyazobenzene derivative linked to BSA by the mixed anhydride procedure. Among the low molecular weight haptens that have been used as determinant groups are substances that are reactive enough to be coupled to proteins directly. Dinitrofluorobenzene has been used to prepare antigens for the stimulation of antidinitrophenyl antibodies. These have been very useful in studies of the binding characteristics and structure of immunoglobulins. Antibodies that react with deoxyribonucleic acid (DNA) have been elicited by immunization with the product of the reaction of 6-trichloromethylpurinex21-~3 with BSA. Nucleotide protein conjugates have also been made by using carbodiimide to link the nucleotide to the protein. ~6-~s Antipenicillin antibodies have been produced by immunization 117 R. Benesch and R. E. Benesch, in "Sulfur in Proteins" (R. Benesch, R. E. Benesch, P. D. Boyer, I. M. Kiotz, W. R. Middlebrook, A. G. Szent-Gyrrgyi, and D. R. Schwartz, eds.), pp. 15-24. Academic Press, New York, 1959. 11s C. N. Pang and D. C. Johnson, Steroids 23, 203 (1974). H9 R. M. Bassiri and R. D. Utiger, Endocrinology 90, 722 (1972). 12o N. S. Ranadive and A. H. Sehon, Can. J. Biochem. 48, 1701 (1967). 121 S. Cohen, E. Thom, and A. Bendich, Biochemistry 2, 176 (1963). 1~ S. Cohen, E. Thom, and A. Bendich, J. Org. Chem. 27, 3545 (1962). l~a V. P. Butler, Jr., S. M. Beiser, B. F. Erlanger, S. W. Tanenbaum, S. Cohen, and A. Bendich, Proc. Natl. Acad. Sci. U.S.A. 48, 1597 (1962).

[4]

ANTIGENIC HAPTEN--CARRIER CONJUGATES

103

with penicillin-protein conjugates. The latter were prepared by the reaction o f penicillin with protein under slightly alkaline conditions. 124-12e A t e t r a h y d r o c a n n a b i n o l - B S A conjugate has been made by reaction with 10-iodotetrahydrocannabinol-9-isocyanate. 89 L-Phenylalanine mustard is another example of a reactive hapten,127 C h a r a c t e r i z a t i o n of t h e C o n j u g a t e s Generally, the haptenic group has an absorbance spectrum that can allow one to differentiate it from the protein carder. This is particularly true for azo derivatives, which absorb in the visible range. H o w e v e r , even if there is overlap in the two spectra, reasonably accurate determinations of the number of haptenic molecules per carrier protein can be determined from difference spectra. A more convenient and direct procedure, introduced by Abraham e t a l . , 128 is the incorporation o f some radioactive hapten in the conjugation procedure. A direct estimation of the extent o f substitution can be made by counting undialyzable radioactive material. A procedure introduced by us for s t e r o i d - p r o t e i n conjugates 4 was the estimation of the remaining free amino groups with the dinitrophenylation technique of Sanger.129 e-Dinitrophenyllysine was not isolated but was estimated directly by spectrophotometry after ether extraction of the acid hydrolyzate. A control with unsubstituted c a r d e r was always run concomitantly, and the difference between the two was taken to be the extent o f substitution by hapten. We have also used a procedure of H a b e e b 13° in which trinitrobenzene sulfonic acid is used as the reagent for estimation of free amino groups in the conjugate. Spectrophotometric comparison of theintactprotein conjugate and the original carrier protein is possible; no acid hydrolysis is required. We found this procedure to be convenient and entirely satisfactory, an example being the case of insect juvenile h o r m o n e - p r o t e i n conjugates. 62,63,6~

n4 B. B. Levine, N. Engl. J. Med. 275, 1115 (1966). 125B. B. Levine and Z. Ovary, J. Exp. Med. 114, 875 (1961). ~z6H. Smith, J. M. Dewdney, and A. W. Wheeler, Immunology 21, 527 (1971). 127j. F. Burke, V. H. Mark, A. H. Soloway, and S. Leskowitz, CancerRes. 26, 1893(1966). ~2sG. E. Abraham, R. Swerdloff, D. Tulchinsky, and W. D. Odell, J. Clin. Endocrinol. Metab. 32, 619 (1971). ~29F. Sanger, Biochem. J. 45, 563 (1949). lao A. F. S. A. Habeeb, Anal. Biochem. 14, 328 (1966).

104

PRINCIPLES

AND

METHODS

[5]

General Comments It was not the purpose of this review to present a comprehensive survey of hapten-protein conjugates, but rather to provide sufficient information to guide the researcher in the design of his or her particular experiments. On the other hand, the most practical approaches to the preparation of hapten-protein conjugates were cited. Many of the methods used to prepare immunogenic conjugates have also been used to link drugs to carrier molecules (including antibodies) in order to "target" cytotoxic drugs. Two reviews that are useful in that they describe many of the methods used to make the carder-drug conjugates are those by Trouet T M and by Ghose. 'a2 The information in these reviews should be useful to immunologists as well. ,a, A. T r o u e t , Eur. J. Cancer 14, 105 (1978). ,3z T. G h o s e , J. Natl. Cancer Inst. 61, 657 (1978).

[5] P r o d u c t i o n

of Reagent

Antibodies

By B. A. L. HURN and SHIREEN M. CHANTLER Immunization The explosion of interest in immunoassay procedures during the last two decades has resulted in an enormous volume of literature describing, for the most part, satisfactory results of immunization. During the same period, knowledge of the underlying mechanisms of the immune response has advanced greatly from an original state of almost total ignorance. Perhaps unfortunately, those who have pursued basic understanding have seldom been much concerned with the practical problems of making useful reagent antibodies. As a result, with few exceptions the literature of immunization does no more than describe successful procedures, and the variety of these is legion. In the usual way of things, abortive attempts are seldom mentioned, let alone described, yet anyone with practical experience who has also discussed the matter with colleagues will be well aware that all the successful methods have also, at other times or in other places, singularly failed to give the desired results. Not surprisingly, the failure rate is higher when making antisera for more demanding test systems, such as radioimmunoassay, than for immunoprecipitin methods, for instance. Much of the uncertainty over the outcome of immunization may be ascribed to variations in individual animal response; however, when an METHODS IN ENZYMOLOGY, VOL. 70

Copyrighl © 19~0by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181970-1

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General Comments It was not the purpose of this review to present a comprehensive survey of hapten-protein conjugates, but rather to provide sufficient information to guide the researcher in the design of his or her particular experiments. On the other hand, the most practical approaches to the preparation of hapten-protein conjugates were cited. Many of the methods used to prepare immunogenic conjugates have also been used to link drugs to carrier molecules (including antibodies) in order to "target" cytotoxic drugs. Two reviews that are useful in that they describe many of the methods used to make the carder-drug conjugates are those by Trouet T M and by Ghose. 'a2 The information in these reviews should be useful to immunologists as well. ,a, A. T r o u e t , Eur. J. Cancer 14, 105 (1978). ,3z T. G h o s e , J. Natl. Cancer Inst. 61, 657 (1978).

[5] P r o d u c t i o n

of Reagent

Antibodies

By B. A. L. HURN and SHIREEN M. CHANTLER Immunization The explosion of interest in immunoassay procedures during the last two decades has resulted in an enormous volume of literature describing, for the most part, satisfactory results of immunization. During the same period, knowledge of the underlying mechanisms of the immune response has advanced greatly from an original state of almost total ignorance. Perhaps unfortunately, those who have pursued basic understanding have seldom been much concerned with the practical problems of making useful reagent antibodies. As a result, with few exceptions the literature of immunization does no more than describe successful procedures, and the variety of these is legion. In the usual way of things, abortive attempts are seldom mentioned, let alone described, yet anyone with practical experience who has also discussed the matter with colleagues will be well aware that all the successful methods have also, at other times or in other places, singularly failed to give the desired results. Not surprisingly, the failure rate is higher when making antisera for more demanding test systems, such as radioimmunoassay, than for immunoprecipitin methods, for instance. Much of the uncertainty over the outcome of immunization may be ascribed to variations in individual animal response; however, when an METHODS IN ENZYMOLOGY, VOL. 70

Copyrighl © 19~0by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181970-1

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experimental comparison of different procedures is made in such a way as to overcome the effect of individual animal variation, the results may well be inconclusive or irreproducible despite the considerable effort involved.1 Regrettably, then, it must be said that information concerning methods of immunizing laboratory animals is almost entirely anecdotal. The available evidence strongly suggests that there are influences as yet unrecognized that may be as important to success as any of the factors already known. Nevertheless, while acknowledging the significance of art, green fingers, or even plain luck, it is worth considering the known factors briefly so as to provide some evidence in support of the methods of immunization recommended later; they are related to the immunogen, the adjuvant, the choice of animal, the route of injection, and the dosage schedule. The Immunogen

Particulate (cellular) materials, such as heterologous erythrocytes or bacteria, are usually intensely immunogenic, producing a rapid response when administered without adjuvant of any sort. The major problem likely to be encountered is lack of the desired specificity in the resultant antiserum, since the particles have a complex antigenic structure much of which may be shared with other more or less closely related cell types. Short immunization courses are usually adequate but often give rise to a high proportion of IgM antibody, which may be very satisfactory in agglutination techniques but tends to be less stable during storage than IgG. Most antigens of interest to immunoassayists are soluble materials that vary greatly in their immunogenicity dependent on their chemical structure and molecular size. Since soluble substances are readily cleared from the circulation, either by some metabolic pathway or by excretion, through routes that largely bypass lymph nodes, spleen, and other reservoirs of immunopotent cells, they rarely stimulate the production of effective reagent antibodies unless administered with some sort of adjuvant, as described below. Even then, they vary widely in immunogenicity. Proteins and the larger polypeptides of molecular weight greater than about 5000 will readily stimulate a potent immune response. Many may exist in dimer or polymer form, either naturally or as a result of minor denaturation during purification, and this may increase their immunogenicity (major denaturation may be associated with loss of native antigenic characteristics, however, and should be avoided). The smaller the peptide 1 S. Lader, B. A. L. Hum, and G. Court, in "Radioimmunoassay and Related Procedures in Medicine," p. 31. International Atomic Energy Agency, Vienna, 1974.

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within the molecular weight range of 5000-1000, the more difficult it seems to be to make avid antisera, although the correlation is much less than perfect. In this size range, closely related (or even identical) peptides are found in all the usual species of laboratory animal, so the element of "foreigness" of the antigen is lost. Many small peptides may lack the clearly defined tertiary structure that is presumably necessary for a substance to be recognized as a unique antigen. Finally, degradation of these substances in the tissues and circulation, by specific enzymes and by nonspecific proteases, may well be so brisk as to prevent effective contact with immunopotent cells. With the exception of some of the larger polysaccharide molecules, no substances other than the proteins and larger polypeptides are effective immunogens in themselves. Nevertheless, antisera of high avidity and specificity can be raised to steroids, glycosides, oligopeptides, and the like if they are first chemically bonded to a large carrier molecule, preferably a protein that is in itself immunogenic in the species under immunization. Current immunological theory suggests that the initial stages of immunization require cooperation between T and B lymphocytes, the T lymphocytes first binding with a recognizably "foreign" substance and then presenting the bound antigen to B lymphocytes bearing suitable receptors. This cooperation is impossible if the antigen is too small to be shared between T and B cells, but a complex of the antigen with a suitable carrier becomes fully effective. Small, nonimmunogenic antigens of this type are known as haptens and, in the form of drugs, steroid hormones and small peptides, have been of great interest to immunoassayists during the last decade. The method of coupling carrier and hapten should be carefully chosen so as to avoid unwanted structural alteration of the latter and so that the linkage does not involve the immunochemically distinctive part of the hapten molecule. Antibodies produced in response to immunization with conjugated haptens generally "recognize" that part of the hapten farthest from the point of linkage, which thus determines their specificity. Highly substituted carriers are usually most effective, and molar ratios of 15-30:1 (hapten :carrier) are desirable, when possible. For best results the carrier should be a protein foreign to the immunized species-thyroglobulin and keyhole limpet hemocyanin are used quite widely, but bovine (or other) serum albumin is fully effective and more easily available. The subject has recently been well reviewed in relation to steroid conjugates by Pratt. 2 The purity of the immunogen is of controversial importance. For synthetic substances, however, no argument exists--the likelihood of closely related substances (such as "error peptides") being present in imJ. J. Pratt, Clin. Chem. 24, 1869 (1978).

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pure preparations, subsequently leading to the most objectionable variety of nonspecific antibody, means that maximum possible purity is essential. For particulate antigens, especially bacteria, there is also no reason for lack of purity, but the needs in respect to soluble substances extracted from natural sources are somewhat different. There is no doubt that relatively crude preparations are highly immunogenic, often more so than purer materials, so that many workers have thought of the impurities as having some adjuvant-like activity. The probability, however, is that greater purification has led to concomitant subtle chemical changes (such as deamidation) so that the immunogen stimulates antibodies that fail, to a greater or lesser extent, to " s e e " the native antigen. Despite this, a high degree of purification of immunogen must sometimes be sought in order to eliminate certain types of cross-reactivity in antisera. At the other extreme, gross impurity should be avoided, even when the cross-reactions are unimportant, because antigenic competition may then prevent formation of any specific antibody. In practical terms, about 10% purity is the minimum required to make a significant specific antibody response reasonably likely. T h e Adjuvant A wide variety of substances are known to have the property of potentiating the humoral antibody response to injected immunogen. Among them are inorganic adsorbents, such as aluminum hydroxide gel; mineral oils, such as liquid paraffin; and bacterial cell wall components. The diversity of materials having adjuvant properties, which has been the subject of a recent review by Whitehouse,3 makes it difficult to identify a simple mechanism of action. Three major effects are involved, albeit to different degrees for each adjuvant type. First, the release of immunogen from the site of injection is slowed, either by adsorption to solid particles or by incorporation into an oily emulsion. This leads to a "sustained release" from a depot at the injection site, where labile immunogens are also protected from breakdown by tissue enzymes. A secondary benefit is that any direct toxic effects of the immunogen on the recipient will be , minimized. Second, adjuvants have a stimulatory effect on reticuloendothelial cells, attracting a local infiltration of the injection area by mononuclear cells and stimulating phagocytosis by macrophages presumably by presenting soluble immunogen in a particulate or partially aggregated form. Adjuvant-treated macrophages with antigen inoculated into histocompatible recipients give rise to a higher antibody response than transfer 3 M. W. Whitehouse, in "Immunochemistry: An Advanced Textbook" (L. E. Glynn and M. W. Steward, eds.), p. 571. Wiley, New York, 1977.

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of macrophages containing antigen alone. 4 Third, it has been shown that adjuvants induce an increased circulation of lymphocytes through lymphoid tissues in the drainage area, 5 macrophages being important for the initiation of these lymphocyte traffic changes, e The increased flow of cells in regional lymph nodes is likely to allow greater contact between antigen and antigen-reactive cells, thus facilitating increased antibody production. The local granulomatous lesions formed at the sites of injection may also serve as foci of antibody production. The most important advance in adjuvant technology arose from the observation of Dienes and Schoenheit 7 that antigen injected into tuberculous granulomata stimulated higher antibody titers than did antigen injected at nontuberculous sites. These findings led Freund and co-workers to develop a series of adjuvants containing mycobacteria, mineral oil, and emulsifier, s which to this day remain the most potent tools available to the aspiring immunologist. The mixture most widely used in the preparation of reagent antibodies contains 9 parts of mineral oil to 1 part of detergent. The detergent (usually Arlacel A) contains a high level of both hydrophilic and lipophilic groups, thus facilitating dispersion of the oily and aqueous (immunogen) phases and allowing the formation of a stable emulsion. The simple oil-detergent mixtures are termed "incomplete" Freund's adjuvant; incorporation of heat-killed Mycobacterium tuberculosis or M. butyricum (0.5 mg/ml) into the oily mixture yieldS "complete" Freund's adjuvant. The latter is the more effective, probably as a result of greater stimulation of the local cellular response, and must now be regarded as an essential aid to the production of reagent antibodies against soluble immunogens. Preparation of Freund's Emulsions. For maximum efficiency, it is necessary to obtain a stable, water-in-oil emulsion. Several ways of pre-, paring such emulsions have been described, but there is no doubt that the simplest and most efficient, at least for the relatively small volumes that most people require, is the double-hub connector method described here. It may occasionally be difficult to persuade the phases to combine as water in oil rather than oil in water or mixed emulsions. Cooling the separate phases before mixing may help, but an infallible way of overcoming the problem is to use 2 - 4 volumes of oily adjuvant to 1 volume of aqueous 4 E. R. U n a n u e , B. A. A s k o n a s , and A. C. Allison, J. Immunol. 103, 71 (1969). 5 p. Frost and E. M. Lance, in " I m m u n o p o t e n t i a t i o n , " C I B A Found. Symp. 18 (New series), p. 29. E x c e r p t a Medica, A m s t e r d a m , 1973. 6 p. Frost and E. M. L a n c e , Immunology 26, 175 (1974). 7 L. Dienes and E. W. Schoenheit, J. lmmunol. 19, 41 (1930). s j. F r e u n d and K. M c D e r m o t t , Proc. Soc. Exp. Biol. Med. 49, 548 (1942).

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immunogen. Experience has shown these oil-rich emulsions to be at least as effective (probably more effective) than the 1 : 1 ratio usually recommended. A subsidiary advantage is that they flow more easily, so both mixing and injection are less of a chore. If it is essential to use a 1 : 1 ratio (because of volume restrictions when the immunogen solution is very dilute, for instance), the formation of water in oil emulsions can be reliably achieved by adding the aqueous phase in three increments, mixing after each. The necessary apparatus consists of the double-hub connector and two syringes, each large enough to contain the total emulsion volume without overfilling. The largest practicable volume that can be handled by someone with averagely large, reasonably powerful hands is 14-16 ml, using two 20-ml syringes. The best type of syringe for the purpose is an all-glass, center-hub pattern; metal-and-glass types tend to leak at the piston, and the common plastic syringes become very stiff while making the emulsion. Plastic syringes are reasonably satisfactory in the smaller sizes, however, since less force is needed for small volumes. If Freund' s complete adjuvant is required, shake it very thoroughly to resuspend the bacterial cells immediately before use. Pour out sufficient of the adjuvant into a small beaker (to avoid contaminating the remainder) and draw the required volume up into one of the syringes. Attach the double-hub connector, and carefully expel all air until the oil rises up into the farther end of the connector. Draw the aqueous immunogen into the other syringe, remove the needle, and again expel all air until the syringe hub is full of liquid, then connect it to the open end of the connector: any air left in the apparatus will be trapped in the emulsion and, because of its compressibility, will make injections more difficult. At this stage make sure that both syringes are firmly inserted into the connector, but be careful from now on not to place any bending stress on the rather unwieldly apparatus, especially if the syringes have glass hubs. A little oil will almost certainly be squeezed out of the connector (the whole process is somewhat messy) and it may be as well to wipe the apparatus and fingers with a tissue before proceeding. To form the emulsion, begin by squirting the aqueous phase into the oil as vigorously as possible, then continue squirting the total contents toand-fro from one syringe to the other a minimum of 10 times each way (20 times is better, if your thumbs can stand it). To avoid bending stress at the connections, practise deliberate relaxation of the "receiving" hand so that the filling syringe just rests on the palm as the other hand is grasping and pressing on the plunger. Especially as the hands tire it is tempting to let the receiving hand try to help the other, but this inevitably places strain on the syringe hubs. A fracture of a hub (or sudden falling apart of a

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carelessly made connection) causes an explosive shower of emulsion to contaminate everything with a radius of several feet (including the operatot's face) and is sufficiently unpleasant to encourage more care thereafter. If the aqueous phase is to be added in several aliquots in order to promote the formation of water-in-oil emulsions at the 1 : 1 ratio, it will be necessary to break one of the connections each time more of the water phase is needed. About five each-way strokes of the syringe will be sufficient for the intermediate mixing, after which the next aliquot of immunogen is taken up into the empty syringe, the connection is made again and, as before, mixing begins by squirting the water into the oily emulsion. Repeated disconnection and reconnection make it all the more difficult to exclude air and prevent messy oil from leaking out: it is better to use a larger oil to water ratio and avoid the problem altogether whenever possible. In the authors' experience, the above method will lead infallibly to the proper type of emulsion, and testing is therefore unnecessary. For those who wish to confirm success, however, the simplest way is to take a beaker half full of water and drop two small, separate drops of emulsion onto the water surface. The first drop always spreads somewhat, but the second will remain a discrete, white globule with no spreading at all if the emulsion is, indeed, water in oil. If the second drop disintegrates into bits and pieces that spread around over the surface of the water, the emulsion was oil in water, at least in part, and should be prepared afresh. Read the above instructions again first, though. After use, plastic syringes should be thrown away, but other apparatus must be washed up. The connector can first be pushed into a piece of rubber tubing connected to a hot tap and flushed through for a few minutes. Syringes should be cleaned with washing-up detergent, then soaked, together with the connector, in a decontaminating detergent (such as Decon 70) for a day or two before rinsing and drying. Residues from emulsions are probably difficult to remove completely, and the syringes should never again be used for any purpose other than preparing such materials. T h e Choice of Animal There are few instances in which categorical evidence has shown one species of common laboratory animal to give consistently better responses than another to any particular immunogen. Some fairly well know exceptions are the superiority of guinea pigs for production of antiinsulin sera (presumably because the endogenous hormone in this species is most unlike the other mammalian insulins) and of horses for preparation of antisera for immunoelectrophoresis. The latter preference is due to the

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solubility of horse antibody immune precipitates in excess antibody (all immune precipitates are soluble in antigen excess) yielding unusually narrow, clear-cut precipitin arcs. Apart from these examples, however, the literature abounds with indications of the personal preferences of the authors for which the evidence is apocryphal and often contradictory. In most instances the choice of species may reasonably be made on the basis of what is available and the volume of antiserum required--the larger the animal, the bigger the yield. It will be understood that it is usually sensible to immunize a species that is "foreign" to the antigen in question. If homologous immunogens are used, it should be for a valid reason (production of tissue typing sera, for instance, relies on antibodies produced in the same species as the donor). Homologous immunization, when it produces a result at all, will yield antibodies that recognize fine, interindividual differences in the antigen; by contrast, immunization of a foreign species readily yields much more abundant antibody but any reactivity with the structurally minor, idiotypic variants of the antigen is almost always lost in the reactivity against the gross, interspecies difference. A well provided laboratory may have access to guinea pigs, rabbits, sheep or goats, donkeys, and horses. There is little doubt that rabbits should be the first choice for most purposes unless very large amounts of serum are needed. Rabbits are cheap, easy to care for, robust in the face of quite intensive immunization, and easy to bleed. The other species may best be held in reserve in case of a failure with rabbits. Another reserve species that may be available is the chicken--again quite easy to handle, but producing antibodies that behave differently from those of mammalian species 9 and hence best avoided if possible. Whichever species is chosen, it pays to immunize several individuals (which is a good reason for avoiding the larger, more expensive species to begin with). Individual variation in response is often very striking, especially to the more "difficult" immunogens, and groups of at least four or five animals should be started if any difficulty whatsoever is to be expected in preparing satisfactory antisera. Nonproductive animals can be disposed of once it is clear that they will not improve (this may not be for several months with some immunogens) whereas the better responders can be kept under immunization for a year or more and bled repeatedly. Obviously, such a course cannot be followed where the early antibody is desired, for instance in the production of hemolytic serum with minimal hemagglutinating activity for use in complement fixation tests. 9 A. A. Benedict, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 1, p. 229. Academic Press, New York, 1967.

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Immune responsiveness to certain antigens has been shown to be genetically determined. '° The importance of this in the context of antigens of general interest is not known, but it would seem to be desirable to use random-bred animals whenever possible, to give the best chance of a good response in one or more, unless previous experience has already shown that a particular inbred strain responds well to the immunogen in question. Whatever animals be chosen, they should be kept clean, healthy, and well fed if they are to perform well as antibody factories. The subject of animal husbandry is dealt with in a number of works (see, e.g., Short and Woodnott 11and Chase 12) but is, perhaps, of no direct interest to the readers of this chapter. T h e R o u t e of Injection For soluble immunogens, it is generally believed that the efficiency of stimulation of the immune response is related to the site of inoculation. A probable series, in order of increasing effect, is intravenous < intramuscular < subcutaneous < intraperitoneal < intradermal < intraarticular < intranodal. The principal reasons for the differences in efficiency are the speed with which antigen is lost from the site of injection and the likelihood of it passing through the lymph nodes or other centers of immunological activity on the way. These considerations, however, are radically affected by the use of adjuvants, especially oily adjuvants, which may stimulate a brisk local cellular reaction and release antigen over a period of several weeks or even months. Using oily adjuvants, then, the injection site can be chosen principally with a view to minimizing discomfort to the animal. Generally this means intramuscular injections in rabbits and larger animals or subcutaneous injections in guinea pigs; note that water in oil emulsions must never be given intravenously because of the virtual certainty of fatal fat embolism. Subcutaneous or intradermal injection of Freund's emulsions almost invariably leads to ulceration, but provided the sites are well chosen (see below) rabbits and guinea pigs show no sign of distress or loss of condition. Some authors (see Herbert 13) have suggested that Freund's emulsions should not be injected subcutaneously since ulceration may lead to 1o I. Green, W. E. Paul, and B. Benacerraf, Proc. Natl. Acad. Sci. U.S.A. 64, 1095 (1969). 11 D. J. Short and D. P. Woodnott, eds., "The I.A.T. Manual of Laboratory Animal Practice and Techniques," 2nd ed. Crosby Lockwood, London, 1969. ,2 M. W. Chase, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 1, p. 254. Academic Press, New York, 1967. ,3 W. J. Herbert, In "Handbook of Experimental Immunology" (D. M. Weir, ed.), 2rid ed., App. 2. Blackwell, Oxford, 1973.

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1 13

loss of the depot: in the experience of the present authors this has never given rise to difficulty. Occasionally deep abscesses form after intramuscular injection and lead to loss of condition. The abscesses are frequently "sterile" and, in our experience, have usually been related to overzealous attempts to improve on sterile injection techniques (cleaning the skin over the injection area, for instance) rather than to the use of unsterile immunogens. Difficulties in preparing antisera against some of the antigens of interest in radioimmunoassay have led people to try a wide variety of methods of immunization. Most of these variations have been irrational (which does not mean to say they have not worked on occasion), but two deserve special mention. By injection of immunogen (angiotensin I, adsorbed on carbon black and emulsified in Freund's adjuvant) directly into rabbit lymph nodes and spleen, Boyd and Peart 14 obtained improved results that they believed to be due to more direct stimulation of the immune system. A subsequent comparative trial gave rather equivocal results, TM however, and the method was too difficult to be widely used. Injection into the Peyer's patches (lymphoid patches in the intestinal wall, quite easily visible in the rabbit) is technically much simpler but has proved no more successful in the authors' hands. Much simpler than the intranodal method, and now quite widely used, is the method of multiple intradermal inoculation introduced by Vaitukaitis e t a l . 16 The immunogen is introduced at 40 or more sites spread widely over the body surface. Antibody response to this primary immunization is much greater than to a first injection given in the usual way, and no more than one booster injection is usually required. Comparison with the usual intramuscular injection schedule ~ showed no great difference in efficiency, although the multiple intradermal technique (with only one booster) required rather less immunogen and yielded effective antisera in a shorter period of time. T h e Dosage of I m m u n o g e n and Timing of Injections Although an animal may be made "tolerant" to soluble antigens given in too low or too high a dose under certain circumstances, the use of a potent adjuvant makes such an outcome extremely unlikely. Nevertheless, the observation that too high a dose can lead to antiserum of rela14G. W. Boydand W. S. Pearl, Lancet 2, 129 (1968). 1~B. A. L. Hurn and J. Landon,in "'RadioimmunoassayMethods" (K. E. Kirkhamand W. M. Hunter, eds.), p.121. Churchill Livingstone,Edinburgh, 1971. 16j. Vaitukaitis,J. B. Robbins, E. Nieschlag, and G. T. Ross,J. Clin. Endocrinol. 33, 988 (1971).

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tively low avidity, 17.18presumably owing to stimulation of lymphocytes bearing low-affinity receptors, may certainly be relevant even when using Freund's adjuvant. Since most sensitive immunoassay techniques of current interest rely on antibody of the highest possible avidity, it is evidently desirable (and economical of immunogen) to use the lowest dose that will be fully effective. This dose is very much smaller than most of the published literature recognizes, and a suitable priming (first) inoculation for rabbits or guinea pigs will generally be of the order of 100 ~g. A range of 50-1000/~g should cover all needs, depending on the purity and immunogenicity of the material in question (but it is sensible to start at the lower end, since an animal showing lack of response after a sufficiently long trial can then be given a larger dose, whereas an animal producing poor antiserum after high dosage is beyond hope of salvage). The dosage required for larger animals does not increase in proportion to body weight: 0.255 mg is satisfactory for sheep and 0.5-10 mg for donkeys. For conjugated haptens, incidentally, these figures refer to total conjugate weight. Booster injections are always needed to obtain antisera of the highest titer and avidity. Practical experience suggests that good results will be obtained using a booster dose about half the size of an effective priming dose, given by the same route (not necessarily at the same site) and using Freund's complete adjuvant on each occasion. It is recognized that these recommendations are somewhat at variance both with immunological theory (which would suggest a progressive increase in dose) and with the advice of other authors to avoid repeated use of Freund's complete adjuvant, especially subcutaneously, because of abscess formation and hypersensitivity reactions. There is some documented evidence in support of the suggested reduction in dose, 1 but the repeated use of complete Freund's adjuvant is a recommendation that stems only from satisfactory, albeit uncontrolled, experience. The repeated booster doses that are usually required for the best arLtiserum should not be given too frequently. It has been shown 19that no further rise in titer results from a second injection given before the response to the first is reaching its peak. At least 4 weeks should pass between injections of Freund's emulsions. After the first booster, or sometimes after the second, antibody response may be quite prolonged and many people believe that a rest of 3-6 months is desirable before the next injection if antiserum of the highest avidity is required; the evidence in favor of this approach is not strong, 1 but in general terms there is little doubt that pa17G. W. Siskindand B. Benacerraf,Adv. lmmunol. 10, 1 (1969). 18E. J. Greeneand J. G. Tew, Cell. Immunol. 26, 1 (1976). 19W. J. Herbert, Immunology 14, 301 (1968).

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tience is desirable when making reagent antibodies. It is not unusual to read descriptions of immunization schedules involving weekly injections of quite large amounts of immunogen in Freund's emulsion; published accounts, not surprisingly, tend to report a successful outcome, but the approach is not to be recommended. Many published immunization procedures terminate with one or more intravenous injections of soluble immunogen given without adjuvant after a course of intramuscular Freund's emulsions. In the authors' experience, this produces a less satisfactory response (about half the final titer of avid antibody) than can be obtained with a final injection of intramuscular emulsion. By contrast with the above, particulate immunogens are normally administered intravenously, frequently (perhaps every other day), in increasing doses and for short periods of time. These materials are usually highly immunogenic, partly because the normal mechanism for their removal brings them into close contact with the immune system and partly because many of them (notably bacterial cells) are antigenically very "foreign" to the immunized animal. Antibody production is rapid, and the early IgM response is excellent for agglutination tests. Initial doses of immunogen are extremely variable, owing to the variable toxicity of the substances concerned (especially bacteria containing endotoxins), and for many of the antigens hypersensitivity reactions to later doses may prove rapidly lethal. Subcutaneous injection, with relatively slow absorption, may ameliorate undesirable acute reactions. Although short immunization courses for particulate antigens are the rule, usually in the belief that antisera will become less specific as immunization proceeds, this is not necessarily the case. Prolonged immunization may result in more stable IgG antibody of higher titer and, because of repeated bleeding over a period of time, in much greater yield.

Practical Immunization Schedules Animals often remain under immunization for many months, even years. You may not be personally responsible for their care during this time, but in your own interests you must ensure that either the individual animals or their cages are properly labeled in a manner compatible with your own records at the time of the first injection so that the individual animals can be identified with certainty thereafter. If the cages alone are labeled, you would also be advised to ensure that the method of animal handling, especially during cage cleaning, is such as to prevent animals being moved accidentally from one cage to another.

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Rabbits Four or more healthy, young adult rabbits should be treated with each immunogen. Soluble I m m u n o g e n s

Either the intramuscular or the multiple intradermal route may be recommended. As examples of representative immunogens for which highavidity antisera are required, consider a crude preparation of human chorionic gonadotropin (hCG) and the beta subunit of hCG (fl-hCG). The former, at a characteristic potency of 1500-3000 IU/mg, is about 20% pure whereas the latter is of necessity highly purified and in short supply. Appropriate doses for primary immunization are 1 mg and 100/zg, respectively. Booster doses should be half these amounts. Dissolve the immunogen in isotonic saline (other immunogens may require slight acidity, alkalinity, or other special condition) to a volume of 0.5 ml per rabbit for the primary injection or 0.25 ml for boosters (i.e., the same concentration for both injections). Emulsify the solution with three volumes of Freund's complete adjuvant, using a double-hub connector and two syringes as described above. The total volume of emulsion will then be 2 ml per rabbit for the primary inoculation or 1 ml for a booster. Use the emulsion within an hour of preparation. Intramuscular Schedule. Do not shave the animals or attempt to prepare the skin in any way prior to injection. A fairly stout needle of medium length (21 gauge x 1 inch) is convenient and need not be changed between animals unless it becomes blunted for any reason. Injections are given into thigh and/or upper foreleg muscle, where thickest, and the hair can be parted by gently blowing down on to the selected site immediately before injection. For the primary injection, give 0.5 ml of emulsion intramuscularly into each of the four limbs of each animal. Now go away and think about other things for at least 4 weeks, or 6 weeks if possible. For booster injections, give 0.5 ml of emulsion intramuscularly either into each hind limb or into each fore limb, alternately. Bleeds (20-40 ml) may be taken for testing on two occasions between 7 and 10 days after each booster and similarly every 3-4 weeks thereafter if the antiserum is satisfactory. Further boosters may be given at minimum intervals of 4 weeks (but preferably not within 2 weeks of a bleed) although it may pay to rest the animal for 3-4 months after the second or third booster. Animals that fail to show a reasonable response after two or three boosters should be disposed of. This decision must be related to the level of response expected for the particular immunogen u s e d - - s o m e animals

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may take several months to respond to "difficult" immunogens, and early responders are not necessarily the best in the end. Multiple Intradermal Method. Shave the hair on the back and on the proximal parts of all four limbs of each rabbit. As a guide to spacing the injections, draw six transverse lines across the shaved area of the back, using a felt-tip marker. The injections should be made with a tuberculin syringe and a fine needle (the syringe holds only enough for one animal but may be loaded repeatedly from the syringe in which the emulsion has been prepared, via the double-hub connector). Make 24 intradermal injections each of 0.05 ml, spaced evenly over the back. Distribute the remainder of the emulsion (about 0.8 ml, or sixteen 0.05 ml injections) over the inner and outer aspects of each upper limb, in the shaved areas. Satisfactory intradermal injections are easily recognized by a characteristic, localized bleb; this is easy to achieve on the back of the animal, where the dermis is quite thick and tough, but very difficult on the limbs, where the skin is much more delicate. Try, but do not be unduly discouraged if you fail. Within a few days of the injections the rabbit will present a horrifying sight, covered as it will be with forty, half-inch ulcers. In the authors' experience, the animals are happily unaware of the aesthetics of the situation and continue to thrive without any specific treatment. Some users of the technique have found otherwise, for no known reason. In the interest of animal welfare, if you find your rabbits are greatly upset by this procedure then please revert to the intramuscular procedure, which can be just as effective. After the multiple injections the animals should be left for at least 10 weeks before boosting. Antibody levels rise to relatively high titers during this time, however, and it is certainly worth taking a large bleed for testing after 8-10 weeks. All booster injections are given by the intramuscular route, and the method of treatment from the tenth week onward is thus exactly the same as for the previous schedule.

Particulate Immunogens These ~intigens are commonly administered by frequent, intravenous injection without adjuvant. Results are obtained quickly, the antisera often containing a high proportion of lgM immunoglobulin. There is a risk both of direct toxicity early in immunization and of severe hypersensitivity reactions as a result of later injections. With some at least of the antigens in question, equally satisfactory results can be obtained by intramuscular injection of Freund's emulsions--immunization is slower but less risky.

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Production of antisera to Escherichia coli, for use as specific typing reagents, furnishes an example of a typical intravenous schedule. Good bacteriological technique and the selection of an appropriate colonial form of the organism is essential to the specificity and reactivity of the antiserum (this is obviously analogous to the purification of a soluble immunogen). Living organisms are required for expression of the important K antigens in this species, but live coli will kill a high proportion of unprotected animals and the early injections are therefore made with heat-killed suspensions. Antisera to most other microbial species can be prepared against killed suspensions throughout. The following schedule should be followed (all suspensions being prepared to an opacity of Brown's tube 4, and all injections given intravenously). Day 1:0.25 ml of killed suspension Day 3:1.0 ml of killed suspension Day 5:3.0 ml of killed suspension Day 9:0.5 ml of freshly prepared living suspension Day 12:1.0 ml of freshly prepared living suspension Day 16:3.0 ml of freshly prepared living suspension Day 22: test bleed for titer Either Day 23 Bleed out if titer is satisfactory Or Continue weekly injections as for day 16 with test bleeds 5-7 days later, until satisfactory titers are obtained. Guinea Pigs Each animal will yield only 3-5 ml of serum by cardiac puncture or 15-25 ml when bled out. For this reason guinea pigs are best reserved for use when only small quantities of antiserum are required (particularly in radioimmunoassay and similar immunoassays) or when other animals are known not to respond well to the immunogen in question (insulin is such a substance, and, in our experience, parathyroid hormone is another). Groups of up to 10 guinea pigs may conveniently be kept in a single large cage, individuals being identified by natural markings or applied pigments (the latter need to be renewed rather frequently). Soluble immunogens should be administered as Freund's emulsions, injected subcutaneously into the abdominal wall just on either side of the midline. The injection sites will usually ulcerate after a week or so, but the animals are apparently free from discomfort, thrive, and make good antibodies. Prepare the inoculum by emulsifying 1 volume of aqueous immunogen

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in 2 - 3 volumes of Freund's complete adjuvant in the usual way, to give a total volume of 0.5 ml per animal. Injections should be given at intervals of not less than 4 weeks although longer rests later in the course of immunization may be desirable. Because of the low yield of serum and the risk of killing the animals when bleeding by cardiac puncture, it is less practicable to bleed guinea pigs repeatedly than it is to bleed rabbits. Since guinea pigs are cheaper to buy and look after, it is probably best to immunize a relatively large number for a comparatively long period of time, then bleed them out and select the best antisera from the result. Our experience has suggested that at least four injections are desirable if this strategy is employed, and six injections may often be better. The decision depends on the purpose for which the antiserum is required and, in particular, whether the highest possible avidity is needed. Sheep The immunization of sheep offers the possibility of obtaining relatively large amounts of antiserum, not only because each individual bleed is larger (150-300 ml of serum, depending on the size of the animal), but also because the animals may be maintained and bled repeatedly for longer than rabbits. This can be a major advantage when antisera are to be prepared for relatively undemanding, insensitive test systems such as immunoprecipitation, when larger volumes of reagent are required but variations in quality over the course of time are unlikely to cause difficulty. The higher cost of buying and keeping a sheep makes it less attractive when the use of a "difficult" immunogen makes it necessary to immunize a large number of animals. Circumstances alter cases, of course, and an Australian laboratory might have a different view of the relative economy of sheep and rabbits. Immunization of a sheep should proceed according to a schedule similar to that described for a rabbit. Intramuscular injections (as usual, always prepared with Freund's complete adjuvant) should be given with a 1½-2-inch needle deeply into the haunch or shoulder (preferably into all four "corners" for the first injection). As has been mentioned before, dosage is not proportional to size and for a relatively good immunogen such as human IgG an initial injection of 0.2-1 rag, followed by booster doses of half that size, should be sufficient. After the first two or three monthly injections, subsequent boosts should be given at longer intervals depending on the quality of antiserum. Bleeds may be collected on a regular schedule throughout the period of immunization, the best yields being obtained if three bleeds (of 300-600 ml, depending on the size and experience of the animal) are taken over a period of 8 - l 0 days followed by

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about 3 weeks rest before the next triple bleed. A healthy animal may remain productive for some considerable time, at least a year or two, but antibody levels will eventually decay and fail to respond to a further booster injection, at which time the animal should be disposed of. Collection and Storage of I m m u n e Serum Animals immunized with Freund's emulsions should be bled 7-10 days after booster injections. If the blood is taken from a vein rather than by cardiac puncture, two or three bleeds can be taken on successive days, but the animal should then be rested for 3 - 4 weeks before further bleeding or before boosting again if the original antiserum was not of satisfactory quality. After intravenous injection antibody levels rise and then fall more rapidly and bleeds should be collected 5-7 days after the last dose. It is often helpful to fast the animals overnight to minimize lipemia, but do not deprive them of drinking water. Blood should be collected in clean, dry, glass bottles and allowed to clot at room temperature or at 37° until the clot has retracted; it may help to "ring" the clot with a glass rod to promote separation. The sample should then be centrifuged and the serum be separated without undue delay in order to avoid unnecessary hemolysis, which looks unaesthetic although it has no obvious deleterious effect on the antibody. When handling large quantities of blood it may be easier to separate serum from the clot by letting it drain through a stainless steel mesh cone supported in a filter funnel--this can even be left to drain overnight in the cold room if the maximum possible yield is required, but in any case a final centrifugation will be required to remove residual red cells. After separation from the clot, antiserum may be stored without significant deterioration for long periods of time under a variety of conditions.Z° A counsel of perfection for reference or otherwise most precious reagents would be to filter sterilize, fill out in appropriate, accurately measured, small amounts (diluting in a suitable carrier medium if necessary), and then freeze-dry prior to storage at 4 ° or below. Experience has shown, however, that IgG antibodies are remarkably robust and that liquid antiserum (even without sterilization) can be kept for many months at 4 ° with 0.1% sodium azide added as an antibacterial agent. Storage at about - 20° in the ordinary laboratory freezer cabinet is, in theory, likely to cause protein denaturation due to the proximity of this temperature to the eutectic of sodium chloride (the complex mixture comprising serum will not be 20K. E. Kirkhamand W. M. Hunter,eds., in "RadioimmunoassayMethods,"pp. 189-193. Churchill Livingstone,Edinburgh, 1971.

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completely frozen at - 20°) but, again in practice, the freezer has proved most convenient and harmless to antibody protein provided that repeated freezing and thawing is avoided. Storage at lower temperature, preferably not in unreliable mechanical refrigerators, is very satisfactory when available. Gas-phase liquid nitrogen is the ideal low-temperature storage medium, being more reliable and convenient than mechanical or CO~ cabinets, not involving the special restrictions on storage vessels imposed by immersion storage in liquid nitrogen yet virtually guaranteeing lifetime stability of precious antisera (the investigator's lifetime, that is to say). Storage of IgM antibodies is far more of a problem and gives very variable results. Most antisera containing IgM can be handled exactly as described above, with only gradual deterioration that would be inapparent in the relatively undemanding test systems in which this class of antibody is generally used. Some, on the other hand, prove much less stable. On occasions, this instability is associated with bacterial growth (which seldom causes much loss of IgG antibody activity although it is embarrassing and should be avoided if possible). For this reason it is strongly recommended that IgM antisera should have 0.1% sodium azide added, be sterilized by filtration at the earliest possible opportunity (before bacterial growth and release of enzymes can occur) and be handled in a cleanly fashion thereafter. Even when collected after overnight fasting of the animal, defatted (see below), sterilized and with a bacteriostat added, serum stored at 4° will gradually become turbid and show a deposit, principally of denatured lipoprotein. This does not lead to any loss of antibody activity although it is easily mistaken for bacterial contamination and causes anxiety for that reason. The only practical disadvantage is seen when the antiserum is used in capillary precipitin reactions, when the turbidity can obscure the result unless the antiserum is first clarified by filtration. Further T r e a t m e n t of Antisera D e f a t t i n g A n t i s e r u m 21 Antisera to be used in capillary precipitin tests must be crystal clear so that the faint ring o f precipitation can be easily seen. Untreated sera become turbid on storage, due to precipitation of denatured lipoprotein; such precipitates can be r e m o v e d by membrane filtration prior to use, but it is usually better to reduce the severity o f the problem by extracting the bulk o f the lipoprotein at the time the serum is first prepared. zl A. S. McFarlane, Nature (London) 149, 439 (1942).

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Materials Diethyl ether, solvent grade Solid CO2-methylated spirit freezing bath Procedure 1. Place the serum in a beaker and add 3 ml of ether for every 10 ml of serum. 2. Place the beaker in the freezing bath. 3. Stir the serum-ether mixture quite briskly with a glass rod until it has frozen solid. The two liquids are completely miscible in these proportions at the freezing point. 4. Allow the frozen mixture to stand in the freezing bath for another 10 min, then remove the beaker and stand it in tepid water until the frozen plug loosens. 5. As soon as possible, tip the still frozen plug into a glass filter funnel (without filter) leading into a cylindrical separating funnel. Make sure the stopcock on the latter is free running and well lubricated with a silicone grease. 6. Allow the frozen material to thaw and run into the separating funnel at room temperature, then remove the filter funnel and close the separating funnel with a rubber stopper covered in metal foil. 7. Allow the separating funnel to stand undisturbed, at 4° if possible, overnight. 8. The next day the serum-ether emulsion will have separated into a lower layer of clear serum shading gradually into an opalescent zone of residual emulsion that has a sharp interface with the uppermost, opaque, fatty layer. Collect the serum by running off the bottom and intermediate layers. 9. Remove the bulk of the residual ether by boiling offunder reduced pressure, ideally with the aid of a rotary evaporator. 10. Add preservative and sterilize the serum by filtration prior to storage. NOTE: Due care should be taken to avoid the risk of fire or explosion when handling ether.

Absorption of Nonspecific Antisera The production of potent antiserum almost always results in a reagent with some degree of reactivity against nonspecific antigens, either because of impurities in the immunogen used or because there are "shared determinants" present in both specific and nonspecific antigens. Whatever the cause of the unwanted reactivity, it is usually necessary to re-

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move it by absorbing the antiserum with an appropriate antigen. Although absorption may be carried out with solutions of the antigen (the immune precipitate being removed afterward by centrifugation or filtration), excess antigen or soluble complexes of antibody and antigen will inevitably remain in the absorbed antiserum. A more satisfactory method, therefore, is the use of a solid phase immunoadsorbent prepared from the appropriate antigen, which can be added in excess and easily recovered for later re-use if required. Such adsorbents may be made from antigen alone by the use of cross-linking reagents (such as glutaraldehyde for protein antigens) or can be more complex reagents prepared by chemical coupling of the antigen to a solid support such as Sepharose. The latter technique is covered in a subsequent section on the use of IgG-Sepharose immunoadsorbent, but the present example describes the preparation of a glutaraldehyde polymer of F(ab')2 suitable for removal of light-chain crossreactivity from class-specific anti-immunoglobulin sera. It should be noted that the pH optimum for efficient polymerization by this method varies considerably depending on the protein to be treated; if a polymer of whole serum is required, for instance, a pH of 4.4 will be optimal.

Preparation of F(ab')2 lmmunoadsorbent by Glutaraldehyde Polymerization 22 Materials F(ab')2 prepared by pepsin digestion of IgG-z3 Phosphate buffer, 0.1 M, pH 7.0 Glutaraldehyde, 2% in saline Glycine-HCl buffer, 0.1 M, pH 2.5 Tris-HCl, 0.1 M, pH 8.0 Phosphate-buffered saline, 10mM, pH 7.5 (PBS) Procedure 1. Dialyze 100 mg of F(ab')2 preparation (20-50 mg/ml) against phosphate buffer at 4° overnight. 2. Place the F(ab'h solution on magnetic stirrer and add 0.4 ml of glutaraldehyde solution dropwise from a Pasteur pipette. 3. Allow the gel that forms to remain at room temperature for 3 hr and then place at 4 ° overnight. 4. Homogenize the gel in phosphate buffer then centrifuge hard in a bench centrifuge and discard the supernatant. 5. Repeat step 4 using glycine-HCl buffer. ~ S. Avramcas and T. Ternynck, lmmunochemistry 6, 53 (1969). 23 L. H. Madsen and L. S. Rodkey, J. Immunol. Methods 9, 355 (1976).

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6. Repeat step 4 using Tris-HCl buffer. 7. Repeat step 4 using PBS. 8. Wash polymer in PBS until the washings have negligible absorption at 280 nm. Tube Absorption Procedure 1. Mix 2 volumes of serum with 1 volume of packed polymer and stir on a magnetic stirrer at 37° for 1 hr. 2. Centrifuge at 4500 rpm for 5 min. 3. Transfer supernatant to another tube and recentrifuge. 4. Remove the supernatant and test it for specificity. NOTE: The polymer can be "regenerated" for further use by washing extensively with PBS followed by incubation with 3 M sodium thiocyanate, pH 6.6, for 30 min at room temperature to elute adsorbed protein. Wash the polymer finally with PBS and store at 4° in PBS containing 0.1% sodium azide. Preparation of Immunoglobulin Fractions from Whole Serum

Precipitation with Rivanol and Ammonium Sulfate Materials Rivanol (2-ethoxy 6,9-diaminoacridine lactate) Activated charcoal Saturated ammonium sulfate solution Isotonic saline Procedure 1. Adjust antiserum to pH 8.5 by careful addition of 0.1 N NaOH. 2. For each 10 ml of antiserum add 35 ml of 0.4% Rivanol solution dropwise from a separating funnel. Stir the serum gently on a magnetic stirrer throughout. 3. Decant the supernatant (containing the immunoglobulins) into universal bottles and centrifuge in a bench centrifuge to remove remaining sediment. 4. Decant the supernatant into a conical flask and add activated charcoal (1 - 1.5 g per 100 ml) to decolorize the solution. Agitate gently for approximately 10 min. 5. Remove charcoal from the protein solution by filtering through a double layer of moistened filter paper (Whatman No. 42) in a Biichner funnel. Transfer filtrate to a beaker. 6. Add an equal volume of saturated ammonium sulfate solution dropwise from a separating funnel, stirring gently on a magnetic stirrer throughout.

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7. When all the ammonium sulfate solution has been added, place the beaker at 4 ° for at least 6 hr to allow the immunoglobulin precipitate to flocculate. 8. Centrifuge at about 4000 g for 20 min, preferably in a refrigerated centrifuge, and discard the supernatant. 9. Dissolve the precipitate in a volume of saline approximately equivalent to half the volume of original antiserum. 10. Place the immunoglobulin solution in Visking tubing and dialyze extensively against several changes of saline to remove sulfate ions. (Alternatively, remove sulfate by chromatography on a Sephadex G-25 column.) 11. Check for residual sulfate ions by adding a few drops of the immunoglobulin solution to a tube containing a small volume of barium chloride solution. Any cloudiness indicates the presence of sulfate ions and the need for further dialysis. 12. Measure the volume of immunoglobulin solution and calculate the protein concentration by measuring the absorbance of a 1 : 25 dilution at a wavelength of 280 nm using a cuvette of 1 cm path length. concentration = (OD~80 × 25)/1.34 mg/ml (The factor 1.34 can be used for the immunoglobulins of most animal species).

Precipitation with Caprylic

A c i d 24

Materials Acetate buffer, 60 mM, pH 4.0 Caprylic acid Isotonic saline Procedure 1. Add 2 volumes of acetate buffer to the antiserum in a beaker. Check and adjust the pH of the mixture to 4.8. 2. For each 10 ml of starting antiserum add 0.74 ml of caprylic acid dropwise. Stir the mixture continuously on a magnetic stirrer at room temperature. 3. Continue stirring for 30 min. 4. Centrifuge at 4000 g to remove the precipitate (or filter on a Biichner funnel). 5. Retain the supernatant (containing the immunoglobulin) and dialyze extensively against saline at 4 °. N. Steinbuch and R. Audran, Arch. Biochem. Biophys. 134, 279 (1969).

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6. Measure the volume of the immunoglobulin solution and calculate the protein content as described above. NOTE. Since the final volume of immunoglobulin solution is approximately three times the volume of starting antiserum, concentration is usually necessary. This may be achieved by ammonium sulfate precipitation as described above, pressure ultrafiltration, or dialysis against hypertonic polyethylene glycol. If the latter procedure is used, the immunoglobulin preparation should subsequently be redialyzed against saline to remove any polyethylene glycol that has diffused into the dialysis bag, thereby contributing to the absorbance at 280 nm. Ion Exchange Chromatography Immunoglobulins, in particular IgG, may be separated from whole serum by ion exchange chromatography. The technique relies upon differences in the net charge of serum proteins: at low ionic strength and neutral pH, IgG carries a neutral or slight net positive charge and will not be adsorbed to diethylaminoethyl (DEAE) cellulose, unlike all other serum proteins. Although the principle of the method remains the same for the serum proteins of different species, the exact conditions of pH and ionic strength required for good separation of IgG will vary. A method for preparation of rabbit IgG by ion exchange chromatography using a batchwise procedure is outlined below. Materials Diethylaminoethyl (DEAE) microgranular preswollen cellulose (Whatman DE-52) Phosphate buffer, 5 mM pH 6.5 Procedure. The batchwise procedure of Stanworth 25 is used. 1. Equilibrate approximately 5 g of DEAE-cellulose with several changes of phosphate buffer. 2. Dialyze 20 ml of serum against phosphate buffer at 4° overnight. 3. Place the cellulose slurry in suitable containers such as universal bottles or large test tubes and centrifuge to sediment the particles. Check the pH of the supernatant buffer against that of the starting buffer to ensure that equilibration is complete. Discard the supernatant. 4. Add dialyzed antiserum to the packed cellulose and mix by gentle rotation for 1 hr at room temperature. 5. Centrifuge gently to sediment the cellulose, then carefully transfer 2s D. R. Stanworth, Nature (London) 185, 156 (1960).

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the supernatant (containing immunoglobulin) to a clean container. Discard the cellulose. 6. Recentrifuge to remove any remaining cellulose, and decant the supernatant immunoglobulin solution. 7. Calculate the protein content as described previously. If this preparation contains serum proteins other than immunoglobulin the process may be repeated using a fresh aliquot of equilibrated DEAEcellulose. Preparation of Immunospecific (Affinity-Purified) Antibody For some purposes it is necessary to use specific antibody rather than whole antiserum or a crude immunoglobulin fraction. Immunospecific antibody can be prepared by passing antiserum or a globulin fraction through an immunoadsorbent column containing antigen chemically coupled to an inert solid phase. Specific antibody combines with the immobilized antigen and can be eluted subsequently with "chaotropic" ions (such as thiocyanate) or low pH buffers. A method for preparation of human IgG immunoadsorbent and the elution of specific anti-IgG antibodies is given here. Use o f lgG-Sepharose Immunoadsorbent Prepared by Periodate Oxidation 2~,27 Materials Sepharose CL4B Sodium metaperiodate Ethanediol Isotonic saline Carbonate-bicarbonate buffer, 0.1 M, pH 9.5 Phosphate-buffered saline 10 AM, pH 7.5 (PBS) Sodium borohydride Sephadex G-50, suspended in PBS Sodium thiocyanate, 3 M, adjusted to pH 6.6 Procedure ACTIVATION OF SEPHAROSE

1. Suck dry some of the Sepharose CL4B slurry. Weigh out 20 g of the gel and wash it with saline in a Biichner funnel containing two Whatman No. 54 filter papers. 26C. J. Sanderson and D. V. Wilson,Immunology 20, 1061 (1971). 27T. J. G. Raybouldand S. M. Chantler,J. lmmunol. Methods 27, 309 (1979).

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2. Make up 40 ml of 10% sodium metaperiodate solution in distilled water. 3. Suck the Sepharose dry on the Biichner funnel and transfer the pad to the periodate solution. Mix or stir gently for 2-4 hr at room temperature. 4. Transfer the Sepharose slurry to a Biichner funnel containing two Whatman No. 54 papers. Wash quickly with saline to remove periodate. 5. Pour on 40 ml of 10% aqueous ethanediol, allowing the liquid to run through the gel very slowly to ensure thorough washing. 6. Wash the activated Sepharose finally with sodium carbonate-bicarbonate buffer and suck dry. C O U P L I N G OF ANTIGEN .

2. 3. 4. 5.

.

Prepare 100 ml of IgG solution at a concentration of 1.0 mg/ml in sodium carbonate-bicarbonate buffer. Add the activated Sepharose to the IgG solution and mix or stir gently for 18 hrs at room temperature (or 4° if preferred). Transfer the slurry to a Btichner funnel containing two Whatman No. 54 papers. Suck dry and wash with PBS. Prepare 20 ml of a 5 mg/ml aqueous sodium borohydride solution. Transfer the Sepharose pad to the borohydride solution and mix or stir gently for 2 hrs at room temperature. (CARE: Borohydride reduction is accompanied by evolution of hydrogen and should be carried out in a loosely stoppered vessel in a well ventilated area). Transfer the gel to a Biichner funnel containing two Whatman No. 54 papers and suck dry. Wash extensively with PBS, and finally resuspend in PBS to desired concentration. The gel is now ready for use.

PREPARATION OF IMMUNOADSORBENT COLUMN

1. Clamp a column (approximately 1.5 cm x 40 cm) to a stand, and with the outlet closed run a small volume of PBS into the column. 2. Pour the Sephadex G-50 suspension into the column and allow to settle until approximately 1 cm of column length is filled. Open the outlet to allow a flow of PBS, which facilitates column packing. Add more Sephadex slurry to give a packed volume of one-third of the column length, with a reasonable depth of PBS above the packed Sephadex. Close the outlet. 3. Pour the IgG-Sepharose slurry into the column carefully so as not to disturb the surface of the Sephadex and allow it to settle in a separate layer on top of the Sephadex. 4. Cut a circle of Whatman No. 54 filter paper the same size as the internal diameter of the column and allow to float onto the settled

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surface of the IgG-Sepharose. This prevents disturbance of the surface of the column during subsequent sample and buffer applications. 5. Open the column outlet to allow excess buffer to run through and wash the column contents by passage of PBS until the absorbance of the effluent at 280 nm is equivalent to that of washing buffer. APPLICATION OF ANTISERUM OR GLOBULIN SAMPLE 1. Dialyze 1 ml of the serum or globulin solution against PBS overnight at 4° . 2. Open the column outlet to allow the head of buffer to pass into the gel. Close the outlet. 3. Apply the dialyzed sample to the top of the column, taking care to avoid disturbance of the Sepharose. 4. Open the outlet and allow the sample to run into the Sepharose column, closing the outlet when all the liquid has been absorbed. 5. Run PBS onto the top of the column and allow to flow through slowly by opening the outlet slightly. Ensure that a head of PBS is always present to avoid drying out. 6. Unadsorbed serum proteins will pass through the column and can be detected by a suitable monitor. When all the protein has emerged allow the remaining head of PBS to pass into the column and then close the outlet. E L U T I O N OF BOUND ANTIBODY

1. Gently apply 5 ml of sodium thiocyanate solution to the column and allow to run into the gel by opening the outlet. 2. As soon as the thiocyanate solution has entered the gel, close the outlet, apply PBS, reopen the outlet and allow PBS to flow continuously through the column as previously. Eluted antibody contained in the thiocyanate solution will pass through the Sepharose and into the lower, Sephadex portion of the column. The molecular sieving properties of the Sephadex will serve to separate antibody rapidly from the thiocyanate and reduce the risk of denaturation. 3. Collect fractions containing the antibody, pool, and concentrate to approximately 5 mg/ml. 4. Measure the volume and protein content. Store frozen or freezedried in suitable size aliquots. NOTE. This method of purification will select all antibodies reacting with the antigen on the immunoadsorbent, including any that may crossreact with other antigens by virtue of shared determinants. If such antibodies are likely to be present (as, for instance, will be the case in antisera raised against whole IgG), they should be removed by straightforward ab-

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sorption as described in a previous section: Absorption of Nonspecific Antisera. Absorption may be carried out before or after preparation of the immunospecific antibody, but the former is to be preferred for logistic reasons. Preparation of Fluorochrome and E n z y m e - L a b e l e d Antibodies

Selection of Antisera for Conjugation Satisfactory conjugates can be prepared only from potent antisera of the required immunological specificity; it is important therefore that the purest available antigen be employed as immunogen and that multiple injections be given to ensure the production of antisera in which the ratio of antibody globulin to nonantibody globulin is high. Ideally, antiserum should be selected on the basis of tests of potency and specificity carried out prior to labeling. This preliminary evaluation may conveniently be performed by titration in conventional gel diffusion and assessment of specificity in immunoelectrophoresis. If the lack of a suitable soluble antigen makes such tests impossible, indirect immunofluorescent or immunoenzyme tests should be done utilizing a range of dilutions of pre- and postimmunization sera as the intermediary layer followed by the appropriate labeled anti-species immunoglobulin. Test samples, which may be histological preparations or cell films should be appropriately prepared (some prior knowledge of the system is almost essential) and should represent both "positive" (antigen containing) and "negative" (non-antigen containing) materials. Antisera exhibiting the highest level of activity and specificity should be selected for labeling. Labeling should be carried out on immunoglobulin preparations derived from the selected antisera, so as to maximize the proportion of specific antibody to total protein and hence reduce non-specific activity in the final reagent. Immunoglobulin can be prepared by any of the methods described above, but only in the most demanding systems will it be necessary to prepare immunospecific antibody rather than a crude immunoglobulin fraction.

Fluorescein Labeling of Antibody Globulins Materials Fluorescein isothiocyanate, isomer I (FITC) Carbonate-bicarbonate buffer, 0.1 M, pH 9.0 Immunoglobulin preparation (10 mg/ml in saline) Phosphate-buffered saline, pH 7.5 (PBS) Sephadex G-50 medium

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Procedure 1. Prepare a solution of FITC in carbonate-bicarbonate buffer to give a solution containing 1 mg of dye per milliliter. 2. Place a measured volume of the immunoglobulin solution in a small beaker and cool to 4°. Place on magnetic stirrer. 3. Add one-tenth volume of carbonate-bicarbonate buffer. 4. Add one-tenth volume of FITC solution dropwise while stirring the immunoglobulin solution at 4° (approximately 1 mg of dye per 100 mg of protein). 5. Check pH after addition of FITC and if necessary adjust to pH 9.0 with 0.1 N NaOH. 6. Cover reaction vessel and stir gently at 4° overnight. (Alternatively the reaction can be carried out at room temperature for 1 - 2 hr if the volume to be labeled is less than 20 ml.) Removal of unreacted free FITC is preferably performed by dialysis followed by gel filtration chromatography on Sephadex G-50 (medium). 7. Dialyze conjugate against several changes of phosphate-buffered saline (PBS). 8. Prepare Sephadex G-50 column equilibrated with PBS such that the packed volume is at least six times the volume of conjugate to be applied. Allow a disc of filter paper, cut to fit the dimensions of the column, to float onto the top of the column. This facilitates the even application of conjugate. 9. Allow the PBS to run through the column until no buffer remains above the top of the column. 10. Stop the flow of buffer and apply the conjugate. 11. Allow the conjugate to flow into the column by opening the tap. When all the conjugate has passed into the column elute with PBS. 12. Collect the first colored peak to emerge (this contains the labeled immunoglobulins) and concentrate to the original conjugate volume. 13. Conjugates can be stored at 4° or in aliquots at - 20° after the addition of a preservative such as 0.1% sodium azide. Repeated freezing and thawing is to be avoided.

Peroxidase Labeling of Antibody Globulins Although a variety of methods can be used for coupling enzymes to antibody, 2s the conjugation procedures most commonly used with horse2s S. Avrameas, T. Ternynck, and J. L. Guesdon, Scand. J. Imrnunol. 8, Suppl. 7, 7 (1978).

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radish peroxidase (HRP) are the two-stage glutaraldehyde 29 and periodate oxidation 3° methods. In the former procedure peroxidase is first mixed with an excess of the dialdehyde glutaraldehyde, which reacts with free amino groups of the enzyme via only one of its active aldehyde groups. After gel filtration chromatography to remove excess glutaraldehyde, the activated enzyme is mixed with the immunoglobulin preparation to allow the free aldehyde group to combine with an amino group of the immunoglobulin. Conjugates prepared in this way have been shown to contain a homogeneous derivative 29~1 with a molecular weight of 90,000, but the coupling efficiency is poor at around 25% and 5% for antibody and enzyme, respectively. 32 The low efficiency in this system appears to be due to the relative paucity of reactive amino groups in HRP. In contrast the periodate oxidation method of conjugation 3°'33 is not dependent on the presence of reactive amino groups but relies upon the generation of active aldehyde groups after periodate oxidation of the carbohydrate moiety of peroxidase. These aldehyde groups combine with the amino groups of added immunoglobulin to form Schiff bases, which are subsequently stabilized by reduction with sodium borohydride. Conjugates prepared by this procedure contain high molecular weight derivatives, 3°-32but the coupling efficiency is increased to approximately 60% for both antibody and enzyme )4 Recent studies using a modification of the method described by Kato et al. 35 have shown that peroxidase can be satisfactorily coupled to antibody by coupling via sulfhydryl groups introduced into both the immunoglobulin and enzyme structures. 36 Conjugates prepared in this way contain active derivatives that are heterogeneous in relation to molecular weight but retain good enzyme and antibody activity. 37 Glutaraldehyde Conjugation M e t h o d 2s Materials

Horseradish peroxidase RZ 3.0 Stock solution of glutaraldehyde, 25% in water 29 S. Avrameas and T. Ternynck, lmmunochemistry 8, 1175 (1971). 3o p. K. Nakane and A. Kawaoi, J. Histochem. Cytochem. 22, 1084 (1974). z~ M. Mannick and W. Downey, J. Imrnunol. Methods 3, 233 (1973). 32 D. M. Boorsma and J. G. Streefkerk, J. Histochem. Cytochem. 24, 481 (1976). 33 M. B. Wilson and P. K. Nakane, in "Immunofluorescence and Related Staining Techniques" (W. Knapp, K. Holubar and G. Wick, eds.), p. 215. Elsevier/North-Holland, Amsterdam, 1978. D. M. Boorsma, J. G. Streefkerk, and N. Kors,J. Histochem. Cytochem. 24, 1017 (1976). 35 K. Kato, Y. Hamaguchi, H. Fukui, and E. Ishikawa, J. Biochem. 78, 423 (1975). ~6 p. D. Weston, J. A. Devries, and R. Wrigglesworth,Biochim. Biophys. Acta 612, 40 (1980). 37 S. M. Chantler and L. S. Cooper, unpublished observations, 1978.

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Phosphate buffer, 0.1 M, pH 6.8 Sephadex G-25 Isotonic saline Immunoglobulin preparation, 5 mg/ml in saline Carbonate-bicarbonate buffer, 0.5 M, pH 9.5 Lysine solution, 1.0 M pH 7 Phosphate-buffered saline, pH 7.5 (PBS) Saturated ammonium sulfate Glycerol Procedure 1. Dissolve 10 mg of peroxidase in 0.2 ml of a freshly prepared 1 : 25 dilution of the stock glutaraldehyde solution in phosphate buffer and allow to stand at room temperature for 18 hr. 2. Pass through Sephadex G-25 column equilibrated with saline to remove excess glutaraldehyde. 3. Collect the brown fractions, which contain the activated peroxidase, pool, and concentrate to 1 ml. 4. Add 1 ml of immunoglobulin solution (previously dialyzed against saline) to the peroxidase solution. 5. Add 0.2 ml of carbonate-bicarbonate buffer and leave for 24 hr at 4 °. 6. Add 0.1 ml of lysine solution and leave the mixture at 4 ° for 2 hr. 7. Dialyze against several changes of PBS at 4°. If desired remove free enzyme by precipitation with saturated ammonium sulfate as described in steps 8-10. 8. Add an equal volume of saturated ammonium sulfate to the conjugate and allow to stand at 4° for 30 min. 9. Centrifuge for 20 min at 4000 g and discard supernatant. 10. Dissolve precipitate in approximately 1 ml of saline and dialyze extensively against several changes of PBS. (Alternatively, sulfate ions may be removed by gel filtration chromatography on Sephadex G-50.) 11. Preserve by adding an equal volume of glycerol, and store at 4 °. Periodate Oxidation Conjugation Method Two procedures have been described by Nakane and co-workers. In the first of these 3° free amino groups on the peroxidase are blocked by fluorodinitrobenzene (FDNB) treatment prior to the production of active aldehyde groups by periodate oxidation. A recent modification of this method, described here, omits FDNB blocking and recommends periodate oxidation of the enzyme at low pH prior to coupling with immunoglobulin .33

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Materials Horseradish peroxidase RZ 3.0 (HRP) Sodium metaperiodate (freshly prepared), 0.1 M Acetate buffer, 1 mM, pH 4.4 Carbonate-bicarbonate buffer, 10 mM, pH 9.5 Immunoglobulin preparation Carbonate-bicarbonate buffer, 0.2 M, pH 9.5 Sodium borohydride Sephacryl S-200 Phosphate-buffered saline pH 7.5 (PBS) Procedure 1. Dissolve 4 mg of HRP in 1 ml of distilled water. 2. Add 0.2 ml of freshly prepared periodate to the enzyme solution and stir for 20 min at room temperature. 3. Dialyze against acetate buffer overnight at 4° . 4. Prepare globulin solution containing 8 mg of protein in 1 ml of 10 mM carbonate-bicarbonate buffer. 5. Adjust activated HRP solution to approximately pH 9 by addition of 20/zl of 0.2 M carbonate-bicarbonate buffer. 6. Immediately add the globulin preparation to the HRP-aldehyde and stir for 2 hr at room temperature. 7. Add 0.1 ml of freshly prepared sodium borohydride solution containing 4 mg/ml and leave at 4 ° for 2 hr. 8. Separate unreacted enzyme from the mixture by chromatography on a column of Sephacryl S-200 equilibrated with PBS or by salt precipitation with ammonium sulfate as described above. 9. If purification of conjugates is performed by gel chromatography, the appropriate fractions should be pooled and concentrated prior to storage at - 20°. Addition of albumin (10 mg per milliliter of conjugate) or an equal volume of glycerol prior to freezing in small aliquots is recommended. Repeated freezing and thawing should be avoided.

Evaluation of Conjugates A variety of tests should be used to determine the efficiency of conjugation and the suitability of the conjugate in use. The extent of the testing performed, particularly with respect to specificity, will vary with the intended use of the reagent. Efficacy of labeling can be determined very simply by measuring the absorbance of the conjugate both at the 280 nm protein peak and at the maximum absorbance wavelength of the label used. For immunohistologi-

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cal studies the ratio of OD495 to OD2s0 for fluorescein-labeled reagents should lie between 0.6 and 0.9; and the ratio of OD4o3 to OD~s0 for peroxidase-labeled conjugates, between 0.3 and 0.6. This test, however, fails to show whether biological activity is present in the conjugate. This should be determined initially by using the conjugate as the antibody in appropriate gel diffusion or immunoelectrophoresis tests (if a suitable soluble antigen preparation is available), followed by testing in the immunofluorescent or immunoenzyme system in which it is to be used. Performance testing by titration (direct method) or chessboard titration (indirect method) is e s s e n t i a l in order to select the optimal working dilution of the reagent and to assess its specificity under working conditions. Tests of immunological specificity carded out by other methods (e.g., gel diffusion) are irrelevant and may even give misleading results because of the widely varying sensitivity shown by different test systems. 3s

Antibody Production by Lymphocyte Hybridomas 3sa Conventional immunization by injection of antigen into an animal stimulates the production of a heterogeneous population of antibodies that differ in respect of both their affinity and their specificity. Although the immunization procedure or prior treatment of the recipient may be manipulated to favor the production of antibodies of predominantly high or low affinity, the specificity of the antibody response is less amenable to control and antibodies directed against each of several antigenic determinants present in the immunogen will usually be present. The extent of this heterogeneity of response will differ not only among members of different species but also in individual animals of the same species despite the use of identical immunogen preparations and immunization schedules. These biological factors influence both the ease and reproducibility with which antisera of the desired immunological specificity can be prepared. The application of cell fusion techniques for in vitro production of antibodies of defined specificity offers a significant potential alternative to conventional methods of reagent antibody production. In 1973, Cotton et al. a9 successfully fused cells of two plasmacytoma lines to produce hybrid cells capable of synthesizing both myeloma proteins. Subsequently, hybrid cells derived by fusion of a murine myeloma with spleen cells from appropriately immunized donors were shown to seas S. M. Chantler and M. Haire, Immunology 23, 7 (1972). aaa The authors wish to acknowledge the helpful criticism of Dr. Jane Hewitt during the preparation of this section. 39 R. G. H. Cotton, D. S. Secherand, and C. Milstein, Eur. J. Immunol. 3, 135 (1973).

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crete antibodies against the immunogen used. 4° These hybrid cells (hybridomas) could be grown in tissue culture, producing antibodies of defined specificity in vitro; alternatively, antibody secretion could be obtained in vivo by inoculation of the hybridoma cells subcutaneously or intraperitoneally into syngeneic recipients. This approach thus offered the possibility of the production of monoclonal antibody of defined specificity by selective cloning procedures, avoiding the need for highly purified immunogen or elaborate antibody purification procedures. Although it is now well established that the fusion of mouse myeloma cells and antibody-secreting splenic lymphocytes is an effective means of producing homogeneous antibody of defined specificity, a number of technical variables remain. The same basic principles are applicable to many systems, but fairly extensive preliminary investigation is required to define the optimal conditions, particularly in relation to the choice of immunization schedule and donor species used. Investigators interested in detailed methodology should refer to the recent proceedings of a workshop on lymphocyte hybridomas. 41 Choice o f Fusion Partners

The myeloma line selected should exhibit good growth characteristics in vitro, a high fusion frequency (one hybrid per 105 to 106 normal cells)

and should be sensitive to the selective medium HAT. If the cell line is lacking in either of the enzymes hypoxanthine guanine phosphoribosyltransferase (HGPRT) or thymidine kinase (TK), growth in this selective medium (which contains hypoxanthine, aminopterin, and thymidine) will be impossible. 4z Only after hybridization with a normal cell containing the enzymes can DNA synthesis and growth occur; thus hybrid cells alone survive in the selective medium. A limited number of myelomas exhibiting these features are available, and the one most commonly used is P3-X63Ag8 of B A L B / c origin. Hybrids obtained by fusion of an antibody-secreting normal cell and a myeloma cell such as the above produce specific antibody together with the myeloma protein and the products of mixed genetic combinations. This heterogeneous immunoglobulin production may not always pose a problem, but in applications where greater purity is necessary the difficulty can be avoided by using a nonsecreting myeloma line that produces 4oG. Krhler and C. Milstein, Nature (London) 256, 495 (1975). 41 F. Melchers, M. Potter, and N. L. Warner, eds., "'Lymphokine Hybridomas,'"in Curr. Top. Microbiol. lmrnunol. 81. (1978). 42j. W. Littlefield, Science 145, 709 (1964).

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no immunoglobulin of its own but still supports the synthesis of spleen cell-derived immunoglobulins. 43,44 The phylogenetic relationship between the cells utilized in hybridization studies determines the functional success of the hybrids produced. Murine myeloma lines have been successfully fused to both syngeneic and allogeneic mouse spleen cells 45,46 and to rat spleen cells, 4r but fusion with human lymphocytes and with cells of rabbit or frog origin has been less successful. 4s Recently Galfre and his colleagues 49 have described a rat myeloma line that has been successfully fused to rat spleen cells, but as yet no suitable human myeloma lines are available. The ontological derivation of potential fusion partners is also important. 5° It appears that optimal results are dependent upon fusion with cells of the B lymphocyte series at an appropriate stage of differentiation. Although the exact characteristics of the cell have not been identified, an activated B lymphocyte at an early stage of differentiation appears to be preferable. It follows therefore that selection of fusion partners of compatible phylogeny and ontogeny together with preselection of suitably differentiated B lymphocytes will increase the success rate of obtaining functional hybridomas. In practice, splenic cells from immunized mice have been most extensively used in experimental work because of the availability of suitable murine myelomas. Although the myeloma line (P3-X63Ag8) commonly used in fusion studies is derived from the B A L B / c mouse strain, it is not essential to use this inbred strain as a source of donor cells. Instead, it is preferable to use a strain that provides the best response to the immunogen in question; however, if it is intended finally to inoculate the hybrid clones into animals in order to produce antibodies in vivo, then clearly the recipient animal must be histocompatible. This can be achieved by using, as recipients, F1 hybrids of B A L B / c and the strain selected for initial immunization.

43 M. Schulman, C. D. Wilde, and G. Krhler, Nature (London) 276, 269 (1978). 44 G. Krhler, S. C. Howe, and C. Milstein, Fur. J. lmmunol. 6, 292 (1976). 45 G. Krhler and C. Miistein, Eur. J. Immunol. 6, 511 (1976). 46 G. Krhler, T. Pearson, and C. Milstein, Somatic Cell Genet. 3, 303 (1977). 47 G. Galfre, S. C. Howe, C. Milstein, G. W. Butcher, and J. C. Howard, Nature (London) 266, 550 (1977). 48 G. Krhler and M. J. Schulman, in "Lymphocyte Hybridomas" (F. Melchers, M. Potter and N. L. Warner, eds.), Curr. Top. Microbiol. Immunol. 81, 143 (1978). 49 G. Galfre, C. Milstein, and B. Wright, Nature (London) 277, 131 (1979). 50 p. Coffino, B. Knowles, S. Nathenson, and M. D. Scharff, Nature (London), New Biol. 231, 87 (1971).

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Immunization Procedure In most somatic cell hybridization studies the potential spleen cell donor is immunized in order to increase the proportion of cells producing specific antibody. This enrichment of functionally active cells has been shown to increase the percentage of hybridomas exhibiting the desired specific antibody activity. The type of immunization schedule adopted will depend upon the physical nature of the antigen and its immunogenicity, so that the variables, such as use of adjuvant, route of injection, and the timing of injections, will differ in different studies. Immunization commonly involves an initial subcutaneous injection of immunogen followed by a booster intravenous injection. The animals are tested 2 - 5 days after the boost, and a good responder is given a second intravenous injection, spleen cells being harvested 2 - 5 days later.

Preparation of Spleen Cells Separation of nucleated cells from red blood cells present in the spleen cell suspension is rarely performed. Spleen cells are washed twice in serum-free medium, the yield from one spleen being approximately 1 x 108 nucleated cells. A ratio of 10 spleen cells : 1 myeloma cell is used for fusion. If it is possible to enrich the proportion of plaque-forming cells in the spleen suspension--for instance, by rosetting with antigen-labeled red blood cells followed by centrifugation in Ficoll-Isopaque--the ratio used for fusion may be reduced to 1 : 1. Such an enrichment procedure not only decreases the number of cells that need to be distributed into individual culture wells after fusion, but also increases the percentage of hybridomas that secrete antibody of the desired specificity, thereby reducing the number of tests performed in the selection of appropriate hybrid clones at a later stage.

Cell Fusion In early studies fusion was promoted by the use of Sendai virus, but more recently polyethylene glycol (PEG) of molecular weight 1000-6000 has been preferred. The sediment of spleen and myeloma cells is gently resuspended in the small volume of washing medium remaining after centrifugation, and approximately 2 ml of 50% PEG solution diluted in the serum-free medium is added. After incubation at 37° for 1 min, the cell mixture is diluted slowly with medium, approximately 5 ml being added over a period of 5 min. The suspension is then centrifuged and resuspended in the selective HAT medium (containing serum) to a final density of approximately 106 cells per milliliter. This procedure yields approximately 100 ml of suspension from one spleen.

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Growth of Hybrid Cells The fused cells, suspended in HAT medium, are seeded into individual tissue culture wells, putting approximately 10n cells into each well. Unfused myeloma cells cannot grow in this selective medium, and normal spleen cells are incapable of prolonged growth, so only the hybrid cells survive. These "microcultures" are examined periodically, and those showing growth visible over approximately 30% of the base of the individual wells are tested for specific antibody activity, this stage being reached in successful wells between 7 and 20 days after seeding. The percentage of wells showing growth will depend on the number of cells originally introduced: approximately 90% of wells exhibit growth when l0 s cells are placed in each culture well. The majority of the wells will contain multiple clones derived from different parent hybrid cells, the products of many of which are irrelevant to the particular study. The proportion of wells containing functional hybrids of the desired specificity will vary considerably, but approximately 5% of those showing growth may contain appropriate hybrids.

Evaluation of Activity of Hybrid Products The supernatants obtained from individual culture wells exhibiting growth must be tested to determine whether any hybrids present in that culture are secreting antibody of the required specificity. Since the level of immunoglobulin secretion is low (approximately 10-50 tzg/ml) and the number of wells to be tested may be relatively large, it is essential that highly sensitive and specific assays that are readily performed on small volumes of supernatants be used for screening. Radioimmunoassays are most widely used, but hemagglutination, hemagglutination inhibition, and (in cases where localization of activity is relevant) immunofluorescence and immunoenzyme procedures have been applied.

Cloning of Active Hybrids As previously mentioned, culture wells containing antibody of the appropriate specificity may contain a heterogeneous population of hybrid cells secreting a variety of products. Individual hybrid cells can be separated only by additional cloning procedures, either by growth in soft agar or by using the limiting dilution method. Cloning by the soft agar method is carried out in petri dishes 3 cm in diameter that contain a layer of normal spleen "feeder" cells (10e per plate) in 5% agar, over which is then layered a dilution of the hybrid cells (obtained from positive wells) suspended in a medium containing 20% fetal calf serum in 2.5% agar. A range of different dilutions of the hybrid

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cells may be treated in this way. After incubation, individual clones of cells are detectable within 1-2 weeks. These discrete colonies are then transferred to microculture wells and their products are again tested for activity of the required specificity. Cultures exhibiting appropriate activity are immediately recloned at least twice in order to select stable functional cell lines, which are stored by freezing in vials or transferred to larger culture vessels. The limiting dilution method of cloning involves culturing serially diluted suspensions of hybrid cells together with normal spleen cells, each dilution being set up in 6-12 wells. The average number of hybrid cells dispensed in each series lies between 240 and 0.1 cells per well. At high cell levels growth is observed in most wells, but statistical considerations suggest that if only one-third of the wells seeded at a particular cell dilution show growth, then it is highly probable that the cells growing within each of the individual wells are derived from a single parent cell. These wells are then tested for antibody activity, and the cellular contents are recloned to establish functional stability in the same way as those derived by soft agar cloning procedures. Antibody Production

Once stable hybrid clones secreting antibody of defined specificity have been isolated, methods of obtaining maximal amounts of antibody become important. These may involve in vitro culture or in vivo growth in a suit~tble recipient. The hybridomas may be maintained in continuous culture in vitro for several months at a cell density within the range of 104 to 4 × 106 cells per milliliter. Under these conditions an antibody yield of 10-100/~g/ml can be obtained, but in most cases loss of functional activity eventually occurs. The reason for such functional instability is not clear, but it is likely to be due to loss of chromosomes during a period of time in culture. For this reason in vitro antibody production is more satisfactorily performed in limited rather than continuous culture, selected stable clones being stored by freezing at an early stage in their life cycle so that a new vial of cells can be thawed when required to initiate a fresh culture. As an alternative, antibody can be produced in vivo. Many cultured hybridomas have been successfully transplanted to genetically compatible recipients, 45,49,51and hybridomas derived from cells of nonmurine origin have been successfully transplanted to athymic nude mice. 52 In vivo antibody production is achieved by inoculating the cloned, hybrid cells 51 T. Pearson, G. Gaifre, A. Ziegler, and C. Milstein, Eur. J. lmmunol. 7, 684 (1977). H. Koprowski, Z. Steplewski, D. Herlyn, and M. Herlyn, Proc. Natl. Acad. Sci. U.S.A. 75, 3405 (1978).

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subcutaneously or into the peritoneal cavity. If the latter route is used, mineral oil is given several days prior to inoculation in order to encourage the production of ascitic fluid. The level of antibody obtained by in vivo culture is reported to be 100- to 1000-fold greater than for in vitro culture .39,45,49,51 In assessing the efficacy of in vitro versus in vivo production of reagent antibody, consideration must be given both to the relative concentrations of antibody and to the volumes obtainable. Subcutaneous inoculation of cells in a mouse yields approximately 1 ml of serum 2 weeks later; the yield of ascitic fluid harvested 7-14 days after intraperitoneal injection is between 5 and 15 ml. As the concentration of antibody produced in this way is at least 100-fold greater than in tissue culture, 10 ml of ascitic fluid from one mouse would be equivalent to at least 1 liter of tissue culture fluid. In this context, the recent description of hybridomas produced by fusion of a rat myeloma line with rat spleen cells is likely to be of considerable practical significance because of the larger volume of serum obtainable following inoculation of hybridomas in these rodents? 9 Nonhybridoma Techniques

The production of a thriving, functional hybridoma is dependent on a close phylogenetic relationship between the two parent cell lines; the only species to have provided suitable cell lines so far are mice and rats. An alternative approach to the production of nonrodent antibodies in cell culture is provided by the transformation of B lymphocytes on exposure to Epstein-Barr virus (EBV). Adult human peripheral blood cells exposed to EBV have been shown to release polyclonal secretory immunoglobulin. 53 Cultures of human peripheral blood lymphocytes exposed to antigen (sheep red blood cells) and EBV produce specific antibody. 54 Preselection of human peripheral blood lymphocytes exhibiting surface binding of tetanus toxoid and the hapten NNP (4-hydroxy-3,5-dinitrophenacetic acid) followed by viral transformation has been shown to yield cells capable of antibody production in vitro, ss.5e Although the cultures have been shown to be active for some months, methods of increasing both the yield of antibody (at present only some 10 ng per milliliter of culture fluid) and longterm stability have yet to be devised. 57 Attempts to establish stable spesa A. Rosen, P. Gergely, M. Jondal, and G. Klein, Nature (London) 267, 52 (1977). 54 A. L. Luzzatti, H. Hengartner, and M. H. Schreier, Nature (London) 269, 419 (1977). ss V. R. Zurawski, E. Haber, and P. H. Black, Science 199, 1439 (1978). M. Steinitz, G. Klein, S. Koskimies, and O. Makel, Nature (London) 269, 420 (1977). s7 V. R. Zurawski, S. E. Spedden, P. H. Black, and E. Haber, in "Lymphocyte Hybridomas" (F. Melchers, M. Potter, and N. L. Warner, eds.), Curr. Top, Microbiol. Immunol. 81, 152 (1978).

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cific antibody-secreting cell lines by somatic hydridization with the murine myeloma P3-X63Ag8 have been unsuccessful. 58

Summary The successful fusion of normal and neoplastic lymphocytes has laid the foundation for the production of a variety of antibody specificities of practical relevance in research and diagnosis. The technical problems associated with this approach should not be underestimated, but one cannot fail to recognize the enormous range of applications that lie ahead once these problems have been overcome. H. Hengartner, A. L. Luzzatti, and M. Schreir, in "Lymphocyte Hybridomas" (F. Melchers, M. Potter, and N. L. Warner, eds.), Curr. Top. Microbiol. Immunol. 81, 92 (1978).

[6] P r e p a r a t i o n o f F a b F r a g m e n t s f r o m I g G s of Different Animal Species By MICHAEL G. MAGE

The light and heavy polypeptide chains of the IgG molecule are folded into a series of globular regions called domains 1 (Fig. 1). The portion of the polypeptide chain between the CT1 and CT2 domains of the heavy chain, known as the "hinge region, ''2 is relatively accessible to proteolytic enzymes. When whole IgG molecules are incubated with the proteolytic enzyme papain, in the presence of low concentrations of sulfhydryl compounds, one or more peptide bonds in the hinge region are split,3 leading to the release of the Fab and Fc fragments (Fig. 1). The Fab fragments of IgG antibodies thus consist of the light chain, and the Vx and CT 1 domains 1of the heavy chain. Fab fragments are univalent, in that each fragment contains a single antibody combining site, composed of parts of the variable regions (VL and Vn) of the light and heavy chains. Because of their univalency, Fab fragments can be used to advantage in procedures where it is desirable to bind antigen to antibody in solution without cross-linking or precipitation or to bind to antigen on cell surfaces without producing "patching" or "capping. T M G. 2 D. 3 S. 4 F.

M. Edelman and W. E. Gall, Annu. Rev. Biochem. 38, 415 (1969). S. Smyth and S. Utsumi, Nature (London) 216, 332 (1967). Zappacosta, A. Nisonoff, and W. J. Mandy, J. lmmunol. 100, 1268 (1968). Loor, L. Forni, and B. Pernis, Eur. J. Immunol. 2, 203 (1972).

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cific antibody-secreting cell lines by somatic hydridization with the murine myeloma P3-X63Ag8 have been unsuccessful. 58

Summary The successful fusion of normal and neoplastic lymphocytes has laid the foundation for the production of a variety of antibody specificities of practical relevance in research and diagnosis. The technical problems associated with this approach should not be underestimated, but one cannot fail to recognize the enormous range of applications that lie ahead once these problems have been overcome. H. Hengartner, A. L. Luzzatti, and M. Schreir, in "Lymphocyte Hybridomas" (F. Melchers, M. Potter, and N. L. Warner, eds.), Curr. Top. Microbiol. Immunol. 81, 92 (1978).

[6] P r e p a r a t i o n o f F a b F r a g m e n t s f r o m I g G s of Different Animal Species By MICHAEL G. MAGE

The light and heavy polypeptide chains of the IgG molecule are folded into a series of globular regions called domains 1 (Fig. 1). The portion of the polypeptide chain between the CT1 and CT2 domains of the heavy chain, known as the "hinge region, ''2 is relatively accessible to proteolytic enzymes. When whole IgG molecules are incubated with the proteolytic enzyme papain, in the presence of low concentrations of sulfhydryl compounds, one or more peptide bonds in the hinge region are split,3 leading to the release of the Fab and Fc fragments (Fig. 1). The Fab fragments of IgG antibodies thus consist of the light chain, and the Vx and CT 1 domains 1of the heavy chain. Fab fragments are univalent, in that each fragment contains a single antibody combining site, composed of parts of the variable regions (VL and Vn) of the light and heavy chains. Because of their univalency, Fab fragments can be used to advantage in procedures where it is desirable to bind antigen to antibody in solution without cross-linking or precipitation or to bind to antigen on cell surfaces without producing "patching" or "capping. T M G. 2 D. 3 S. 4 F.

M. Edelman and W. E. Gall, Annu. Rev. Biochem. 38, 415 (1969). S. Smyth and S. Utsumi, Nature (London) 216, 332 (1967). Zappacosta, A. Nisonoff, and W. J. Mandy, J. lmmunol. 100, 1268 (1968). Loor, L. Forni, and B. Pernis, Eur. J. Immunol. 2, 203 (1972).

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PREPARATION OF FAB FRAGMENTS

"Hinge" "

~ ~ ~ 1

/ Regi°n~

\CL \ J -- ~

//

""; //

VL

Papain

)

/

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143

Fab

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//

FIG. 1. A schematicdiagram of an IgG molecule, showingthe relationship of the Fab and Fc fragments to the intact ]gO molecule, and the cleavage by papain of the heavy chain between the C71andC72domains [modified from W. E. Gall and P. D'Eustachio,Biochemistry 11, 4621 (1972)]. Because they lack the Fc region (the C3,2 and C73 domains of the heavy chain1), Fab fragments are also useful where antigen binding is desired in the absence of effector functions, such as complement fixation, or where whole IgG molecules, particularly if complexed to antigen or if aggregated, could bind via the Fc portion of the heavy chain to cellular receptors for Fc. ~ This is of particular importance in studies using fluorescent antibody to surface antigens of cells that also have Fc receptors. Fab fragments have also been used therapeutically for the specific binding and excretion of small toxic molecules, 6 taking advantage of the Fab fragment's smaller molecular size, rapid clearance from the circulation, and lesser immunogenicity than whole IgG molecules. Fab fragments are usually prepared from whole IgG molecules by digestion with papain, as originally described by PorterF There are distinct subclasses of IgG, that in some animal species (e.g., mouse,a guinea pig, 9 sheep1°), in addition to having antigenically distinct Fc portions, differ with respect to electrophoretic mobility and binding to ion-exchange H. B. Dickler, Adv. lrnmunol. 24, t67 (1976). 8 V. P. Butler, Jr., D. H. Schmidt, T. W. Smith, E. Haber, B. D. Raynor, and P. Demartini, J. Clin. Invest. 59, 345 (1977). 7 R. R. Porter, Biochem. J. 73, 119 (1959). 8 M. Potter, Methods Cancer Res. 2, 105 (1967). B. Benacerraf, Z. Ovary, K. J. Bloch, and E. C. Franklin, J. Exp. Med. 117, 937 (1963). 10 E. T. Harrison and M. G. Mage, Biochim. Biophys. Acta 147, 52 (1967).

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media. The more acidic subclass, usually called IgG1, binds to DEAE-cellulose under conditions of low ionic strength, and can be eluted with a gradient of increasing salt concentration. TM However, it may be contaminated by small amounts of other serum proteins. The more basic class, IgG2, does not bind to DEAE-cellulose under these conditions, or elutes at the start of the gradient. Subsequent purification of the Fab fragments by ion-exchange chromatography is facilitated if the starting material is not a mixture of two IgG subclasses of different electrophoretic mobility. After purification of an immune IgG, one should check both subclasses for antibody activity, as antibody activity is not infrequently found to be predominantly in one subclass of IgG. ~1 The following procedure, modified from Keckwick TM and from Sober and Peterson, 13 can be used to prepare IgG from immune serum. To 100 ml of immune serum are added 75 ml of "36%" (36 g of Na2SO4, anhydrous, plus 100 g of H20) Na2SO4 solution slowly, with stirring. (Phenol, to a final concentration of 0.25%, can be added to Na2SO4 solution, as a preservative.) After standing for 1 hr at room temperature, the suspension is centrifuged for 20 min at 8000 rpm. The precipitate is redissolved in 20 ml of 0.15 M NaC1 and reprecipitated with 15 ml of the Na2SO4 solution. After 1 hr, the suspension is recentrifuged for 20 min at 8000 rpm. The pellet is redissolved and dialyzed against phosphate buffer, 10 mm phosphate, pH 7.6. Following dialysis, the retentate is passed through a column of DEAE cellulose (200 ml bed volume) equilibrated with the same buffer. The effluent consists of the more basic molecules of IgG (IgG2). Following emergence of the unbound IgG, an exponential gradient to 1 M NaCI can be used to elute the more acidic IgG molecules (IgG0, which emerge at the start of the gradient. The following procedure, modified from Porter, 1° can be used for proteolysis of IgG by papain. One milligram of crystalline papain or mercuripapain (Worthington Biochemical Corp., Freehold, New Jersey) is added to a solution of 100 mg of IgG in 10 ml of phosphate-buffered (10 mM phosphate, pH 7.3) 0.15 M NaCI, with 1 mM EDTA and 25 mM mercaptoethanol. The mixture is incubated for 1 hr at 37°. Further proteolysis is ended, and sulfhydryl groups are alkylated by adding iodoacetamide to a final concentration of 30 mM and incubating for an additional 15 min at 37°. 11 W. K. Ashe, M. Mage, R. Mage, and A. L. Notkins, J. Immunol. 101, 500 (1968). 12 R. A. Kekwick, Biochern. J. 34, 1248 (1940). la H. A. Sober and E. A. Peterson, Fed. Proc. 17, 1116 (1958).

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PREPARATION OF FAB FRAGMENTS

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FIG. 2. Immunoelectrophoresis of undigested goat IgG (upper well), and of Fab and Fc from papain digestion of goat IgG (lower well), showing the electrophoretic difference and the reaction of nonidentity between the Fab and the Fc fragments. The slot contains antibody to goat IgG.

After digestion, especially if preparing fragments from IgG of a previously uncharacterized animal species, the extent of fragmentation can be conveniently monitored by immunoelectrophoresis of the digest, TM where the electrophoretically separated fragments are visualized by precipitation with antiserum to the whole IgG. Since Fab and Fc are derived from different portions of the IgG molecule and share no antigenic determinants, there will be two arcs of precipitation that cross each other in a reaction of nonidentity, whereas whole IgG gives a single arc of precipitation (Fig. 2). 14 Purification of the Fab fragments is most conveniently done by ion-exchange chromatography. Choice of ion-exchange media depends on the animal species and subclass of IgG. In the rabbit, the Fc fragments are more basic than the Fab fragments, 7 and the separation is done with CMcellulose, the Fc fragment being most tightly bound and eluting last. In most other species (goat, 14 sheep, l°,H mouse, s human, ~5 horse, TM guinea 14 A. L. Notkins, M. Mage, W. K. Ashe, and S. Mahar, J. lmmunol. 100, 314 (1968). 15 B. Frangione and E. C. Franklin, J. Exp. reed. 12,4, 715 (1966). le j. H. Rockey, J. Exp. Med. 125, 249 (1967).

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FIG. 3. Immunoelectrophoresis of separated Fab (upper three wells) and Fc (lower three wells), following DEAE chromatography of a papain digest of goat IgG. The slots contain antibody to goat IgG. pig, 9,1r r a t , TM c o w , 19,2° c h i c k e n 21) t h e F c f r a g m e n t is m o r e a c i d i c t h a n t h e F a b f r a g m e n t , a n d D E A E - c e l l u l o s e c a n b e used.14 Z o n e e l e c t r o p h o r e s i s in s t a r c h gel, 22,2a P e v i k o n , 24 o r a g a r 25 h a s a l s o b e e n u s e d to p u r i f y h o r s e 16,~4 a n d h u m a n 26 F a b f r a g m e n t s . S e p a r a t i o n o f F a b b y i o n - e x c h a n g e c h r o m a tography can be done as follows: T h e d i s g e s t is d i a l y z e d a g a i n s t t h e s t a r t i n g b u f f e r f o r t h e s u b s e q u e n t c h r o m a t o g r a p h y ( a c e t a t e buffer, 10 m M a c e t a t e , p H 5.5 f o r C M - c e l l u l o s e , o r 10 m M p h o s p h a t e b u f f e r , p H 7.6, f o r D E A E - c e l l u l o s e ) . 17 R. C. Q. Leslie, M. D. Melamed, and S. Cohen, Biochem. J. 121, 829 (1971). ~8 V. Nussenzweig and R. A. Binaghi, Int. Arch. Allergy Appl. Imrnunol. 27, 355 (1965). 19 N. E. Kuchinskaya, A. Ya. Kulberg, and V. S. Tsvetskova, Biokhimia 30, 1065 (1965). 30 S. I. Wie, J. Immunol. 121, 98 (1978). 31 G. Dreesman and A. A. Benedict, J. Imrnunol. 98, 855 (1965). 33 H. G. Kunkel and R. J. Slater, Proc. Soc. Exp Biol. Med. SO, 42 (1952). 3a O. Smithies, Biochem. J. 71, 585 (1959). 3, H. J. Miiller-Eberhard, Scand. J. Clin. Lab. Invest. 12, 33 (1960). 25 D. V. Stefani arid A. Ya. Kulberg, Vopr. Med. Khim. 10, 279 (1964). 3s H. G. Kunkel, F. G. Joslin, G. M. Penn, and J. B. Natvig, J. Exp. Med. 132, 508 (1970).

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PREPARATION OF FAB FRAGMENTS

147

FIG. 4. Rabbit Fab (well 1) and Fc (well 2) fragments, separated by chromatography on CM cellulose, showing reaction of nonidentity between Fab and Fc. Wells 3, 4, and 5 have antibody to rabbit IgG.

After dialysis, the retentate is placed on the appropriate ion-exchange column and unbound material (Fab) is eluted with starting buffer before starting the gradient. A gradient with limit buffer of 1 M sodium acetate, pH 5.5, is used for CM-cellulose. For DEAE-cellulose the limit buffer can be 1 M phosphate, pH 7.6. On applying the gradient, the first material to elute is additional Fab, followed by Fc. Chromatography can be done at room temperature or at 4°. Figure 3 shows the immunoelectrophoresis of goat Fab and Fc fragments separated by chromatography on DEAE-cellulose. Figure 4 shows the reaction of nonidentity of rabbit Fab and Fc fragments, separated by chromatography on CM-cellulose. After separation of the fragments, when preparing Fab fragments from a new animal species, the Fab fragment can be identified by antigen-binding activity, for example by inhibition by Fab of the precipitation reaction between antigen and whole antibody molecules (Fig. 5). T M For commonly used animal species, Fab fragments from nonimmune IgG can be detected

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FIG. 5. Precipitation of antigen (mouse lgG1) (well 1) by goat antibody to the same antigen (wells 2, 5) is inhibited by the Fab fragment of antibody to the same antigen (well 7), but not by Fab from another antibody (to mouse lymphoma EL4) (well 3). Fc from antibody to mouse IgGl (well 6) and rabbit IgG (well 4) likewise fail to inhibit the precipitation.

by precipitation in gel with anti-light chain antibodies, which are commercially available. Fab fragments purified by ion-exchange chromatography may still contain undigested or partly digested IgG molecules that still have some or all of the Fc portion of the IgG molecule attachedY "z8These contaminants can be detected by double diffusion in gel against antibody to the Fc fragment or to whole IgG. When the Fab is destined for use in a procedure ~r j. W. Goodman, Biochemistry 4, 2350 (1965). 38 T. E. Michaelson and J. B. Natvig, Scand. J. lmmunol. 1, 255 (1972).

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FIG. 6. After chromatography on DEAE cellulose, a preparation of Fab from goat IgG (welt 2) still had material reacting with anti-Fc (well 1). Following passage through a column of protein A-Sepharose, the purified Fab (well 3) no longer had material reacting with the anti-Fc (well 1) but still reacted with anti-Fab (well 4).

where the absence of Fc is important, the Fc-containing fragments can be removed by passing the partially purified Fab fraction through an affinity column containing insolubilized anti-Fc antibodies. 29 Fragments containing Fc bind to the column, and the Fab fragments emerge unbound in the effluent. A procedure for preparing such an anti-Fc affinity column, quoted with permission za and modified, follows. One milliliter of horse anti-Fc serum (Behring-Werke Inst., Frankfurt, GFR) is added to 25 ml of 0.5 M NaCI-0.1 M NaHCOa buffer, pH 29 F. DeLaFarge and P. Valdiguie, J. Chromatogr. 123, 247 (1976).

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PRINCIPLES AND METHODS

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8.3, and the mixture is stirred (for 2 hr at room temperature) with 15 g of CNBr-activated Sepharose 4B (Pharmacia): This couples the antiserum to the CNBr activated Sepharose, and the activated Sepharose (with bound anti-Fc serum) is placed in a column (21 × 1.6 cm i.d.). Unbound material is washed from the column with the coupling buffer, and any remaining groups are allowed to react with 1 M ethanolamine at pH 8 for 1-2 hr. Three washing cycles are then used to remove noncovalently absorbed protein, each cycle consisting of a wash at pH 4 (0.1 M acetate buffer containing 1 M NaCl), and at pH 8 (0.1 M acetate buffer containing 1 M NaCl). When the IgG is of a subclass whose Fc binds to S t a p h y l o c o c c u s protein A, 3° an affinity column of insolubilized protein A can be used to remove Fc-containing fragments31 as follows.

aureus

Five grams of protein A-Sepharose (Pharmacia Fine Chemicals, AB, Uppsala, Sweden) are rehydrated according to the manufacturer's directions, packed into a column, and washed with l0 mM phosphate-buffered 0.15 M NaC1, pH 7.4. Goat Fab (23 mg in 13 ml) containing some molecules with Fc determinants still present (Fig. 6) was passed through the column. The effluent contained Fab but no longer reacted with anti-Fc (Fig. 6). When the Fc-containing fragments are sufficiently larger in size than Fab fragments, they can be removed by gel filtration on Sephadex G-150. 6"a2Fab fragments have been directly purified by binding to insolubilized antigen, followed by specific elution with hapten, e or with acetic or propionic acid. 33 They have also been purified by binding to an affinity column of insolubilized anti-Fab, followed by elution with 0.1 M glycineHC1 buffer, pH 2.8. 29 It should be noted, however, that such affinity purification by specific binding of the Fab fragment cannot be expected to remove undigested IgG molecules or partly digested fragments containing both Fab and Fc.

30H. Spiegelberg,Adv. ImmunoL 19, 259 (1974). al j. W. Goding,J. Immunol. Methods 20, 241 (1978). ~ H. G. Van Eyck, Biochim. Biophys. Acta 127, 241 (1966). 33j. L. Spratt and S. B. Jones, Life Sci. 18, 1013(1976).

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C A R B O D I I M I D E S FOR I M M U N I Z I N G C O N J U G A T E S

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[7] The Use of Carbodiimides in the Preparation of Immunizing Conjugates By S A R A B A U M I N G E R a n d M E I R W I L C H E K

Introduction and Principle Carbodiimides comprise a group of compounds whose general formula is R - - N ~ - - - C ~ N m R ', where R and R' are aliphatic, such as diethylcarbodiimide (C~H5--N~C~---N--CzHs), or aromatic, such as diphenylcarbodiimide

Sheehan and Hess ~ introduced the use of carbodiimides for the synthesis of peptide bonds. The preparative procedure is simple and easy to perform, and therefore it became the most important coupling method. The reaction may be represented as a dehydration and expressed as shown in Eq. (1).

R,

R2

RNHCHCOOH + H2NCHCOORs+R4N=C=NR 4

RNHCHCONHCHCOOR 3

+ R4NHCONHR 4

(1) The mechanism of the reaction is not yet fully understood. It is postulated that an intermediate is formed that can react either with an amine to give the desired peptide or to rearrange to an acyl urea [Eq. (2)].

J. C. Shcehan and J. P. Hess, J. Am. Chem. Soc. 77, 1067 (1955).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1960by Academic Press, Inc. All dightsof reproduction in any form reserved. ISBN 0-12-181970-1

152

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PRINCIPLES AND METHODS RI I

RNHCHCONR 4 I

CO I

NHR4 (Acyl Ureo)

RI

NR 4 II RNHCHCO0-C I

(2)

I

NHR 4

Ri

R2

I

I

RNHCHCONHCHCOOR 5

+ R4NHCONHR 4

The acyl ureas are the main side products at elevated temperatures, and in order to shift the reaction toward peptide bond formation, temperatures around 0° should be used. Presence of an amino group during the reaction, also reduces the formation of acyl urea. The most commonly used carbodiimide for peptide synthesis performed in organic solvents is

urea, which is the product of the reaction, is very insoluble and precipitates in most solvents. Therefore it is easy to remove it by filtration. On the other hand, when the reaction is performed in aqueous solutions, the carbodiimides used are usually water soluble. The most useful water-soluble carbodiimides are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (I) and 1-cyclohexyl-3-[2-morpholinyl-(4)-ethyl]carbodiimide(II). /CH~ CH3CH2 N = C = N CH2 CH2 CH2 N H(~)CI(-) CH3

(~)

CH3

@

N=C= NCH2CHPN

0

(3)

(rr)

The urea formed during the reaction is water soluble and can be extracted with water if the peptide synthesis is performed in organic solvents. It can be removed by dialysis or by gel filtration when used to couple haptens to high molecular weight carriers. In addition to the use of carbodiimides for the direct formation of peptide bonds, they can also be applied for the preparation of active esters, such as hydroxysuccinimide

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orp-nitrophenyl esters. These esters can then be used for the formation of amide bonds according to Eq. 4.

0,,\ RCOOH+HO- N ~

0,,,, DCC • RCOON~

+ H2N-P

~RCONHP

(4) In the field of immunology, the main use of carbodiimides has been in the conjugation of weakly immunogenic or nonimmunogenic compounds to larger carder proteins or to synthetic antigens, thus enhancing their immunogenicity. Among the immunogens synthesized are complexes containing low molecular weight peptides, such as protein fragments (e.g., loop peptide of lysozyme 2) or hormones (e.g., ACTH, a bradykinin, 4 and gonadotropin-releasing hormoneS), steroidal hormones (e.g., estrogens, progestins, and androgense), prostaglandins, 7,8 cyclic nucleotides (e.g., adenosine 3',5'-cyclic phosphatea), and plant hormones (e.g., genistein 1° and gibberellic acidH). The conjugation of two compounds by the carbodiimide method requires the presence of an amino and a carboxyl group. In most cases the amino groups involved in the reaction are lysyl residues of the protein carrier 3-e or lysyl and alanyl residues of synthetic polypeptide carriers. 24° The carboxyl groups are, in most cases, contributed by the hapten. These either are originally present in the hapten or may be introduced into the molecule using a variety of chemical procedures. The introduction of such reactive groups has been performed either when the native molecule lacks such groups, a or when the native functional groups are also responsible for biological activity of the compound, and coupling through one of these groups may lead to their masking as antigenic determinants. 6,12The latter case relates in particular to steroid hormones, where methods have been 2 R. Arnon and M. Sela, Proc. Natl. Acad. Sci. U.S.A. 62, 163 (1969). 3 j. McGuire, R. McGili, S. Leeman, and T. L. Goodfriend, J. Clin. Invest. 44, 1672 (1965). 4 T. L. Goodfriend, L. Levine, and G. D. Fasman, Science 144, 1344 (1964). 5 y . Koch, M. Wilchek, M. Fridkin, P. Chobsieng, U, Zor, and H. R. Lindner, Biochem. Biophys. Res. Comrnun. 55, 616 (1973). e S. Bauminger, F. Kohen, and H. R. Lindner, J. Steroid Biochem. 5, 739 (1974). r L. Levine and H. Van Vunakis, Biochem. Biophys. Res. Commun. 41, 1171 (1970). s S. Bauminger, U. Zor, and H. R. Lindner, Prostaglandins 4, 313 (1973). 9 A. L. Steiner, D. M. Kipnis, R. Utiger, and C. Parker, Proc. Natl. Acad. Sci. U.S.A. 64, 367 (1969). 10 S. Bauminger, H. R. Lindner, E. Perel, and R. Arnon, J. Endocrinol. 44, 567 (1969). 11 S. Fuchs and Y. Fuchs, Biochim. Biophys. Acta 192, 528 (1969). l= S. L. Jeffcoate and J. E. Searle, Steroids 19, 181 (1972).

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devised for the insertion of chemical handles at different positions into the steroid molecule.6,12 Insertion of carboxyl groups to peptide hormones for similar reasons has been reported. 5,13 Among the mary methods for introducing carboxyl groups to molecules are succinylation,9 preparation of azo derivatives, 5 formation of chloroformate, hemisuccinate, or O-carboxymethyl oxime, TM and formation of thioether alkanoic acid derivatives.15'16 The compounds containing the native or inserted carboxyl groups are then attached to the macromolecular carrier using carbodiimide as the coupling reagent. Experimental

Methods for Introduction of Carboxyl Groups A carboxyl group can be introduced in almost any compound by various methods. In cases where a free thiol is present, the carboxyl group can be introduced easily by reaction with bromo- or iodoacetic acid. a7 This reaction is very mild and is usually performed at pH around 8-9. When the hapten contains a hydroxyl group, carboxylic acid may be introduced by one of the following methods: (a) carboxymethylation of the hydroxyl group with bromo- or iodoacetic acid; 17 (b) esterification with dicarboxylic acid anhydrides, such as succinic anhydride, to yield hemisuccinates, 14 which are unstable above pH 9; (c) reaction with phosgene, which results in the formation of chlorocarbonates. TM Haptens containing amino groups can be coupled directly to carriers containing carboxyl groups, or the amino group can be converted to a carboxylic acid by reaction with succinic anhydride, a'ls When a keto or an aldehyde group is present in the hapten, it can be converted to a carboxyl via reaction with O-(carboxymethyl)hydroxylamine15 or with hydrazides. Haptens containing double bonds can be made to react directly with mercaptoacetic or mercaptopropionic acid, TM or a two-step reaction may be performed: bromination followed by reaction with mercaptocarboxylic acid. a When a phenol or an imidazole is present in the hapten, the carboxylic 1~ y . Koch, T. Baram, and M. Fridkin, FEBS Lett. 63, 295 (1976). 14 B. F. Erlanger, F. Borek, S. M. Beiser, and S. Lieberman, J. Biol. Chem. 234, 1090 (1959). 15 H. R. Lindner, E. Perel, A. Friedlander, and A. Zeitlin, Steroids 19, 357 (1972). le A. Weinstein, H. R. Lindner, A. Friedlander, and S. Bauminger, Steroids 20, 789 (1972). 17 F. R. N. Gurd, This series, Vol. 11, p. 532. is I. M. Klotz, This series, Vol. II, p. 576. 19 R. Wagner and H. G. Gassen, Biochem. Biophys. Res. Commun. 65, 519 (1975).

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acid is most easily introduced via a reaction with diazonium salts, such as diazophenylacetic acid. 5 Haptens containing guanido groups may be made to react with p-carboxyphenylglyoxal to yield carboxyl groups. Typical examples for introduction of carboxylic group are described below. ll~-Hydroxyprogesterone Hernisuccinate. This compound is prepared essentially by the method of Buzby et al. 2° l la-Hydroxyprogesterone dissolved in dry pyridine is refluxed for 20 hr with a fivefold excess of succinic anhydride. The solution is then poured into iced water and extracted with 1 ml of ether. After washing with water, the hemisuccinate is extracted from the ether with bicarbonate solution and precipitated with 10% hydrochloric acid. The compound is usually recrystallized from methylene chloride-hexane. Testosterone-3-(O-carboxymethyl) Oxime. This compound is prepared according to the general method of Erlanger et al.~4 Testosterone dissolved in ethanol is refluxed for 3 hr with a solution of O-(carboxymethyl)hydroxylamine in 2 N KOH. The ethanol is then removed by evaporation, water is added, and the mixture is washed with ethyl acetate. The aqueous solution is acidified to pH 2 with hydrochloric acid. The precipitate formed is filtered, washed with water, and recrystallized from ethyl acetate-petroleum ether. Gonadotropin-Releasing Hormone-Azophenylacetic Acid (LH-RH Azophenylacetic Acid). 5 L H - R H has blocked N and C terminals, and therefore a carboxyl group has to be introduced in order to make it available for conjugation to protein. This is performed by attaching p-diazophenylacetic acid to synthetic LH-RH, which results in the formation of an azo derivative. p-Aminophenylacetic acid in cold 2 N HCI is diazotized by the addition of nitrite in cold (4°) water. After 8 min at 4 °, the solution is brought to pH 8.5 with a cold solution of sodium bicarbonate and immediately made to react with a solution of LH-RH in 60% aqueous dimethylformamide (DMF) containing 20% NaHCOa. The reaction mixture turns orangebrown within a few minutes, and the reaction is allowed to proceed for 12 hr at 4 °. The mixture is acidified to pH 2 with 2 N HCI and washed with ether, The aqueous phase is adjusted with 0.5 N NaHCOa to neutral pH and used as such after lyophilization or further purification by chromatography. Since LH-RH contains one histidine and one tyrosine, the product is a mixture of azohistidyl and azotyrosyl derivatives. 20

G. C. Buzby, Jr., D. Hartley, G. A. Hughes, H. Smith, B. W. Gadsby, and A. B. Jansen, J. Med. Chem. 10, 199 (1967).

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PRINCIPLES AND METHODS

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Synthesis of Compounds The coupling of carboxyl-containing compounds to amino groups of the polypeptide carrier is usually performed with an excess of carboxyl groups and an equivalent amount of the water-soluble carbodiimide around pH 5 at room temperature. In some cases where the hapten is not water soluble, it is usually dissolved in DMF or dioxane. In such cases it is also possible to prepare the hydroxysuccinimide ester using dicyclohexylcarbodiimide and couple this ester directly to the carrier. The experimental procedure will be demonstrated by two examples. Genistein-poly(DL-alanine) (Genistein-p DLAla--pLys). lo Genistein2-carboxylic acid, prepared according to Baker and Robinson, 21 is attached to pDLAla--pLys (MW 175,000)22 through the o~-amino groups of alanine as follows: 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (Ott Chemical Co.) is dissolved in 4 ml of H,O and added to a mixture of 1 g of pDLAla--pLys in 10 ml of H20 and 0.4 g of genistein-2carboxylic acid in 10 ml of DMF. After 20 hr at room temperature, the solution is dialyzed against 50% aqueous DMF for 24 hr, then against H20 for 48 hr and finally lyophilized. Spectrophotometric analysis indicated an average of 22 residues of genistein coupled per molecule of polymer.

Prostaglandin E~--Bovine Serum Albumin (PGE~-BSA). 8 PGE~ (100 mg, Upjohn Company, Kalamazoo) and 1.5 × 10-8 Ci of [3H]PGE~ (New England Nuclear Corp., 100 Ci/mmol) are dissolved in 1 ml of DMF. Then 60 mg of dicyclohexylcarbodiimide and 70 mg of N-hydroxysuccinimide are added. The reaction mixture is stirred at room temperature for 30 min. The precipitated dicyclohexylurea is removed by centrifugation, and the supernatant is added to a solution of 250 mg of BSA in 10 ml of 0.1 N sodium bicarbonate. The mixture is stirred at 4° for 2 hr, dialyzed against 50 mM sodium phosphate buffer, pH 8.0, and stored at - 2 0 ° . Measurement of the radioactivity in the conjugate indicated that an average of 15 residues of PGE~ were bound to each molecule of BSA.

Characterization of Conjugates Before characterizing the conjugate, it is essential to strip the complex of any molecule not covalently bound to the carrier. This is usually done by exhaustive dialysis TM or by gel filtration. 5 After these processes, the number of hapten molecules linked to each molecule of the macromolecular carrier can be determined by several ways. 21 W. Baker and R. Robinson, J. Chem. Soc. London 1926, 2713 (1926). 22 M. Sela, E. Katchalski, and M. Gehatia, J. A m . Chem. Soc. 78, 746 (1956).

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1. The easiest way is to introduce a radioactive tracer. The number of residues attached to each molecule of the carrier can then be determined by comparing the specific activity of the hapten and the conjugate.~5 2. If the bound compound absorbs light at a different region from the cartier, the analysis can be done spectrophotometrically. 14 3. If the attached groups are not labeled and do not contain chromophoric groups, they can be quantitated by dinitrophenylation or deamination of the unoccupied lysines of the carrier. 23 The amino acid analysis of the conjugate before and after deamination or dinitrophenylation enables determination of the number of side chains per carder molecule. Usually only a fraction of the lysine residues present in the carder molecule is accessible for coupling, e.g., about 30-35 out of 59 lysine residues of BSA. In general, for a compound to yield a good antiserum, 15-30 residues of hapten molecules have to be attached to each molecule of BSA.

Immunization The methods of immunization used by various investigators are numerous, and therefore we shall describe only the method we have used for the raising of antibodies to steroid hormones. This method may be applied also to other immunogens. The antigen (1 mg per milliliter of saline) is emulsified with an equal volume of complete Freund's adjuvant and of a vaccine against Bordetella pertussis (Pertussis Vaccine fluid, USP, Eli Lilly & Co., Indianapolis, Indiana; 1.6 units per animal) and injected into multiple intradermal sites of the rabbits. Booster injections are given at monthly intervals, and the rabbits are bled 10-14 days after each booster injection. The same schedule, using only 100-200/~g of antigen, may be applied to rats.

Specificity of the Antibodies Produced The specificity of the antibodies produced to the hapten is affected mainly by the site of attachment of the hapten to the protein carrier; recently it has also been reported that different species of animals may produce antibodies differing in specificity when challenged with the same antigen (prostaglandin-protein conjugate). 24 The influence that the site of attachment of the hapten to the peptide carrier has on the specificity of the antibodies produced was studied in great detail, mainly in the field of antibodies to steroid hormones. When 23 C. B. Anfinsen, M. Sela, and J. P. Cooke, J. Biol. Chem. 237, 1825 (1962). 24 S. Bauminger, J. lmmunol. Methods 13, 253 (1976).

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PRINCIPLES AND METHODS SPECIFICITY OF ANTISERA RAISED IN RABBITS WITH DIFFERENT CONJUGATES OF PROGESTERONE Cross-reaction (%) antisera to Compound

P-20- BSA a

P-7 - BSA b

P- 11- B SAc

Progesterone 11-Deoxycorticosterone Testosterone 17-Hydroxyprogesterone 20a-Hydroxy-4-pregnen-3-one 20fl-Hydroxy-4-pregnen-3-one Estradioi-17fl

100 96 95 98 34 96 <0.01

100 7 <0.1 15 1 2 <0.1

100 8.5 2 <0.1 <0.1 <0.3

a Progesterone-20-bovine serum albumin (P-20-BSA), from Midgley and Niswender. ~5 Progesterone-7-bovine serum albumin (P-7-BSA), from Bauminger e t al. z8 c Progesterone-1 l - b o v i n e serum albumin (P-11-BSA), from Lindner e t al. 15

preparing antibodies to steroid hormones, one is faced with the difficulty that the steroids share a common skeleton and often differ from each other only by the nature of a single functional group. If the steroid is attached to the protein via this position, the coupling may lead to the masking of distinctive features of the steroid hapten and thus to diminished specificity of the antibodies produced. Indeed most of the antibodies produced to steroids until the late 1960s utilized one of the functional groups present in the native hormone, and the antibodies formed with such antigens cross-react to a significant extent with each other. In the early 1970s Lindner e t a l . ~5 and Midgley and Niswender 25 used a different approach whereby the coupling of steroids to proteins was performed through reactive groups introduced into the steroid at positions remote from the existing functional groups. In this way it was possible to produce antisera that discriminate more efficiently between closely related hormones. A comparison of antisera to three conjugates of progesterone is shown in the table, a5,25,2eIt can be seen that sera produced by immunization with progesterone conjugated to the cartier via ring B (position 7), or ring C (position 11), recognize changes in the D ring better than does antiserum raised with progesterone conjugated to the carrier via ring D (position 20): they exhibit only negligible cross-reaction with testosterone, 17a-hydroxyprogesterone, deoxycorticosterone, and 20t~- and 20/3-dihydroprogesterone. On the other hand, all the anti2s A. R. Midgley and G. D. Niswender, in "Steroid Assay by Protein Binding" (E. Diczfalusy, ¢d.), p. 320. Karolinska Sjukhuset, Stockholm, 1970. 2~ S. Bauminger, H. R. Lindner, and A. Weinstein, S t e r o i d s 21, 847 (1973).

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bodies are equally efficient in discriminating estrogens, which differ from progestagens in the structure of ring A, since conjugation via ring B, C, or D leaves ring A unmasked and able to serve as an antigenic determinant. Antibodies specific to other groups of steroids, such as estrogens or androgens, 6 and to peptide hormones, such as LH-RH, s were produced by a similar approach. Comments The carbodiimide method is the most popular method for coupling hapten to carriers because it is carried out under mild conditions and the reaction is relatively fast. It is preferable to use, in addition to carbodiimide, the hydroxysuccinimide ester, which binds only to the amino groups. Most of the carriers contain carboxyl groups and therefore crosslinking may occur when using carbodiimides directly. Such a cross-linking may not, however, disturb the formation of antibodies as long as the conjugate remains soluble. By using the carbodiimide method a wide range of compounds were conjugated to carriers and used for production of antibodies. The antibodies produced were useful in a variety of immunological and biological studies, such as determination of the immunochemical properties ofhaptens and antibodies, 27of the physiological role of hormones 2s and their cytological localization, za The main contribution of the antibodies produced has been the development of radioimmunoassay procedures for various hormones and biologically active compounds, z° 27 S. M. Beiser, V. P. Butler, Jr., and B. F. Erlanger, in "Textbook of Immunopathology" (P. A. Miescher and H. J. Muller-Eberhard, eds.), p. 15. Grune & Stratton, New York, 1968. A. Kaushansky, S. Bauminger, Y. Koch, and H. R. Lindner, Acta Endocrinol. (Copenhagen) 84, 795 (1977). 29 G. K. Hargis, G. A. Williams, A. Tenenhouse, and C. D. Arnauld, Science 152, 73 (1966). zo "Radioimmunoassay and Saturation Analysis," Br. Med. Bull. 30, No. 1 (1974).

[8] U s e o f G l u t a r a l d e h y d e as a Coupling for Proteins and Peptides

Agent

B y MORRIS REICHLIN

On numerous occasions in immunochemical study the need arises to link proteins to particles, to polymerize proteins or to form covalent conjugates of proteins and smaller peptides. The availability of reagents that are simple and effective while largely preserving the native antigenicity of METHODS IN ENZYMOLOGY, VOL. 70

Copyright© 1~0 by AcademicPress,Inc. All rightsof reproductionin any form reserved. ISEN O-12-181970-1

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bodies are equally efficient in discriminating estrogens, which differ from progestagens in the structure of ring A, since conjugation via ring B, C, or D leaves ring A unmasked and able to serve as an antigenic determinant. Antibodies specific to other groups of steroids, such as estrogens or androgens, 6 and to peptide hormones, such as LH-RH, s were produced by a similar approach. Comments The carbodiimide method is the most popular method for coupling hapten to carriers because it is carried out under mild conditions and the reaction is relatively fast. It is preferable to use, in addition to carbodiimide, the hydroxysuccinimide ester, which binds only to the amino groups. Most of the carriers contain carboxyl groups and therefore crosslinking may occur when using carbodiimides directly. Such a cross-linking may not, however, disturb the formation of antibodies as long as the conjugate remains soluble. By using the carbodiimide method a wide range of compounds were conjugated to carriers and used for production of antibodies. The antibodies produced were useful in a variety of immunological and biological studies, such as determination of the immunochemical properties ofhaptens and antibodies, 27of the physiological role of hormones 2s and their cytological localization, za The main contribution of the antibodies produced has been the development of radioimmunoassay procedures for various hormones and biologically active compounds, z° 27 S. M. Beiser, V. P. Butler, Jr., and B. F. Erlanger, in "Textbook of Immunopathology" (P. A. Miescher and H. J. Muller-Eberhard, eds.), p. 15. Grune & Stratton, New York, 1968. A. Kaushansky, S. Bauminger, Y. Koch, and H. R. Lindner, Acta Endocrinol. (Copenhagen) 84, 795 (1977). 29 G. K. Hargis, G. A. Williams, A. Tenenhouse, and C. D. Arnauld, Science 152, 73 (1966). zo "Radioimmunoassay and Saturation Analysis," Br. Med. Bull. 30, No. 1 (1974).

[8] U s e o f G l u t a r a l d e h y d e as a Coupling for Proteins and Peptides

Agent

B y MORRIS REICHLIN

On numerous occasions in immunochemical study the need arises to link proteins to particles, to polymerize proteins or to form covalent conjugates of proteins and smaller peptides. The availability of reagents that are simple and effective while largely preserving the native antigenicity of METHODS IN ENZYMOLOGY, VOL. 70

Copyright© 1~0 by AcademicPress,Inc. All rightsof reproductionin any form reserved. ISEN O-12-181970-1

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PRINCIPLES AND METHODS

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the proteins and peptides under study is of considerable utility. Glutaraldehyde is such a reagent and has been successfully used for all the above applications. This chapter describes the usefulness of glutaraldehyde in the preparation of such materials. T h e Reactions of Glutaraldehyde with Proteins The chemistry of the reaction of glutaraldehyde with proteins has not been definitively elucidated; it is likely that several reactions occur, giving rise to a number of products. It is known that the reaction gives rise to a product(s) that is stable to acid hydrolysis and a chromophore with an absorption maximum at 265 nm. The stability of the cross-linkages to acid hydrolysis rules out simple Schiff base formation. The relationship of 4 mol of glutaraldehyde that react for the total moles of lysine plus hydroxylysine consumed in protein and the demonstration that model compounds, such as N~-carbobenzoxy-L-lysine and poly(L-lysine), react with glutaraldehyde to yield a 265 nm chromophore indicates that the e-amino group of lysine is the principal side chain in proteins reacting with glutaraldehyde. 1 Studies of the amino acid composition of carboxypeptidase A allowed to react with glutaraldehyde had also suggested that lysine was the primary amino acid not recovered in acid hydrolyzates.2 There have been recent attempts to identify the reaction products of glutaraldehyde with the model compounds 6-aminohexanoic acid and aN-acetyllysine.3 The ultraviolet, infrared, and nuclear magnetic resonance spectra of a purified product from the reaction of glutaraldehyde with 6-aminohexanoic acid has led to the postulate that compounds of the type illustrated below that have a polymeric quaternary pyridinium structure are a major type of product seen in proteins. More work will be needed to identify with certainty all the various reaction products that occur in proteins. Coupling of Peptides to Proteins Radioimmunoassay has proved to be such a powerful tool for the quantitative determination of peptides in biological fluids that assays have been developed for the great majority of peptide hormones and for many nonhormone peptides of biological interest. Since with rare exceptions the free peptides are not immunogenic, numerous coupling strategies A. H. Korn, S. H. Feairheller, and E. M. Filachione,J. Mol. Biol. 65, 525 (1972). z F. A. Quiochoand F. M. Richards,Biochemistry 5, 4062 (1966). 3 p. M. Hardy, A. C. Nicholls, and H. N. Rydon,J. Chem. Soc., Perkin Trans 1, 9, 958 (1976).

[8]

GLUTARALDEHYDEIN PROTEIN-PEPTIDE COUPLING [CH215"CO2H

161

[CH2]5"C02H

[CH2]5 "CO~H

L

R[CH2]2-/~ "CHe-------'~ :H2]s R[CH2]2~

yC

[CH~]~

~ HF-----------~

[CHz]5"CO2H

;H2]a

CH~--------~I~

[CH~]~ • C % H

].

~

[CH~]2R

[CH,]o • C % H

R = CHO or CH2OH

have been employed to prepare immunogenic peptide-protein conjugates. Glutaraldehyde has been employed for coupling adenocorticotropic hormone 4 (ACTH) and glucagon 5 to larger proteins. The resulting conjugates were uniformly antigenic in rabbits. A description of the coupling techniques is presented. ACTH-BSA

A solution was made containing 20 mg of bovine serum albumin (BSA) and 6 mg of porcine ACTH dissolved in 2 ml of 0.1 M phosphate buffer at pH 7.0. This represented 0.3/zmol of BSA and 1.3/~mol of ACTH so that the average A C T H - B S A ratio was 4.3: 1. One milliliter of 21 mM glutaraldehyde solution (also in 0.1 M, pH 7.0, phosphate buffer) was added dropwise to the B S A - A C T H solution with constant stirring. The reaction was allowed to proceed for 24 hr at room temperature, and the solution was then dialyzed against 0.1 M phosphate buffer, pH 7.0. Under these conditions the ACTH is quantitatively coupled to BSA, and this was demonstrated by ultracentrifugation and gel filtration on calibrated Sephadex columns, which revealed disappearance of the slowly sedimenting ACTH and disappearance of the retarded ACTH fraction, respectively, by the two methods. This conjugate was highly immunogenic in rabbits, and in eight of eight animals antisera were obtained in which antibodies to ACTH were easily demonstrable. After absorption with BSA, the com-

4 M. Reichlin,J. J. Schnure,and V. K. Vance,Proc. Soc. Exp. Biol. Med. 128,347 (1968). 5 L. A. Frohman, M. Reichlin,and J. E. Sokal,Endocrinology 87, 1055(1970).

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plement fixation reactivity of antiserum with B S A - A C T H was completely inhibited by ACTH, which did not itself fix complement with absorbed antiserum. Glucagon-RSA Glucagon was conjugated to rabbit serum albumin (RSA) by a technique quite analogous to the work described above for ACTH and BSA. Two modifications were introduced in this system. Owing to the low solubility of glucagon at neutral pH, the reaction was carried out at pH 10.0. To circumvent a vigorous immune response to the carrier protein, glucagon was conjugated to the autologous RSA. The RSA 15 mg and pancreatic glucagon (5 mg) were dissolved in 2.0 ml of pH 10.0 borate buffer; 1.0 ml of a 21 mM glutaraldehyde solution in borate buffer was added dropwise to the RSA-glucagon mixture with constant stirring. As in the preceding coupling procedure, the reaction was allowed to proceed 24 hr at room temperature and was then dialyzed against 0.1 M phosphate buffer. Direct evidence of coupling was obtained by ultracentrifugation of the complex, which demonstrated disappearance of the slowly sedimenting glucagon. In this case the glucagon-RSA ratio was 6.2: 1. Immunization with this conjugate gave rise to antibody in four of four rabbits, and the sera fixed complement with RSA-glucagon but not with free RSA or free glucagon. Free glucagon, but not free RSA, completely inhibited the complement fixation reaction between RSA-glucagon and antiserum. Of the four rabbits injected, three produced antibodies in titer and affinity sufficiently great for use in radioimmunoassay. The binding capacity for glucagon was calculated to be 10/zg/ml. With this serum as little as 0.05 ng of glucagon could be detected in radioimmunoassay. These two examples illustrate the ease and flexibility of the use of glutaraldehyde for the preparation of peptide-protein conjugates. In the two instances described the conjugates were uniformly immunogenic in rabbits. Preparation of Protein Polymers The immunogenicity of some small proteins can be considerably enhanced by polymerization. This is illustrated by the case of cytochrome c polymers prepared with glutaraldehyde. 6 Mammalian cytochromes c are only weakly immunogenic as monomers, and polymerization results in the production of potent immunogens. The procedure for polymerization of cytochrome c follows. Horse cytochrome c solutions (0.75 mM; 0.1 M phosphate, sodium M. Reichlin, A. Nisonoff, and E. Margoliash, J. Biol. Chem. 245, 947 (1970).

[8]

163

GLUTARALDEHYDE IN PROTEIN--PEPTIDE COUPLING TABLE I IMMUNOGENICITY OF CYTOCHROME C POLYMERS IN RABBITSa

Cytochrome c source Horse Human Tuna Macaca mulatta

Kangaroo Rabbit

Type of polymer injected

Precipitin content of antisera b (tLg of antibody N/ml serum)

Ratio of No. of rabbits responding to No. of rabbits receiving injection

Soluble Insoluble Soluble Soluble Soluble Soluble Soluble

192, 0, 51,585 40, 25, 15 232,262, 174, 214 415, 112, 106, 250 170, 163, 229, 80, 198 161, 85, 67 30, 0, 65

3:4 3 :3 4:4 4:4 5:5 3 :3 2 :3

a Data taken from Reichlin et al. 6 b Determined with homologous polymer preparations.

salts; pH 7.0; room temperature) were treated with glutaraldehyde at concentrations varying from 237 mM to 0.98 mM. At the highest glutaraldehyde concentrations (237 to 26 mM), the cytochrome c had completely precipitated from solution after 1 hr. At concentrations of 8.7 mM or less, corresponding to glutaraldehyde to cytochrome c molar ratios of 11 or less, the product was completely soluble. Excess L-lysine (0.1 M) was added to bind any residual aldehyde groups, and the reaction was allowed to proceed for one additional hour. The solutions were then dialyzed against 0.1 M sodium phosphate buffer, pH 7.0, and stored at 4°. Gel filtration studies on Sephadex G-150 of the materials prepared with a cytochrome c to glutaraldehyde molar ratio of 1 : 11 revealed that about 70% of the protein was excluded and the remaining polymers appeared to be quite heterogeneous. With a protein to glutaraldehyde molar ratio of 1 • 3, smaller, less heterogeneous polymers were obtained, as might be expected from a smaller degree of cross-linking. Polymeric mixtures were prepared from horse, human, rabbit, monkey (Macaca mulatta), turkey, tuna, cow, and kangaroo (Macropus canguru) cytochromes c at protein to glutaraldehyde molar ratios of 1 : 11. All yielded similar gel filtration elution patterns. The polymeric materials emulsified in an equal volume of Freund's adjuvant were potent and reliable immunogens in rabbits, as listed in Table I. Of the cytochromes c, only the human protein was a consistent immunogen in rabbits as the monomer as detailed in a previous publication. 7 No polymer-specific antibodies were found in these antisera, as r E. Margoliash, A. Nisonoff, and M. Reichlin, J. Biol. Chem. 245, 931 (1970).

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PRINCIPLES AND METHODS

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monomeric cytochromes c were equivalent to polymeric cytochromes c in competitive binding assays even though the monomeric material was antigenically deficient in assays depending on antigenic multivalence, such as precipitation and complement fixation. ~ Interestingly, once the animals were immunized with polymers, excellent anamnestic response could be achieved with monomeric material. The use of glutaraldehyde to prepare more immunogenic forms of weak antigens by cross-linking has also been successfully used to elicit potent antisera to human myoglobins and to induce reliably an anti-idiotypic response to certain rabbit antihapten Fab fragments in rabbits, a It is our impression from a limited study that soluble polymers are more immunogenic than insoluble polymers. Protein-Particle Conjugation Immobilized enzymes, antigens, and antibodies have found wide application in biochemistry and immunochemistry, and glutaraldehyde has been effectively used for their preparation. Several enzymes have been coupled to polyacrylamide beads with glutaraldehyde, and the optimum conditions of activation and linkage have been determined by Weston and Avrameas. 1° BioGel P300 minus 400 mesh was used, and pH 6.9 was found to be the optimum pH for activation with 6% glutaraldehyde. The most extensive studies were carried out with wheat germ acid phosphatase. Activation time was 17 hr at 37°, and protein uptake at 37° was complete in 18 hr. Although more enzyme was bound at higher pH levels, the beads first aggregated and then became a white powdery product. However acceptable levels of protein conjugation were achieved with beads activated at pH 6.9, under which condition the beads maintained their original physical nature. Uptake of protein to the washed activated beads was comparable over the pH range 6.9 to 9.0 and varied from 26 to 36 mg of protein per gram of dry gel. Using a pH of activation of 6.9 and a coupling pH of 7.7, the following enzymes were coupled to the polyacrylamide beads: ribonuclease, glucose oxidase, trypsin, and chymotrypsin. Table II lists the protein bound and the enzyme activity of such protein-particle conjugates. As can be seen, reasonable preservation of enzymic activity is achieved after conjugation and the particle-bound enzyme was stable to lyophilization and no diminution of activity was demonstrable after 16 days of storage at 4°. s M. Reichlin,unpublished results. 9 H. Dangharty,J. E. Hopper, A. B. MacDonald,and A. Nisonoff,J. Exp. Med. 130, 1047 (1969). 10p. D. Weston and S. Avrameas,Biochem. Biophys. Res. Commun. 45, 1574(1971).

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TABLE IIa Enzyme linked

Protein bound (p.g/g dry gel)

Protein active a s enzyme (%)

Ribonuclease Glucose oxidase Trypsin Chymotrypsin

27 7 18 19

30 56 58 65

a Data from Weston and Avrameas.10 In another study a similar technique was used to couple several antigens and antibodies to polyacrylamide beads with glutaraldehyde. 11 Among the antigens studied were rabbit and mouse IgG, BSA, trypsin, and peroxidase, all of which were effectively coupled to the activated beads. The corresponding sheep and rabbit antibodies were quantitatively absorbed to these immunoabsorbents and desorbed with 0.2 M glycine HCI buffer, pH 2.8. Yields of antibody ranged from 67 to 96.7% of the calculated added antibody as determined by quantitative precipitin analysis. Such specifically purified antibody was also coupled to activated polyacrylamide beads, leading to materials that could bind specific antigen. These antigen-particle or antibody-particle preparations were stable to acid elution, and repeated desorption steps did not seem to diminish their binding capacities. Comments The use of glutaraldehyde for the applications cited in this article is well established. The disadvantages of this reagent as a cross-linking reagent are few and are shared with all other cross-linking reagents. Any chemical modification related to cross-linking will have some altering effect on the conformation of the protein. This has a variable effect on the biological activities of proteins. The advantages are many and include simplicity, generality of application, and reasonable specificity for ~amino groups of lysine (and sulfhydryl groups as well). These properties lead to highly efficient coupling between peptides and proteins, easily controlled polymerization with the formation of soluble products, and efficient coupling of enzymes, protein antigens, and antibodies to polyacrylamide beads. All these materials have nearly complete biological activity. This reagent should gain an even wider usage as a cross-linking reagent in the future. 11T. Ternyckand S. Avrameas,FEBS Lett. 23, 24 (1972).

166

PRINCIPLES AND

METHODS

[9]

[9] I m m u n o c h e m i c a l Analysis by Antigen-Antibody P r e c i p i t a t i o n in G e l s By JACQUES OUD1N

The author's aims are (a) to classify as rationally as possible the techniques of immunochemical analysis by antigen-antibody precipitation in gels; (b) to state the more or less general principles and laws which hold true for all or part of the techniques; (c) to help the reader in understanding the bases of the different techniques in a way that does not require any special knowledge of physics; and (d) to give a few examples to illustrate that this understanding of the principles and laws, plus an analysis of the reactions may raise, and eventually solve, problems of appreciable importance. The author has tried to offer a synthetic kind of presentation that has rarely, if ever, been attempted by others. Although antigen-antibody precipitation in gels had been performed long before, it was only in early 1946 that it was applied to the immunochemical analysis of antigen mixtures. ~ Examples were given (a) of one antigen (ovalbumin or Pneumococcus polysaccharide) giving rise to a single precipitation zone when it was allowed to diffuse from its solution into a gel containing specifically precipitating antibodies; and (b) of the same two unrelated antigens giving rise to two precipitation zones that evolved quite independently when they were allowed to diffuse from a mixed solution into a gel containing specifically precipitating antibodies against both antigens. As early as 1905 (less than 10 years after the first observation of the antigen-antibody precipitation 2 and the description of the so-called Liesegang phenomenon3), antigen-antibody precipitation performed in a gel had been mentioned in a paper devoted to Strukturbildung in Gallerten (structure formation in gels).4 The observation was briefly reported (without special comment) of a reaction of a rabbit serum anti-goat serum with a goat serum in a gelatin gel giving rise (in the ice box) to two opaque rings. No information was given on their movement or immobility. 4 An important characteristic of the Liesegang phenomenon is that each of the rings or bands that appear successively at greater and greater distances J. Oudin, C. R. Acad. Sci. 222, 115 (1946). 2 R. Kraus, Wien. Klin. Wochenschr. 736 (1897). "a R. Ed. L iesegang, Naturwiss. Wochenschr. 11, 353 (1896). 4 H. Bechhold, Z. Phys. Chem. 52, 185 (1905).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright @ 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

[9]

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167

from the source of diffusion is immobile, so that a continuous progression of the precipitation fronts is sufficient definitely to rule out a phenomenon of this kind. This point is often not explicitly stated in papers dealing with this subject. Prior to 1946/antigen-antibody precipitation in gels (chiefly in plates) had been reported by a fairly small number of researchers, who used it mainly for obtaining a Liesegang-like phenomenon or for the recognition of bacteria or bacterial variants. 5-11 Multiplicity of the reacting antigens had never been proposed as the explanation for the multiple precipitation zones that had been sometimes observed, but they were usually considered to be "Liesegang rings." The author is unaware of a single, unquestionable case of a phenomenon of the Liesegang type in antigen-antibody precipitation in gels. After 1946 the Liesegang phenomenon was referred to less frequently in discussions on antigen-antibody precipitation in gels (never considering the mobility of the separate bands), but the rational explanation proposed for multiple precipitation zones (that of the multiplicity of the reacting antigens) was nearly always accepted. Therefore, this particular type of precipitation rapidly proved to be the most suitable means of immunochemical analysis of antigen mixtures.

Definitions The word antigen will be used, in agreement with Landsteiner, TM to designate a molecular s p e c i e s - - o r a category of molecules--defined by its antigenic specificity, the latter being the ability of each molecule to combine with a given population of antibodies. Too frequently (and confusingly) the word antigen has been used to designate either a mixture of antigens or an antigenic determinant or group of determinants. In this chapter, an antigenic determinant, or an epitope 13 (on the surface of an antigen molecule), will be defined as the elementary structure which can combine with only one antibody molecule (or one antibody site). 5 L. Reiner and H. Kopp, Kolloid Z. 42, 335 (1927). 6 R. H. Sia and S. F. Chung, Proc. Soc. Exp. Biol. Med. 29, 792 (1932). 7 G. F. Petrie, Br. J. Exp. Pathol. 13, 380 (1932). B. G. Maegraith, Br. J. Exp. Pathol. 14, 227 (1933). a M. B. Kirkbride and S. M. Cohen, Am. J. Hyg. 20, 444 (1934). l0 R. Brown, Proc. Soc. Exp. Biol. Med. 45, 93 (1940). H G. F. Petrie and D. Steabben, Br. Med. J. 1, 377 (1943). lz K. Landsteiner, "The Specificity of Serological Reactions," Rev. Ed., pp. 7, 8. Dover, New York, 1962. 13 N. Jerne, Annu. Rev. Microbiol. 14, 341 (1960).

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PRINCIPLES AND METHODS

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Aims of Immunochemical Analysis The aims of immunochemical analysis are the enumeration, identification, and eventually the titration of the various antigens contained in an unknown mixture. The concentration of antibodies in antisera against these antigens may also be determined. It should be emphasized that no antigen can be detected unless the antiserum contains a sufficient concentration of antibodies to precipitate it; and, similarly, two antigens of a mixture cannot be distinguished from one another if, in the antiserum used, there is not a sufficient concentration of antibodies to precipitate one but not the other. Although certain nonimmunochemical characteristics of the antigens (e.g., evaluation of the diffusion coefficients, electrophoretic mobility, chemical or enzymic reactions) may help in the identification, the actual identification depends upon the antigenic specificity, which does not have any numerical expression. Therefore, this identification consists in establishing whether a given antigen that is present in the mixture to be analyzed (e.g., biological fluid) is present or not in another given solution. This identification is, in fact, that of the precipitation zone 14 due to a given antigen when one of the reagents (antigen solution, antiserum, or antibody preparation) is replaced by another. Classification of Techniques Approximately 2 years after antigen-antibody precipitation in gels was first proposed for immunochemical analysis,1 a number of techniques began to appear in the literature. No attempt is made to describe or enumerate all these techniques or their variations, which are described elsewhere. 15 Since antigen(s) and antibodies are located in different sites (layers, reservoirs) at the beginning of the experiment, in all cases one or both have to move so that they meet in the gel in concentrations and proportions suitable to give a visible precipitate. The cause of this movement may merely be the thermal molecular agitation which results in diffusion. Alternatively, one or two of the reagents may migrate as a result of an electric field (electrophoresis). In the latter case, diffusion, which cannot 14 The term "precipitation zone" is used instead of"precipitate zone" in order to emphasize that, in contrast with the precipitation zone that usually moves (laws of this movement will be considered below), the precipitate itself does not move in the gel under any circumstance (but possibly becomes denser or less dense). ~s C. A. Williams and M. W. Chase, eds., "Methods in Immunology and Immunochemistry," Vol. 3, pp. 103-374. Academic Press, New York, 1971.

[9]

IMMUNOCHEMICAL

A N A L Y S I S IN GELS

169

be avoided at the usual temperatures, becomes a somewhat parasitic phenomenon, but can usually be ignored. In either case, it may be assumed that the nature of the medium (agar or agarose, gelatin, polyacrylamide, cellulose acetate membranes) does not change the general principles. In the discussion that follows, the medium will be a gel of agar-agar.

Diffusion Techniques The techniques in which the reacting molecules are merely moved by diffusion may be classified according to two criteria. In so-called simple diffusion the antigen solution and the antiserum are in direct contact, whereas in double diffusion the two reagents are separated by a layer containing neither reagent. While convenient, these terms are not quite satisfactory, as none of the reagents ever escapes diffusion. In the latter case, the reagents have to diffuse toward one another to attain the conditions favorable for p?ecipitation (usually in the intermediate layer). In the former case, the conditions are most frequently such that the antigen(s), with concentrations in sufficient excess compared to that of the corresponding antibodies, diffuse into the layer containing the antibodies. Another criterion for classification is based upon the number of dimensions (one or two) in which the diffusion takes place. The one-dimension techniques are mainly those that make use of cylindrical tubes or of cells with parallel walls in which the antigen and the antibody layers usually occupy the whole section of the vessel with a height that can be varied along the dimension parallel to the axis of this vessel. In these conditions, the diffusion can be considered only along the latter dimension. In the two-dimension technique, the reservoir (at best circular) of one reagent is surrounded by a uniformly thick layer of gel placed at the surface of a glass slide (for example, in a Petri dish). A thin layer contains the other reagent (simple diffusion). Alternatively, both reagents diffuse from similar reservoirs into a gel layer containing neither of them (double diffu-

sion). Taking these two criteria into account, four types of techniques can be distinguished. In all of them, it is advisable to add a suitable antiseptic, such as Merthiolate 0.02% or sodium azide 0.05 to 0.1%, to the reagents, and to what will be the gel without reagent (in double diffusion). Simple diffusion in one dimension (Fig. 1A) (in tubes or in cells with parallel walls) was proposed in the first paper on immunochemical analysis I and later described in greater detail. TM A layer of antiserum mixed with a gelling substance (nearly always agar-agar) is covered with a layer ~6 j. Oudin, Ann. Inst. Pasteur 75, 30, 109 (1948).

170

PRINCIPLES

AND

[9]

METHODS

double diffusion

simple diffusion

2:::.:~i!iL

D

C

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1"

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~..

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F

-........_..~

FIG. 1. Schematic representation of six techniques of antigen-antibody precipitation in gels in which the two reagents--antigen(s) and antibodies--are not moved toward one another by any motor other than molecular agitation in diffusion. The latter is represented by dotted-line arrows. The solid-line arrows (in the first step, ofelectrophoresis, in boxes E and F), represent the effect of an electric field. The dotted areas represent the layers or reservoirs that contain an antigen solution (AgS, AgS 1, AgS2); the hatched areas represent those that contain an antiserum (or immune serum: IS). The precipitation zones, or rather their leading edges, are each represented by a more or less thick line. The following are shown, side by side, in the four upper boxes: on the left a reaction between one antigen solution and one antiserum; on the right, compared reactions of one antiserum with two antigen solutions AgS 1 and AgS2. o f a n t i g e n s o l u t i o n in t h e liquid o r g e l l e d s t a t e . T h e c o n d i t i o n s u s u a l l y a r e s u c h t h a t t h e a n t i g e n , diffusing into t h e g e l c o n t a i n i n g t h e a n t i s e r u m , g i v e s r i s e to a p r e c i p i t a t i o n z o n e . S i n c e a g a r - a g a r d o e s n o t s t i c k to g l a s s , p r e c a u t i o n m u s t b e t a k e n to p r e v e n t t h e r e a g e n t s f r o m s l i p p i n g b e t w e e n t h e gel a n d t h e g l a s s wall, t h u s d i s t u r b i n g t h e r e a c t i o n . T h e i n n e r w a l l o f t h e v e s s e l s h o u l d b e c o a t e d w i t h a thin l a y e r o f d r i e d agar. TM W h e n t u b e s a r e u s e d , t h e y a r e filled w i t h a 0.2 t o 0 . 3 % a g a r s o l u t i o n h e a t e d to a b o u t 60°C a n d a r e e m p t i e d at o n c e w i t h the s a m e p i p e t t e , t h e n i m m e d i a t e l y d i p p e d i n t o a b a t h o f m e l t i n g i c e , a n d finally d r i e d u n d e r v a c u u m o v e r P~O~. I f t h e a g a r s o l u t i o n w a s m a d e u p in n o r m a l s a l i n e , s m a l l salt c r y s t a l s f o r m a n

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opalescent layer that disappears when the reagents are poured into the tube. When cells (made as described below) are used, the same agar solution may be smeared on the inner glass walls (which have been moderately flame heated) to be used for the cell, and allowed to dry before the parts of the cell are assembled. These precautions do not have to be taken with gelatin, since it sticks to the glass walls. The advantages of agar over gelatin are a somewhat greater sensitivity of the reactions and a more rapid progression of the precipitation zones, probably correlated with the smaller concentration of the gel substance. Tubes with a small diameter are advisable; for example, somewhat less than 2 mm are used in the author's laboratory, and 1 mm when reagents must be spared. The first layer usually contains antiserum or, less often, antibody preparations. The antiserum (or a suitable dilution of it), or the antibody preparation, is heated in a water bath adjusted between 46° and 50°C and is mixed, a short time before its distribution in the tubes or cells, with an agar solution at the same temperature; the proportions are such that the antibody solution, brought to the chosen, final concentration, contains a final agar concentration in the range of 0.3%. A higher agar concentration would be advisable if wider tubes are used. A higher agar concentration (0.6 to 0.8%) is also advisable in cells. This layer is placed in the tubes using Pasteur pipettes which were suitably stretched so that their tips reach the bottoms of the tubes. For the distribution of the layers with agar, the pipettes are moderately flame heated. The delivery is mouth controlled with the help of a rubber tube attached to the pipette, care being taken to avoid air bubbles. The advisable height (preferably uniform) of this layer is in the range of 35 to 45 mm. Once the lower layer has gelled, and while the upper surface of the tube is still moist with vapor, the upper layer--either liquid or, more rarely, gelled in the same way as the lower layer--is poured above it. The height of this layer, usually about 35 mm or much less, if necessary (e.g., 10 mm), has to be uniform in all the tubes to be compared, especially for quantitative purposes. The tubes are hermetically sealed, for example, with a mixture of beeswax and vacuum seal or with modeling clay. Temperature changes, especially when rapid, are to be avoided during the progress of the reactions for reasons stated below. If quantitative determinations are made, the temperature should be the same for all the tubes to be compared, especially for the standards and the experimental samples. The best, but not necessary, conditions are realized in a constant temperature room. For quantitative purposes, measurements should be made after a uniform time. If time permits, we make them after 7 days, although essential data

172

PRINCIPLES AND M E T H O D S

[9]

can often be obtained in 1 or 2 days. The measurements may be made by very simple procedures (for example, with a caliper); our very numerous measurements are made very conveniently and accurately on photographic plates using a microscope (with a very limited magnification) built for this purpose. 16a Photographs are useful also for qualitative analysis; up to 15 tubes with not too thick wails can be photographed together, twice magnified, on a 9 × 12 cm plate. The principle of double diffusion in one dimension (Fig. 1B) had been proposed1: "Antigen and antiserum placed at either end of a gel column"; but this technique was practiced only later. 17 Double diffusion in tubes is accomplished in a manner similar to simple diffusion, except that between the layers containing the reagents a layer containing only agar of a suitably chosen height is placed. Cells with parallel walls may be used instead of tubes for either simple or double diffusion in one dimension. 5~ In the reactions in cells shown on the right of Fig. 1A and 1B, diffusion in one dimension may be considered to be achieved far enough from the region of the gel, which is in front of the interface between the two antigen solutions to be compared using the same antiserum. The cells (primarily composed of glass or plastic) are easily made using a sheet of flexible plastic, or rubber of suitable thickness, ~Tacut to the shape of the hatched part of Fig. 2 and slightly greased with silicone, the glass walls being held on either side of it by clamps. It is advisable that the width of these intermediate parts (hatched) be in a range of 2 cm. In the author's laboratory, the thickness of this intermediate part is usually about 1 mm, and sometimes even 0.5 mm. Simple diffusion in two dimensions (Fig. 1C) (on a glass slide or plate) had been the most common procedure used for eliciting the conventional Liesegang phenomenon (hence the term "Liesegang rings"), also studied sometimes with simple diffusion in tubes. This technique has been used (in Petri dishes) for antigen-antibody precipitation, TM and is now extensively and nearly exclusively used for quantitative determinations (see below67). For this purpose, the gel layer, made of a mixture of equal parts of antiserum brought to the desired concentration and agar at 3% in barbiturate buffer (pH 8.6; ionic strength 0.1) has to be of quite uniform thickness. This can be conveniently achieved in a manner somewhat similar to that used for the preparation of cells with parallel walls described above. The freshly prepared liquid mixture is poured into a cell made of l~a I. Riva and J. Oudin, Ann. Inst. Pasteur 121, 625 (1971). 17 C. L. Oakley and A. 1. Fulthorpe, J. Pathol. Bacteriol. as, 49 (1953). ira A. Bussard, Int. Congr. Microbiol., 7th Stockholm Abstr. 9c, 152 (1953). 18 ~ . Ouchterlony, Acta Pathol. Microbiol. Scand. 25, 186 (1948).

[9]

IMMUNOCHEMICAL ANALYSIS IN GELS

B

173

A

@ G

I)

FIG. 2. Double diffusion, or simple diffusion, in a cell with parallel walls. The hatched area represents the intermediate part, cut in a sheet of plastic or of rubber, and placed between two glass plates held together with clamps. The first layer, usually of antiserum (AS) with agar, is poured as shown by arrow 1. When this layer has solidified, the second layer, made of pure gel (AG) when double diffusion is involved, is poured as shown by arrow 2. When the latter layer has solidified, the cell is rotated onto its side BC, and the layers of antigen solutions with agar (S1, $2) are successively poured as shown by the arrows 3 and 4, each layer having to be solid before the next layer is poured. The procedure is the same for simple diffusion, except that there is no layer AG.

two glass plates (10 × 7 cm) held together with clamps on each side of a brass piece (1 mm thick) having a U shape along the one larger and the two smaller dimensions of the glass plate, the width of the U piece being 1 cm along these three sides. Once the gel has solidified, one of the glass walls, which had been previously siliconized, is removed. Round wells are dug at suitable places using a needle of 2 mm bore which is attached to a rubber tube that is used to suck out the gel inside the needle. The volume of antigen solution put into each well has to be accurately measured (for example, 2/zl). This may be performed with a microsyringe and a needle. 67 Double diffusion in two dimensions (Fig. 1D) has been used independently by two researchers for the recognition of toxicogenic bacterial strains. This gave them the opportunity of observing several precipitation zones, which they considered to be due to several reacting antigens. 1s-22 Both authors had used, as the antigen source, bacteria seeded in streaks, perpendicular to the antibody reservoir, on the surface of a medium suit1~ S. 2o 0 . 21 S. 22 S.

D. Elek, Br. Med. J. 1,493 (1948). Ouchterlony, Ark. Kem. Mineral. Geol. 26B, 141 (1949). D. Elek, J. Clin. Pathol. 2, 250 (1949). D. Elek, Br. J. Exp. Pathol. 30, 484 (1949).

174

PRINCIPLES

AND METHODS

[9]

able for bacterial growth in a Petri dish. The difference between the techniques used by these two authors was in the nature of the antibody reservoir: either a trough dug in the medium and containing the antiserum, TM or a strip of filter paper moistened with the antiserum and placed on (or into) the layer of agar. 19 Related techniques were later applied by these two researchers to the antigens contained in antigen solutions. ~°,2~ The reservoirs of antigen solutions were either holes in the agar medium or filter paper strips. The technique using holes has been used much more widely than the one with filter paper strips. The shape, position, and size of these holes may be endlessly varied (see examples, Fig. 3). Certain variants (rectangular reservoirs parallel to each other) may be closer to diffusion in one than in two dimensions, the latter being realized best with circular reservoirs. Two ways of making these holes may be considered: either the gel is prevented from forming in the reservoirs or is removed after it forms. In the first case, after a thin layer of agar gel has been placed on the glass bottom of a Petri dish, metallic molds, previously coated with a very thin layer of paraffin, with the desired shape of the reservoirs are placed on the surface of the gel, and a second amount of melted agar is poured around them so as to make a second gel layer about 3 mm thick. Once this second layer is solid, the molds are carefully removed (a hole in the middle of each mold may make its removal easier). Frames with molds of the different reservoirs in a fixed, desired position (such as those of Fig. 3) may be used. Alternatively, a single gel layer of suitable thickness may be poured in a single step, and holes, usually cylindrical, dug in it, for example, with a cork borer. After the gel is removed, one drop (or a few) of hot, melted agar is dropped into the bottom of each hole in order to prevent the reagents from slipping between the agar gel and the glass. This technique, as the others, may be employed on a micro scale, using small reservoirs such as those used in simple diffusion (see above). Once the reservoirs have been filled with the reagents, precautions should be taken to avoid desiccation, for example, by closing the Petri dishes hermetically. Refilling the reservoirs in order to compensate for the lowering of the liquid level, although mentioned in the first description of the technique, 2° is not advisable, as it may involve the introduction of artifacts as discussed below. 32 Electrophoresis followed by diffusion has been done on filter paper in cells with parallel walls,2a and in a layer of agar placed on the surface of a glass slide~4 (Fig. 1F). The latter technique is most frequently used. After the antigenic components of the mixture have undergone electrophoretic 23 M. D. Poulik, Can J. Med. Sci. 30, 417 (1952). ~4 p. Grabar and C. A. Williams, Biochim. Biophys. Acta 10, 193 (1953).

[9]

175

IMMUNOCHEMICAL ANALYSIS IN GELS

[]

[]

O0 0

[] C

D

&

0

0 0 K

0

0 000 0 F

0 O0 0 ©0 0 Q

00000 0000

00000 0000 00000 N

FIG. 3. A - H are examples in double diffusion in two dimensions of shape and position of a various number of reservoirs in a gel plate. Other examples may be found in Oudin, *'L'analyse immunochimique par la m6thode des gels" in "Techniques de laboratoire" (Loiseleur, secr6taire de r6daction), p. 1415. Masson, Paris, 1963, and in the references given therein.

separation, in a second step they diffuse from the place in the gel layer to which they have migrated toward antibodies diffusing from an antiserum placed in a trench parallel to the direction of the previous electric field. They give rise to precipitation zones (or arcs) at different places along this previous electric field. Double diffusion in two dimensions is performed in this second step (Fig. IF). Simple diffusion has also been carried out in a second step (Fig. 1E) to determine the concentrations of the reacting antigens 25 (see below). In this technique, a sufficiently large concentration of an antigen mixture is used, and a layer of antiserum mixed with agar is placed on the glass slide in contact with the antigens. Techniques Using Electric Fields for Immunochemical Analysis In contrast to the techniques described, several procedures use antigen-antibody precipitation in gels as a means of immunochemical analysis with an electric field as the motor o f the reagents. The antigens may be moved by an electric field into a gel containing the suitably buffered antiserum (Fig. 4A). The antigen molecules move with a constant speed until they combine with the corresponding antibodies, giving rise to a precipitation zone. When all the molecules of an 25 R. Backhausz, Ann. Immunol. Hung. 10, 133 (1967).

176

[9]

PRINCIPLES A N D M E T H O D S A 9 - Ab precipitation durin9 electrophoresis in Ab at uniform C on --

~ -~

in buffered 9el A

B

X Is,

-

+

+

+ FIG. 4. Schematic representation (see legend to Fig. 1) of three techniques in each o f which the motor o f the reagents is an electric field represented by arrows and by + or signs.

antigen have so combined and precipitated, the precipitation zone remains fixed. 26,27 Electrosyneresis (or immunoelectroosmosis) consists of placing in two layers in a tube, ~s or into two reservoirs in an agar layer on a glass slide 29 (Fig. 4B), the antiserum mixed with agar toward the anode, and the antigen solution mixed with agar toward the cathode, with buffered agar between them. Many antigens will migrate toward the anode in contrast with the immunoglobulins, which migrate toward the cathode. Under such conditions, the precipitation zones will appear in a much shorter time than if there were no electric field but merely double diffusion. This technique is particularly useful for antigens of high molecular weight (e.g., viruses). Indeed, it is difficult not to point out a partial similarity (a) between the preceding technique shown in Fig. 4A and simple diffusion since, in both cases, the antigen molecules enter a layer containing the antiserum; and 2a C. ~7 M. ~s A. 2u A.

B. Laurell, Anal. Biochem. 15, 45 (1966). Lopez, T. Tsu, and N. E. Hyslop, Jr., lmmunochemistry 6, 513 (1969). J. Crowle, J. Lab. Clin. Med. 48, 642 (1956). Bussard, Biochim. Biophys. Acta 34, 258 (1959).

[9]

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A N A L Y S I S IN G ELS

177

(b) between electrosyneresis and double diffusion since, in both cases, the reagents are moved toward each other in a layer of initially pure gel. A third technique (Fig. 4C) uses two s u c c e s s i v e electric fields to m o v e the a n t i g e n s . 3° In the first step, the antigens are separated by electrophoresis. In the second step, a strip of gel, cut after the electrophoretic separation, is transferred onto a gel layer containing the antiserum, and a second electric field, at right angles to the first, is applied to move the antigens into a gel layer containing the antiserum (similar to Fig. 4A). When the electroendosmotic flow is slow, certain proteins (yG-globulins), in contrast with most others, may move toward the cathode. Principles and Laws Three principles will be discussed. The first two hold true for all the techniques that have been summarized. 1. A single antigen gives rise to a single precipitation zone. Causes of error have led to erroneous assertions which have been discussed by the author. 31 The causes of error to be considered are as follows: Nonspecific precipitation which would also occur if the immune serum were replaced by a nonimmune serum. Changes in temperature, which cause, in diffusion techniques, changes in the number of collisions between molecules of antigen and antibody and, therefore, in the density of the precipitate in a very thin gel layer. These artifacts differ from the mobile precipitation zones not only in their appearance (thinness) but also in their necessary immobility. 16'al For the latter reason, they do not occur when the precipitation zone itself is immobile but mainly when its displacement is fairly rapid, as is frequently the case in simple diffusion (see below). Artifacts with somewhat similar properties may also occur when the concentration of the reagents is changed in the reservoirs, for example, by refilling them in double diffusion in two dimensions. 32 Leakage of a reagent between the gel and the glass wall can greatly disturb the reaction. A technical precaution to prevent such an accident with agar gels is always used in the one dimension diffusion techniques TM and has been described above. It has been reported that two precipitation zones for a particular kind 3oC. B. Laurell,Anal. Biochem. 10, 358 (1965). al j. Oudin, "Allergology,"pp. 319-338. Pergamon, 1962. a~j. Oudin, "Methods Med. Res. 5, 335 (1952).

178

PRINCIPLES AND METHODS

[9]

of antigen (a hapten coupled with a protein) may be caused by complement, which precipitates the soluble antigen-antibody complexes formed in antibody excessY The observation of " d u a l " precipitation zones was not made with protein antigens. The ability of complement to give rise to a second precipitation zone in the reaction of a single antigen would have been more clearly demonstrated with protein antigens, especially with preparations known to be homogeneous. Whenever there may be some reason to expect a similar result, it seems reasonable to decomplement the antiserum, and eventually the antigen solution. It has sometimes been claimed that a single antigen might give rise to more than one precipitation zone, an alleged reason being "that multiple streaks may be formed by a single antigen which reacts at different points in the diffusion medium with various antibodies with different diffusion rates and reactivity.'2° A priori, this would not lead to the assumed consequence. There is, in liquid media, a single equivalence ratio if the multiple epitopes against which the various antibodies may be directed are located on the same molecule. It is a reasonable assumption that there is, in the gel medium, a place, or rather a surface, where the equivalence ratio is realized, and that the molecules of one reagent (antigen or antibodies) that cross this surface will meet molecules of the other reagent and combine with them. Such reasoning is not a definitive demonstration of the principle under discussion, but of the conclusion that there is no a p r i o r i reason to expect the alleged consequence. It is certain that a l l - - o r nearly all--antigens, especially proteins, carry more than one epitope against which the corresponding precipitating antiserum contains specific antibodies. Therefore, if this were a sufficient reason for each antigen to give rise to several precipitation zones, multiple zones would a l w a y s - - o r at least frequently--be elicited by single antigens, even in the pure state. This expectation has by no means been verified. It might be added that antigen-antibody precipitation in gels had not been proposed as a means of immunochemical analysis before the principle "one antigen : one precipitation zone" had been verified with several antigens. 1 Overwhelming evidence has been supplied by the experimental results of several investigators. It is possible enzymically to break down the serum albumin molecule into three fragments. The reaction in gels (double diffusion in two dimensions) of the digestion products gives rise to three distinct precipitation zones with anti-serum albumin sera that give a single precipitation zone with the undigested serum albumin. 34 Other workers have coupled two simple haptens to the same protein and have allowed this artificial antigen aa W. E. Paul and B. Benacerraf,J. lmmunol. 95, 1067(1965). a4C. Lapresle,Ann. Inst. Pasteur 89, 654 (1955).

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to react with varying proportions of two precipitating antisera, respectively directed against the two haptens; they observed a single precipitation zone in double diffusion in two dimensions a5,36or in simple diffusion in one dimension. 37 The problem seems to be definitely solved. It might be added that in the literature very large numbers of antigens, each giving rise to a single precipitation zone, have been reported, but not a single reliable example of an undoubtedly single antigen giving rise to more than one precipitation zone if the causes of error are excluded. 2. A second principle, which should hold true whatever the motor of the reagents, is that the precipitation zones which are due to quite distinct antigens, i.e., to antigens showing no cross-reactivity detectable by the reacting antisera, appear and evolve independently from one another. 1,16There may be exceptions, such as in the case when the concentration of the antibodies against one antigen is so large that their precipitate stops the diffusion of the other antigens. Such exceptions seem to be extremely rare; only one such example has been observed by the author with an extremely strong goat antiserum (unpublished). It is implicit in the above statement of this principle that every time there are, in the antiserum, antibodies capable of combining with more than one antigen of the reacting mixture, the precipitation zones of these cross-reacting antigens no longer behave independently of one another. Precipitating systems of that kind have been studied qualitatively and quantitatively in the author's laboratory under the name of "complex systems."16'38 In the less complicated complex systems, only a part of the antibodies precipitated by an antigen of the mixture is also precipitated by another. For example, the antiserum is directed against hen ovalbumin, and this homologous antigen is contained in the antigen solution together with a heterologous antigen, duck ovalbumin. When these two antigens give rise to two distinct precipitation zones, the zone farther from the interface is necessarily that of the heterologous antigen. In the region of the gel located between the source of antigen and the precipitation zone of the homologous antigen, there is no free antibody able to combine with this homologous antigen and, even more so, with the heterologous one. 3. The third principle applies only to those techniques using diffusion as the motor of the reagents, and is concerned with the immobility or 35 H. Fujio, Y. Noma, and T. Amano, Biken J. 2, 35 (1959). 3e The same authors also coupled one given hapten to two proteins of different molecular weights, and therefore o f different diffusion coefficients; the reaction of the mixture gave a single precipitation zone with the precipitating antihapten serum. 37 M. Richter, B. Rose, and A. H. Sehon, Can. J. Biochem. Physiol. 36, 1105 (1958), 3s D. J. Buchanan-Davidson and J. Oudin, J. lmmunol. 81, 484 (1958).

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PRINCIPLES A N D M E T H O D S

[9]

movement of the precipitation zones. It will introduce the laws of the movement of the precipitation zones and those of their maximum density of precipitate. Mathematical treatments based on the physical theory of free diffusion have been published for simple diffusion in one dimension, 39 for double diffusion in one dimension, 4° and for double diffusion in two dimensions. 41 When the conditions of the reaction are quite symmetrical for a given antigen and the corresponding antibodies (namely, the shape and dimensions of their layers or reservoirs, the density and viscosity of their solutions, the fact that they are in the liquid state or in a gel, their diffusion coefficients), the precipitation zone is immobile when the ratio of the initial concentrations of antigen and antibody is close enough to the equivalence ratio determined by reactions in liquid media. When the ratio of the initial concentrations of the antigen and of the corresponding antibodies is sufficiently different from the equivalence ratio, the precipitation zone moves farther and farther from the source of diffusion of the reagent which is in excess. Limits to this movement in time are described below. All other things being equal, including the initial concentration of the antibodies, the greater the distance between the source of diffusion of the antigen and the precipitation zone, the greater the initial antigen concentration. All other things being equal, including the initial antigen concentration, the greater the distance between the source of diffusion of the antigen and the precipitation zone, the smaller the initial concentration of the antibodies. The preceding conclusions may be tentatively adapted so that they apply to all techniques, including those in which the motor of the antigen, and eventually of the antibodies, is an electric field, and those in which, because of the small volume of the reservoirs, the total amount of the antigen combines with the antibodies. A greater concentration--or a m o u n t - of the antigen and a smaller concentration of the antibodies both increase the distance between the starting point of the antigen and the extreme point reached by the precipitation zone either in a given time or when the movement of the precipitation zone has ceased. The laws of the movement of the precipitation zones were studied, especially in the case of simple diffusion in one dimension. As a function of the time t elapsed since antigen and antibodies were put into contact, the distance h (also called "penetration") between the interface and the leading edge of the precipitation zone is proportional to the square root of 39 j. A. Spiers and R. Augustin, Trans. Faraday Soc. 54, 287 (1958). 40 j. Engelberg, J. Immunol. 82, 467 (1959). 41 F. Aladjem, R. W. Jaross, R. L. Paldino, and J. A. Lackner, J. Immunol. 83, 221 (1959).

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t. 42 This law is related, using a simplifying hypothesis, 4s to the equations derived by integration of Fick's law. It is verified with a very satisfying accuracy for a long time (in the range of several weeks), provided that the height of the layers of antigen solution and antiserum are great enough. In simple diffusion in one dimension also, when the ratio h/x/~(or h in the unit of time) is studied as a function of the initial concentrations g of antigens, or a of antibodies, this ratio is related to the logarithms (log g or log a) of these concentrations by linear laws over a fairly large range of these concentrations. 42 In its linear portion, the slope of the curve of h/x/t against log g is proportional to the square root of the diffusion coefficient of the antigen. 45 The same slope is inversely proportional to the square root of the viscosity of the liquid mixed with the gelled substance. 45 For high values of the antigen concentration and low values of the antibody concentration, the above law is no longer valid, but is replaced by another. 46 The ratio of antigen concentration extrapolated to a zero value of h/x/~to the antibody concentration is that which entails the immobility of the precipitation zone, the latter remaining in close vicinity of the interface. In symmetrical conditions, especially when the two layers are in the gelled state, the above principle holds true. When the diffusion coefficients of antigen and antibodies are sufficiently different from one another, the immobility no longer occurs for a ratio of antigen and antibody concentrations equal to the equivalence ratio. It has been derived from the theoretical laws that if R is the antigen/antibody equivalence ratio, the precipitation zone is immobile for a g/a ratio equal to RX/DA/D~,where 4~ j. Oudin, C. R. Acad. Sci. 228, 1890 (1949). 43 This simplifying hypothesis consists in assuming that in the antibody-containing gel, at the successive levels that correspond to the leading edge of the precipitation zone, the diffusing antigen finds the same antibody concentration. One equation, derived by integration of Fick's law of free diffusion,44 gives the concentration c of the diffusible substance of diffusion coefficient D in a column of solvent of infinite length at time t and at a distance x from its level in contact with a solution at a constant concentration Co: c = co

1- ~

e - ~ dy

, w h e r e y = 2X/-D-t

Another equation that applies to the case of a column of infinite length of solution in contact with a column of infinite length of solvent is similar to the preceding equation, except that co is replaced by co/2. It may be seen that if the successive levels in which c has the same value in time are considered, only x and t are not constant, and the ratio x / X / t is constant. The above hypothesis is also helpful in visualizing the laws of maximum density of the precipitation zone (see below). 44 j. Crank, "The Mathematics of Diffusion." Oxford Univ. Press (Clarendon), London and New York, 1956. 45 j. C. Neff and E. L. Becker, J. Immunol. 78, 5 (1957). 46 E. L. Becker, J. Munoz, C. Lapresle, and L. J. Le Beau, J. Immunol. 67 (6), 501 (1951).

182

PRINCIPLES AND METHODS

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D~ is the diffusion coefficient of the antigen and D A that of the antibodies. 3a When the upper layer containing the antigen(s) is liquid, the h value may be significantly different for uniform concentrations o f antigen and antibodies when the total concentration (in weight per milliliter) of the substances dissolved in the upper layer is larger (h larger) or smaller (h smaller) in the upper than in the lower layer. 47 This p h e n o m e n o n has been tentatively explained by reference to the theoretical equations. 48 The fact that it is not observed when the two layers are in the gelled state agrees with the role played by convection in its mechanism. Not only the h distance, but also the maximum density of precipitate may be measured. Provided that the antigen concentration is in sufficiently large excess, the maximum density does not significantly vary with time and with antigen concentration. On the contrary, with suitable corrections for the optical density of the medium, the maximum density of precipitate is, for one given antigen, proportional to the concentration of the precipitated antibodies. 4a This is in agreement with theoretical reasoning. a9 In the techniques that make use of double diffusion, the " p o s i t i o n " o f the precipitation zone, i.e., the ratio of the distance between its level o f maximum density of precipitate and the antigen source to the total distance between the sources o f antigens and antibodies, has to be considered instead of the penetration. This position does not seem to have been related to time by simple laws. In double diffusion in one dimension, the position o f the precipitation zone varies almost linearly against the logarithm of the initial antigen concentration g and in an approximate linear fashion against log g / a . 5° It had been previously derived from the theoretical equations of free diffusion that when the precipitation zone does not move the ratio o f its distance to the respective sources of antigen and antibodies is equal to that of the square roots of the diffusion coefficients o f these reagents. 5x In double diffusion in two dimensions from circular reservoirs, the shape o f the precipitation zone depends on the relative diffusion coefficients of the antigen and the antibodies. When these diffusion coefficients are sufficiently different from one another, the shape o f the precipitation zone is that o f an arc with its convexity toward the reservoir o f the reagent with the smaller diffusion coefficient. 52 47j. Oudin, Discuss. Faraday Soc. 18, 351 (1954). 4s j. R. Preer, Jr. and W. H. Telfer, J. Immunol. 79, 288 (1957). 49A. R. Hayden and E. L. Becker, J. Immunol. 85, 591 (1960). 5oj. R. Preer, Jr., J. Immunol. 77 52 (1956). 5x0. Ouchterlony, Ark. Kern. 1 (7), 43 (1950). 52L. Korngold and G. Van Leeuwen, J. lmmunol. 78, 172 (1957).

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183

The maximum density of precipitate in the precipitation zone in double diffusion does not seem to obey simple laws as a function of time and the concentration of the reagents. In contrast with simple diffusion, in which the time necessary for the antigen and the antibodies to attain the minimum value required for precipitation is zero, this time is far from being negligible in double diffusion. According to the laws of free diffusion (see equations in footnote 43), this time is proportional to the square of the distance between the sources of diffusion of the two reagents. 3z Application of the above Principles and Laws to Immunochemical Analysis

Determination of the Number of Reacting Antigens This number is equal to that of the precipitation zones, provided that what one is counting is actually antigen-antibody precipitation zones and not something else (see discussion of sources of error). The number of antigens determined is a minimum number because the concentration of the antibodies against one antigen (or of an antigen) may be below the threshold detectable value and because a precipitation zone may be hidden by others, especially when they are numerous.

Identification of the Precipitation Zones This identification in the reaction of one given antiserum with several antigen solutions may be obtained by several means. The Appearance of the Precipitation Zones in Simple Diffusion. When one antigen reacts with the antibodies of one given antiserum, in simple diffusion, the precipitation zone is mainly characterized by its maximum density of precipitate and by the appearance of its leading edge, which is more or less sharp depending upon the proportion of nonprecipitating antibodies in the antiserum (see below). Since, in sufficient antigen excess, the antigen concentration does not influence the maximum density, these two features should not vary much when the same antigen is contained (at different, but sufficient, concentrations) in two different solutions that one wants to compare. Therefore, the appearance of the precipitation zone of one antigen, when this appearance is sufficiently different from that of the other zones, may be a very useful indication for identification in reactions with the same antiserum. 53

The Superimposition o f the "Profiles" in Simple Diffusion in One Dimension. This rests on the laws, summarized above, of the movement of ~aj. Oudin,Ann. Inst. Pasteur 89, 531 (1955).

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$I

SZ

SI

I

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S?_ hbz

hbl

o

Fl~. 5. In the two tubes containing the same antiserum, a, in the lower layer, the penetrations hal and ha2 of the precipitation zone of a given antigen diffusing from two different solutions S~ and $2 (at different concentrations) are different. The same is true for antiserum b. But so long as the laws that define the penetration h in a given time as a function of the logarithms of the antigen and antibody concentrations are linear, the difference between the penetrations does not vary when the concentration of the antibodies is varied (in antisera such as a and b).

the precipitation zones. It follows from the linear laws of the penetration h of the precipitation zone in a constant time, as a function of the logarithms of the concentration of the antigen and the antibodies, that the difference between the penetrations of the zones of the same antigen at two different initial concentrations should not vary when the concentration of the antibodies is varied (Fig. 5). Therefore, the difference between the penetrations of the precipitation zones of the same antigen diffusing from two antigen solutions should be the same with different antisera. Since only the difference between the penetrations, but not the penetrations themselves, are the same, this superimposition of the leading edges does not entail that of the interfaces. In addition, this superimposition may only be an approximation, as are the laws from which it follows, and also because certain minor variables are neglected. It is nevertheless sufficient to formulate a procedure for identification from the comparison (and superimposition) of the "profiles" (or of the trace copies) of the leading edges of the precipitation zones of a given antigen in the reactions, with different antisera, of the same set of antigen solutions (in the same order) after a constant time, for example, using photographs ~3 (see examples in Fig. 6). This procedure does not require extra reactions. Its certainty increases with the number of antigen solutions and antisera involved in the reactions that are compared. The preceding means of identification is helpful for the establishment of tentative profiles. This procedure has been extensively used in the author's laboratory in the immunochemical analysis of human serum and of its numerous fractions. Confirmation may be obtained by suitable absorption (see below).

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g

FIG. 6. The profile of the precipitation zones of one antigen in the reactions of the various antigen solutions with one antiserum in row A (white dashed line) superimposes on precipitation zones due to the same antigen in rows B and C (different antisera reacting with the same antigen solutions in the same order; photographs were taken after the same length of time). The profile of the precipitation zones of another antigen in all but two tubes of row B (dotted line) superimposes on the denser zones of the same antigen in row C. The identification of the precipitation zones of a third antigen in row C, which has not as yet been identified, could now be surmised from their appearance. The analysis of the reactions in a fourth row with the same antigen solutions and another antiserum might confirm this supposition. From J. Oudin. ~

The Modification of the Concentration of a Part of the Antigens or Antibodies; Absorption of Antibodies. T h e i d e n t i f i c a t i o n o f the p r e c i p i t a t i o n z o n e s b y a b s o r p t i o n o f the a n t i b o d i e s c o r r e s p o n d i n g to a g i v e n a n t i g e n is the m o s t u s u a l a n d c o n v e n i e n t p r o c e d u r e o f i d e n t i f i c a t i o n that rests o n the i n d e p e n d e n c e o f the p r e c i p i t a t i o n z o n e s o f n o n - c r o s s - r e a c t i n g a n t i g e n s . S e v e r a l p o i n t s s h o u l d be e m p h a s i z e d . T h i s p r o c e d u r e m a y be u s e d with a n y t e c h n i q u e , w h a t e v e r the m o t o r o f the r e a g e n t s . A s long as the a n t i b o d i e s that h a v e b e e n r e m o v e d r e a c t e d

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with only one antigen of the mixture, only one precipitation zone will disappear. The great number of precipitation zones (and therefore of reacting antigens) that may obscure the results of other procedures is no objection since the appearance of the reaction will be simplified by it. The antigen solution to be mixed with the antiserum for this absorption does not need to be a pure solution of one antigen and does not even have to contain only one antigen able to react with the antiserum. When, in simple diffusion, especially in one dimension, the distance between two precipitation zones is sufficiently large, it is almost certain (although the difference between the diffusion coefficients of the two antigens plays a role in this distance) that a suitable stepwise absorption may make the zone farther from the interface disappear without significantly changing the closer one. ~ Other procedures for identification also depend on the principle of the independence of the precipitation zones of distinct antigens and can be used with all techniques. They consist, using schematically a pure antigen solution or a unispecific antiserum, in increasing the initial concentration (or the initial amount in small reservoirs) of one antigen or of the antibodies against one antigen. Only one precipitation zone will be modified and reach, in a given time or when its movement stops, a point farther (antigen concentration or amount increased) or closer (antibody concentration increased) from the starting point of the antigen. Neighboring Reactions in the Gel Medium. The procedure of identification which is perhaps the most commonly employed in the techniques using diffusion without electrophoresis or eventually in electrosyneresis consists in allowing one antiserum to react with two antigen solutions in two neighboring regions of the same gel medium. Double diffusion in two dimensions was the first technique in which this procedure was applied 2° (Fig. 1D). The same kind of procedure was also used in simple and in double diffusion in one dimension in cells with parallel walls 55 (Fig. 1B). In double diffusion in two dimensions, there are primarily two precipitation zones for the same antigen diffusing from two reservoirs. When the position of the reservoirs is favorable, these two zones, after growing in length, coalesce, while the zones of two distinct, non-cross-reacting antigens would, all other things being similar, keep growing independently from one another. In the one-dimension technique, an antiserum placed in a gel layer at the bottom of a cell with parallel walls is allowed to react with the antigens J. Oudin, J. Immunol. 81, 376 (1958). 5s j. Oudin, J. Imrnunol. 114, 143 (1960).

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of two solutions placed in two adjacent gel layers. The latter are either in contact with the antiserum layer (simple diffusion) or separated from it by a layer of gel without reagents (double diffusion) (Figs. 1A and 1B). When an antigen is present in the two antigen solutions, it will give rise to a precipitation zone continuous from the start in front of the two layers. It is only when the concentration of the antigen is considerably different in the two solutions that the continuity may not be quite immediate and perhaps not quite perfect. This continuity will obviously not occur between the precipitation zones of two distinct--non-cross-reacting--antigens.

The Particular Case of Neighboring Reactions of Two Antigens Cross-Reacting with the Antiserum. The results, whatever the technique, are obviously different when two cross-reacting antigens are present, respectively, in the two antigen solutions and only a part of the antibodies that are precipitated by one of them is precipitated by the other. This is the case when one antigen is homologous and the other heterologous (to use Landsteiner's terminology 12) in relation to the antiserum. Then the coalescence 56 or the continuity ~ is only partiaP 7 (Fig. 7). Part of the antibodies does not distinguish the two antigens, and reacts with them as if they were reacting with the same antigen: coalescence or continuity. Another part of the antibodies that are precipitated by one antigen are not at all by the other: no coalescence or no continuity. Of course, these two antibody populations give rise not to two precipitation zones, but to a single one in front of the homologous antigen. The reaction is more complicated when the antiserum is homologous in relation to the two antigens (in the two solutions). Then if the two antigens are very different in relation to the antiserum (for example, in the case of human serum albumin and

O. Ouchterlony, Acta Pathol. Microbiol. Scand. 32, 231 (1953). 57 The term "partial identity," sometimes used to designate this situation, is quite unsatisfactory because identity is complete by definition. Even the term "identity," used to compare the antigen(s) responsible for a complete coalescence of the precipitation zones is not satisfactory for several reasons. One is that two naturally occurring antigens may seem "identical" in their reaction with one given antiserum, while another antiserum does not detect anything common to both. As an example, the IgGs of two rabbits reacting, on the one hand, with a suitable antiallotypic serum (nothing common) and, on the other hand, with an antiisotypic serum (no difference: the two antigens seem to be identical). ~ Another reason is that even when a single antiserum was used, in the author's laboratory two antigens (idiotypes) gave quite a continuous precipitation zone with a given antiserum, although precise absorption of the same antiserum made it possible to distinguish the two idiotypes by precipitation in liquid media. 59 For these reasons, among others, it is better to use a descriptive terminology instead of one which implies a conclusion that should be preceded by careful discussion, eventually after suitable complementary reactions. J. Oudin, J. Exp. Med. 112, 125 (1960). 59 G. Bordenave, Eur. J. Immunol. 3, 718 (1973).

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B

C FIG. 7. Cross-reaction of anti-human serum albumin rabbit serum (IS) with the homologous antigen (human serum albumin in solution $2), and with a heterologous antigen (horse serum albumin in solution S1) in three of the techniques illustrated in Fig. 1: (A) simple diffusion in one dimension in a cell with parallel walls (photographed after 2 hr); (B) double diffusion in one dimension in a cell with parallel walls (photographed after 2 days). In A and B the interfaces between the different layers are indicated by white dashes. (C) Double diffusion in two dimensions in a plate with square reservoirs (photographed after 3 days). For further explanations see text. From J. Oudin? 1

horse serum albumin reacting with a mixture o f rabbit antisera against both), the reactions can hardly be distinguished from those o f two antigens that are not related at all. When the two antigens are very similar (for example, hen and guinea hen ovalbumin reacting with a mixture o f rabbit antisera against both), the antibodies that are precipitated by one o f the antigens but not by the other are in small (or very small) minority, so that it is sometimes necessary to observe the reactions very carefully in order to distinguish them from those o f one antigen diffusing from the two antigen mixtures. The same means o f identification is also used to compare the reactions o f one antigen solution with two antisera. In this case, coalescence or continuity is observed e v e r y time there are, in the two antisera, respectively, antibodies that are precipitated by the same antigen, even if the reactions are homologous for one antiserum and heterologous for the other.

Supplementary Procedures Certain procedures are used in order to make the precipitation zones more visible (such as protein staining) or detectable (for example by auto-

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radiography6°), or to characterize certain antigenic components (e.g., carbohydrate or lipid staining, enzymic reactions 61) when the gel is accessible (gel layer on a glass slide) or when it can be made accessible by removing one of the glass walls of a cell. See also, below, the procedures used to increase the sensitivity of the antigen determinations in simple diffusion in two dimensions.

Quantitative Determinations Among a number of procedures that were proposed for quantitative determinations, only some which make use of the measurements of a variable and its comparison to a calibration curve will be considered. The results of all but one of the procedures below are expressed in arbitrary units: the concentration of the antigen in a solution chosen as standard. The concentration determined may be corrected by calculation and expressed in weight of antigen per unit volume if such a concentration in the standard solution is known. In simple diffusion in one dimension, the usual conditions are such that the antigen, in excess, diffuses into the antibody layer. Then, the penetration h, i.e., the distance between the interface and the leading edge of the precipitation zone, may be measured with precision so long as this leading edge is sharp enough. This is usually the case with protein antigens. This measurement is made after a uniform time, conveniently, using photographs (see above). A suitable solution of the antigen to be titrated, containing this antigen at as high a concentration as possible, is used for the calibration curve. The reactions of the chosen antiserum with the dilutions of this standard solution and with the test antigen solutions must be performed under uniform conditions and at a uniform temperature. In order to avoid a source of error mentioned above, care should be taken that the density (weight per unit volume) of the upper (antigen) liquid layer be, in all the reactions to be compared, larger than that of the antiserum (or antiserum dilution) in the lower (gel) layer. This should be taken into account when choosing the diluents of the antigen or antiserum. The density of the antigen solutions might be, in all reactions, smaller than that of the antiserum or antiserum dilution without introducing errors, but the smallest antigen concentrations that could be determined would then be larger. The same would be true if the same source of error were avoided by also gelling the antigen layer; this would introduce an unnecG. J. Thorbecke, G. M. Hochwald, and C. A. Williams, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 3, pp. 343-357, Academic Press, New York, 1971. el j. Uriel, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 3, pp. 295-321. Academic Press, New York, 1971. e0

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essary extra manipulation. These conditions are easy to fulfill when the antigen solution is, for example, blood serum, 62-64 and when the antiserum in the gel layer is suitably diluted (for example Aor 41in normal saline, or still less concentrated). For reasons stated above, it is convenient to draw the calibration curves on semilogarithmic paper. The reservation mentioned above about the expression of the results in arbitrary units is not true for an elaborate method of titration, also with simple diffusion in one dimension, 65 that makes use (a) of measurements of the maximum density of precipitate, from which the amount of precipitate at the level of the gel that corresponds to the equivalence ratio is evaluated, using a calibration curve; (b) of an evaluation, from the slope of the curve of h/vrf against the logarithms of the dilution of the antigen solution, of the diffusion coefficient of the antigen that leads, using a calibration curve, to the evaluation of the proportions of antigen and antibody in the precipitate (and therefore the antibody concentration in the antiserum); and (c) of measurements of the penetration h that lead to the evaluation of the excess, and therefore the concentration, of the antigen in the test antigen solution. Antigen determinations to be made using double diffusion in tubes with calibration curves having the shape mentioned above 5° and for double diffusion in plates 6e have been proposed. The principle of antigen determinations by simple diffusion in two dimensions is quite different from that in simple diffusion in one dimension, in spite of the similarities and relationships between the two techniques. In contrast with simple diffusion in one dimension, the volume into which the antigen diffuses is much greater than that of the reservoir from which it diffuses. Consequently, the time after which the precipitation zone ceases to progress is very far from being infinite. Practically, in the conditions described, 67 this time consists of just a few days. In contrast with simple diffusion in one dimension, the variable that is determined is not the initial antigen concentration but the amount of antigen that has been placed into the reservoir. This does not eliminate the role of the initial antigen concentration because, here also, if this concentration is not in sufficient excess to that of the antibodies, no precipitation zone will appear in the gel of the antiserum. The volume of antigen solution placed into the reservoir has to be measured with sufficient accuracy; this volume is in the range of a few microliters. ez j. Oudin and F. Stoltz, Ann. Inst. Pasteur. 121,581 (1971). C. Brezin, P. Lazar, and J. Oudin, Ann. Inst. Pasteur 121,603 (1971). m C. Brezin, P. Lazar, and J. Oudin, C. R. Acad. Sci. Ser. D 278, 401 (1974). 65 E. L. Becker, Arch. Biochem. Biophys. 93, 617 (1961). D. A. Darcy, Immunology 3, 325 (1960). 67 G. Mancini, A. O. Carbonara, and J. F. Heremans, Immunochemistry 2, 235 (1965).

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A simple law which was given for both this technique 67 and the following one, which uses an electric field instead of diffusion, 27 is that the area delimited by the stopped precipitation zone (or the height of the peak in the electrophoretic techniques) is directly proportional to the amount of antigen put into the reservoir, and inversely proportional to the concentration of the antibodies in the uniformly thick gel. It may be noted that this is what would be expected as a consequence of the simplifying hypothesis on simple diffusion in one dimension (see above and footnote 43). Other laws have been given for simple diffusion in plates 6s,69 and for electrophoresis of the antigens into a gel containing the antibodies, a° The sensitivity of these techniques may be increased by a factor ranging from 8 to 20 or more, as compared with the results obtained after protein staining, by several procedures that give legible results with purified antibodies--or immunoglobulin fractions--at an antibody concentration divided by the same factor. These procedures consist of labeling antibodies with radioactive iodine, 7°'71 with fluorescein, 72 with peroxidase, 73 or with glucose oxidase, alkaline phosphatase, or peroxidase.74 This labeling may be that of the antibodies of the reaction (direct method) or of the antibodies of another animal species directed against the above, and used after the reaction in order to label the specific precipitate in the washed gel (indirect method). As already pointed out, the technique in which the antigens are moved by an electric field into a gel layer containing the antiserum has certain similarities with simple diffusion. Here also, the displacement of the precipitation zone will cease after a finite time that is variable and measured in hours z~,27 when the antigen has been entirely absorbed in a precipitate with the antibodies. A consequence of the role of the electric field is that it may also entail movement of the antibodies in the gel. It has been pointed out that if antigen and antibodies "move in the same direction and at a similar rate, immune precipitation may not occur or bizarre patterns may b e s e e n . ''27

The principle of the antigen determination in the second step of the technique successively using two electric fields at 90 ° from one another a° is not different from that of the latter technique. Antibody determinations may usually be made using procedures more or less similar to the ones above. 68 j. L. Fahey and E. M. McKelvey, J. lmmunol. 94, 84 (1965). 69 G. Sandor and Z. Urosevic, C. R. Acad. Sci. Ser. D 276, 2753 (1973). 70 D. S. Rowe, Bull. WHO 40, 613 (1969). 71 R. Jalanti and C. Henney, J. lmmunol. Methods 1, 123 (1972). 72 y . M. Centifano and H. E. Kaufman, J. lmrnunol. 107, 608 (1971). 7a M. Stanislavski, C. R. Acad. Sci. Ser. D 271, 1452 (1970). 74 j. L. Guesdon and S. Avrameas, Irnmunochemistry 11,595 (1974).

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J FIG. 8. The same antigen solution (horse serum albumin) was placed in the upper layer of these two simple diffusion tubes. The antiserum used in the right-hand tube was that of the left-hand tube (i.e., anti-horse serum albumin) that had been partially absorbed by the homologous antigen, so that not only the antibody concentration had been decreased, but the proportion of precipitating to nonprecipitating antibodies had also been greatly decreased; hence the difference in the appearance of the precipitation zone: smaller density and fuzzy (instead of sharp) leading edge. The antiserum in the left-hand tube had been diluted in norreal rabbit serum in order to ensure a similar penetration h in the two tubes. From J. Oudin, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 3, p. 125. Academic Press, New York, 1971.

P a r t i c i p a t i o n in t h e P r e c i p i t a t i o n R e a c t i o n s in G e l s of A n t i b o d i e s P r e s e n t in A n t i s e r a T h a t W o u l d U s u a l l y N o t P r e c i p i t a t e Such antibodies can be suspected or e v e n detected by their qualitative or quantitative influence on the precipitation zones of w h a t could be t e r m e d normally precipitating antibodies. T h e y can affect the a p p e a r a n c e o f the precipitation zone in simple diffusion (Fig. 8) and its penetration. Their presence in an antiserum that does not precipitate the corresponding antigen, possibly after a suitable absorption o f the precipitating antibodies, m a y at least be suspected f r o m the reaction, in a cell with parallel walls (double diffusion), o f the same antigen solution with this antiserum and with a precipitating one (Fig. 9A and C). This is the cas¢ w h e n the precipitation zone that appCars in front of the layer o f the latter antiserum ends abruptly in front o f the layer o f the f o r m e r instead of bending inside this layer as it would if this layer contained a n o n i m m u n e serum.

In m a n y cases, the absence o f precipitation of a protein antigen with an antiserum (or a preparation) containing antibodies against it is not due to a particular p r o p e r t y o f these antibodies, such as a low affinity. It m a y be sufficient that the antibodies are specific for an insufficient n u m b e r o f epitopes of the antigen molecule to f o r m a three-dimensional lattice. The

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C FIG. 9. The white dashes in A and B indicate the interfaces between the adjacent layers of gel in two cells with parallel walls (double diffusion). (A) The upper layer contained a solution of hen ovalbumin (Ov P). The layer below was initially made of pure agar. The three lower layers contained (in A and in B), respectively, from left to fight: the serum o f a nonimmunized rabbit (SLN), a precipitating anti-ovalbumin rabbit serum (IS), an anti-ovalbumin rabbit serum partially absorbed so as no longer to precipitate the antigen, but still containing antibodies able to combine with it (IS ep). (B) The upper layer contained soluble polymers of hen ovalbumin (poly Ov. P). The other layers were the same as in the cell of(A). (C) The six round wells in a gel plate (double diffusion in two dimensions) contained, respectively, the same reagents as in (A) and (B) in the following order: the two upper wells, hen ovalbumin (Ov P); the three wells on the line below, the serum of a nonimmunized rabbit (SLN), an anti-ovalbumin rabbit serum (IS), and antiserum absorbed as in (A) and (B) (IS ep); the two wells below, a solution of polymerized ovaibumin (poly and poly Ov P). For further explanation, see text. Reactions similar to those photographed in this figure and in Fig. l0 were executed by students of immunology at the Institut Pasteur until 1978 in a series of practicals organized by the author with the help of Mademoiselle Michel.

mixture of two antisera, each of which is not precipitating for this reason, may be able to precipitate the antigen. If the antibodies of the two component antisera are not specific for the same epitopes, the total number of epitopes (of the same antigen) that combine with the sum of the antibodies

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FIG. 10. In the lower part of this cell with parallel walls (double diffusion), on the photograph of which white dashes indicate the interfaces between the adjacent layers, two antisera (anti-Aa3 and anti-xg) were reacting with the same antigen solution: nonimmunized rabbit serum in the upper layer. Each antiserum alone does not precipitate with the antigen solution, but a mixture of them, which is produced by diffusion above their interface, does. For further explanation see text.

may be sufficient to form a three-dimensional lattice with them. In the reaction of two such nonprecipitating antisera with the antigen in a cell with parallel walls, these antisera will b e c o m e mixed by diffusion in front o f the interface between them and there give rise to a precipitation zone such as that shown in Fig. 10. When the antigen is a protein, a suitable procedure makes it possible to obtain a specific precipitate with a single antiserum that is not precipitating for the above reason. The procedure consists in the mild polymerization of the protein antigen with glutaraldehyde, such that the polymers remain soluble. The precipitation becomes possible (Fig. 9B and C) if the epitope(s) against which the antiserum contains antibodies are numerous enough in p o l y m e r s / 5 It is possible that an antiserum contain antibodies, nonprecipitable for the same reason, against several proteins contained in a c o m m o n antigen solution. In this case, there will be several kinds o f polymers, each composed o f one or several of the protein antigens. A complex precipitating system (see above) will probably be realized. The number o f precipitation zones in the reaction of the polymers with the antiserum will give a minimum value to the number o f distinct antigens that were present in the antigen solution before the polymerization. Applications a n d Possibilities The immunochemical analysis by a n t i g e n - a n t i b o d y precipitation in gels, as a whole, has been applied to an extremely large n u m b e r o f materials in many laboratories, either as a tool o f research or in routine work. 75P.-A. Cazenave, FEBS

Lett.

31, 348 (1973).

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Other types of applications, chosen in order to show that there are possibilities which have rarely been used, will be described. One of the first applications, which remained one of the best, was that to the blood of Cecropia silkworms and to the quantitative evolution of six antigens among the nine detected during the metamorphosis of the silkworm. TM Examples will also be given of problems that were raised and/or solved owing to knowledge of the principles and laws stated above. The reason for such examples is that they strongly suggest that other more or less similar possibilities exist and have probably been overlooked in the past.

Examples of Consequences Drawn from the Analysis of Reactions The first example is concerned with allotypy of rabbit immunoglobulins, or rather with the first two systems of allotypic specificities or of aUotypic patterns later called " a " and " b . " The author wondered if in the reactions of normal rabbit sera with rabbit anti-allotypic precipitating sera according to the technique of simple diffusion in one dimension (in tubes) the procedure of identification by superimposition of profiles would apply to allotypes. Indeed, the procedure applied to the reactions involving antisera against a single allotypic pattern (or family of allotypic patterns) such as Ab5. But this was not true when the reactions of one such antiserum were compared to those of an antiserum directed against both an allotypic pattern of the b system (Ab5) and one of the a system (Aal). The profiles of the precipitation zones observed in the reactions of an anti-Ab5 serum with 11 normal rabbit sera superimposed satisfactorily with that of the reaction of an antiserum both anti-Ab5 and anti-Aal when the normal rabbit sera contained Ab5 without Aal (Fig. 11). With the two Ab5 ÷ normal sera that were also Aal ÷, the Ab5 precipitation zone was found not at the expected level, but at a definitely higher level when it could be distinguished from the Aal precipitation zone. The most likely explanation of this observation was the presence, on the same molecules, of the Ab5 and Aal allotypic patterns. It follows from what has been stated above that if, in a normal rabbit serum, all the Ab5 ÷ molecules were also Aal ÷, the reaction of this normal serum with the bispecific antiserum would give rise to a single precipitation zone. If only a part of the Ab5 ÷ molecules carried also the Aal + pattern, then two distinct precipitation zones might be observed, but the Ab5 precipitation zone would not be at the level expected from the Ab5 profile, where it should be if there were no joint Ab5 ÷ Aal ÷ molecules. This interpretation was not obvious when the two polypeptide chains that constitute the im7e W. H. Teller and C. W. Williams, J. Gen. Physiol. 36, 389 (1953).

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iii

7i t ii 1 Fic. 11. The same 11 antigen solutions (sera of nonimmunized rabbits) were placed, in the same order, at the upper layers of the simple diffusion tubes of these two rows. They reacted, respectively, in the two rows, with the two antisera (IS) indicated on the left. The less dense precipitation zone of the second row was due to anti-Ab5, and the most dense to anti-Aal antibodies. There are two tubes in which both zones might have been expected to be observed; they are clearly distinct from one another in the tenth, but not in the fifth tube. The white dashes indicate the levels at which a precipitation zone similar to those of the first three tubes of the second row would have been expected to be found if their profile had superimposed that of the first row. These reactions were presented at a lecture, which was later published. For further explanation see text. From J. Oudin, in "L'Antig~nicit6" (B. Halpern, ed.), Editions M6dicales Flammarion, Paris, 1963.

munoglobulins had not yet been separated. It was in agreement with other experimental evidence supplied later by the author, r~a With the second example, which is concerned with idiotypy of rabbit antibodies, it is intended to show how, in a careful analysis of the reactions between an antiserum and two antigen solutions, a seemingly small detail that might otherwise have been overlooked led to an important working hypothesis later confirmed by other experiments. The lower layer of the cell with parallel walls (Fig. 12) contained an anti-idiotypic rabbit serum (855) against the anti-Salmonella typhi antibodies of another rabbit. 77 The two upper layers (above an intermediate layer of initially pure agar) contained, respectively, on the right (S1) the anti-S, typhi serum against the antibodies of which the above anti-idiotypic serum had been prepared, and on the left (S1 abs. PS), the same anti-S, typhi serum at the same concentration but previously absorbed by the polysaccharide ofS. typhi. It had been known long ago that this absorption, made by mixing an antibacterial seruro and a polysaccharide solution in slight excess, removes a part--and only a p a r t - - o f the antibodies that can be precipiCsa j. Oudin, C. R. Acad. Sci. 254, 2977 (1962). ¢7 J. Oudin and M. Michel, J. Exp. Med. 130, 619 (1969).

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F1G. 12. In this cell with parallel walls (double diffusion), the white dashes indicate the interfaces between the adjacent gel layers. See text for explanation. From J. Oudin and M. Michel. 77

tated by the complete antigen. It will be just mentioned, incidentally, that one precipitation zone disappeared as a consequence of this absorption, as can be seen on Fig. 12. The authors' attention was focused on the other precipitation zone that remained after removing the antibodies precipitable by the polysaccharide. As can be seen on the figure, this precipitation zone is definitely (even though not considerably) closer to the source of diffusion of the antigen in front of the absorbed rather than in front of the nonabsorbed antibacterial serum. This definitely showed that the concentration of the antigen (an idiotype) responsible for this precipitation zone had been decreased in the anti-S, typhi serum by the absorption. The presence of the same idiotype both in the absorbed and the nonabsorbed anti-S, typhi serum and the decrease of its concentration as a consequence of the absorption indicated that this idiotype was present both among the anti-S, typhi antibodies that are precipitated by the polysaccharide and among those that are not. Confirmation was supplied by a comparative reaction, with the anti-idiotypic serum, of the anti-S, typhi serum absorbed by the polysaccharide and of the antibodies recovered from the precipitate formed in this absorption. These observations led the authors to think that the same idiotypic pattern might be shared by antibodies with different functions r7 against the same antigen in a given antiserum. On the basis of this working hypothesis, a number of experiments were then carried out with protein antigens more suitable for this purpose. However unexpected is the above possibility deduced from the careful observation of a comparative reaction in the gel medium of a cell with parallel walls, it was definitely confirmed by these experiments. Indeed, they permitted observation of the same idiotypic determinants or the same idiotypic patterns on antibodies responsible for cross-reactions with different antigens TM and, moreover, on antibodies directed against distinct r8 j. Oudin and P.-A. Cazenave, Proc. Natl. Acad. Sci. U.S.A. 68, 2616 (1971).

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epitopes, devoid of cross-reactivity, located on distinct parts--enzymically separable--of the same antigen (i.e., of the same molecule; see definition above). 75'79 The author will be satisfied if these few examples have helped the reader understand that in scientific research the choice of a technique should sometimes be influenced by the extent and importance of the consequences that can possibly be drawn from its results through the principles and laws that it involves rather than by the ease with which it can be executed or by the number of those who used it. I would like to conclude this section with a statement suggested by a comparison between the method we have been concerned with and methods more recently described using, for example, radioactive labeling of antigens (or antibodies) and immunoadsorbants or indirect precipitation. Such methods can also detect antigens that are not precipitated (but we have seen procedures for precipitating or detecting such antigens); their main advantage is in greater sensitivity, even taking into account the possibility of using such labelings in the precipitation in gels. But it should be noted that the information they may supply concerns a population of epitopes, and not their molecular distribution, i.e., on a single or on several molecules, and how many. At present, it does not seem possible to obtain such information of interest by means other than those summarized above. Acknowledgment The author wishes to thank Dr. Peter David for his help in correcting the English version of this paper.

~a P.-A. Cazenave and J. Oudin, C. R. Acad. Sci. 276, 243 (1973).

[10]

RADIOIMMUNOASSAYS" AN OVERVIEW

[10] Radioimmunoassays:

201

An Overview

By HELEN VAN VUNAKIS

Since Berson and Yalow first described the radioimmunoassay for insulin, 1 similar assays have appeared in the literature for several hundred substances that differ markedly in chemical structure and biological activity. These represent only a small fraction of the biologically important molecules that could be analyzed by this technique. This overview is intended primarily for the investigator who wishes to develop a new radioimmunoassay for a specific substance. It directs his attention to papers that provide background information and necessary procedural details. The principles that govern the competitive binding of ligands to specific receptors are pertinent in radioimmunoassays (Fig. 1). 1-e The antibody, produced in response to an antigen, serves as the receptor. The ligand in the test sample competes with a constant amount of labeled ligand (i.e., the antigen or suitable derivative) for a limited number of combining sites on the antibody molecules. After equilibration, free labeled antigen is separated from antibody-bound labeled antigen and the radioactivity present in the free or bound state is measured? ,e The extent of the competition between the labeled and unlabeled ligand for the antibody is compared to the inhibitory effectiveness observed with known concentrations of standards. Antibodies can have distinct advantages over the natural receptor molecules that are used in some competitive binding assays. They are stable, soluble proteins with known chemical and physical properties. ~,7,8 Procedures for their purification are available,a-~° but usually diluted antisera is used in fluid phase radioimmunoassays. It has been estimated that an individual animal has the potential to produce antibodies specific for approximately 107 diverse immunodominant moieties? These molecules may possess binding constants for individual antigens on the order of R. S. Yalow and S. A. Berson, J. Clin. Invest. 39, 1157 (1960). 2 R. P. Ekins, Br. Med. Bull. 30, 3 (1967). a E. A. Kabat, this volume [1]. 4 D. N. Orth, this series, Vol. XXXVII [2]. 5 D. Rodbard and H. A. Feldman, this series, Vol. XXXVI [1]. e D. Rodbard and G. R. Frazier, this series, Vol. XXXVII [1]. r M. G. Mage, this volume [6]. s C. N. Hales and J. S. Woodhead, this volume [24]. 9 M. Wilchek and W. B. Jakoby, this series, Vol. XXXIV [1]. 1o j. B. Robbins and R. Schneerson, this series, Vol. XXXIV [90].

METHODS IN ENZYMOLOGY, VOL. 70

Copyright ~) 19e0 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

202

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS Ag* + Ab

~

+ Ag II Ag-Ab

Ag*'Ab

[10]

Ag* = free labeled antigen Ab = antibody Ag = free unlabeled antigen (in standard solutions or in samples to be analyzed)

Ag*. Ab = labeled antigen-antibody complex FIG. 1.

104-101° M -1. With ligands of high specific activity, the sensitivity of radioimmunoassays approaches that required to quantify compounds in the picogram to nanogram level. Often it is possible to assay test samples directly by suitable dilution of the physiological fluids, tissue or bacterial extracts, enzyme reaction mixtures, etc. that are being examined. Antibody Specificities Low Molecular weight substances as antigens. Small molecules (haptens) with molecular weights lower than 5,000-10,000 usually cannot elicit the production of antibodies in experimental animals unless they are covalently attached to large immunogenic molecules (usually proteins) prior to immunization. 3,1H4 Generally, the antibodies recognize structures on the antigen that are free and not involved in the attachment to the carrier macromolecule. With haptens, the specificities of the antibodies can be predetermined to some extent by choosing the functional group that will be covalently bound to the carrier. The choice of utilizing a functional group already on the molecule or of introducing one for the specific purpose of forming the hapten-carrier conjugate depends upon (a) the necessity of retaining the integrity of the original functional group(s) so that it can contribute to antibody specificity and (b) the feasibility of introducing a group at the desired position in the hapten by synthetic procedures. To illustrate how it is possible to "develop an antibody to perform a particular analytical task," Niswender et al.15 immunized animals with four different conjugates prepared by attaching progesterone via carbons ~1 K. Landsteiner, "The Specificity of Serological Reactions." Harvard Univ. Press, Cambridge, 1945. 12 B. F. Erlanger, this volume [4]. 13 p. H. Maurer and H. J. Cailahan, this volume [2]. 14 B. D. Stollar, this volume [3]. 15 G. D. Niswender, A. M. Akbar, and T. M. Nett, this series, Vol. XXXVI [2].

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RADIOIMMUNOASSAYS" AN OVERVIEW

PERCENTAGE CROSS REACTIONS OF METHYLATED AND DEMETHYLATED DRUGS COMPARED TO INHIBITION OBSERVED WITH THE HOMOLOGOUS ANTIGEN AT IN Antigen

% Cross reaction

Derivative

% Cross reaction

Nicotine Cotinine Nornicotine N,N-Dimethyltryptamine

100 100 100 100

Normeperidine l-a-Acetylmethadol (LAAM)

100 100

Nornicotine Desmethylcotinine Nicotine N-Methyltryptamine Tryptamine Meperidine N o r LAAM Dinor LAAM

0.9 0.3 0.19 2.3 0.06 0.01 8.5 2.4

3, 6B, 1 la, or 20 to carrier molecules. Each [all]progesterone-antibody reaction was inhibited with progesterone and 17 other steroids that differed in structure and activities. In general, steroids that possessed distinct substituent groups at positions well separated from the linkage site were poor inhibitors of the specific antigen-antibody reaction. Steroids that had structural modifications at or near the linkage site could effectively inhibit the same antigen-antibody reaction. The review by Butler le gives data on the inhibitory effectiveness of drugs and their metabolites for a large number of radioimmunoassays. The information gleaned by studying the effect a given structural group can have on the ability of the antigen to bind to an antibody can be valuable. In the table are shown data from our laboratory in which drugs and their demethylated metabolites are compared as inhibitors of several antigen-antibody reactions. The immunodominance of the methyl group is apparent since its presence or absence can alter the binding by two to three orders of magnitude. Also, specific antibodies to the methylated bases can often distinguish the modified base in the presence of large amounts of its nonmethylated precursor. Because of the multiplicity of metabolites that can be formed during some biotransformation reactions, it is not always possible to obtain a specific antibody to a hapten. Antibodies that recognize a family of molecules (i.e., parent drug and closely related metabolites) are useful to determine total immunologically reactive material (i.e., for screening purposes). Such antisera can also be used to quantify different drugs and metabolites after separation by an efficient chromatographic procedure (e.g., high-pressure liquid chromatography). In such procedures, the specificity of the antibody permits quantification of compounds at the pile V. P. Butler, Jr., Pharmacol. Rev. 29, 103 (1977).

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cogram to nanogram level even in the presence of large amounts of extraneous materials which can contribute to other detection systems (e.g., absorption or fluorescence spectra). 17"1s With larger hapten molecules (and with some macromolecules) novel synthetic approaches are also being taken to obtain specific antibodies. Parathyroid hormone (MW = 9500) is not a highly structured molecule.19 Like several other circulating polypeptide hormones, it can exist in multiple fornls. 19-21 Segre e t a l . 19 synthesized small peptides with amino acid sequences identical to those contained in limited portions of the hormone. Animals immunized with these peptide-carrier conjugates produced antibodies that recognized defined areas of the hormone. In another study,~l a radioimmunoassay for C-peptide was developed and used to measure proinsulin-like components and C-peptide after separation of the immunologically reactive components by gel filtration. As expected, the antibodies produced by immunizing animals with this peptide showed no reaction with insulin. When insulin antibodies were used to analyze plasma samples after they had been fractionated by gel filtration, the presence of "big" insulin was revealed. 21 This prohormone possessed no biological activity but was serologically active. Synthetic procedures used to prepare antigenic hapten-carrier conjugates are reviewed by Erlanger. 1~Procedures that have proven successful with hapten molecules that possess carboxyl, amino, hydroxyl, and carbonyl groups are fairly routine. 1~ The preparation of hapten-carrier conjugates by other synthetic routes is also considered. Individual chapters describe the use of carbodiimide, 22 glutaraldehyde, 23 and other bifunctional reagents 24,25to prepare hapten-carrier conjugates. Information on ligand coupling via the azo-linkage is also presented. 26 Detailed procedures for preparing purine and pyrimidine protein conjugates, 27 nucleoside-specific synthetic antigens, 2s antigenic vitamin

17 j. j. Langone and H. Van Vunakis, Biochem. Med. 12, 283 (1975). is L. J. Riceberg and H. Van Vunakis, J. Pharrn. Exp. Ther. 206, 158 (1978). is G. V. Segre, G. W. Tregear, and J. T. Potts, Jr., this series, Voi. XXXVII [3]. 20 j. F. Habener and J. T. Potts, this series, Vol. XXXVII [29]. 21 H. S. Tager, A. H. Rubenstein, and D. F. Steiner, this series, Vol. XXXVII [28]. 22 S. Bauminger and M. Wilchek, this volume [7]. 23 M. Reichlin, this volume [8]. F. Wold, this series, Vol. XI [75]. 2s F. Wold, this series, Voi. XXV [57]. 2e L. A. Cohen, this series, Vol. XXXIV [7]. 27 S. M. Beiser, S. W. Tannenbaum, and B. F. Erlanger, this series, Vol. XIIB [173]. 2s M. Sela and H. Ungar-Waron, this series, Vol. XIIB [175].

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and coenzyme derivatives, ~a,3° carbohydrate antigens, 3~-3s cyclic nucleotides, 39 steroids, TM and prostaglandins 4° have been described in other volumes of this Series. Macromolecules as antigens. An antibody-combining site can accommodate molecules having dimensions ranging between 4 - 6 and 34 A and molecular weights between 200 and 1000. 3 Several antigenic determinants can therefore exist on the surface of macromolecules and give rise to a heterogeneous population of antibodies with differing specificities. 3 Differences also exist in the association constants between antibodies and labeled monovalent antigens and between antibodies and labeled multivalent antigens. From inhibition experiments with small fragments it has been possible to define the antigenic determinants of some linear polymers with repeating sequences. 3,13,14 Since the antigenic determinants on proteins are dependent upon the primary, secondary, tertiary, and (if the protein is made up of subunits) quarternary structure that the molecule possesses in its native state, the elucidation of the structures of the antigenic determinants has been more difficult. 3 The specificities of antibodies to proteins and polysaccharides, 3 proteins, and polyamino acids, 13 and nucleic acid and synthetic polymers 14 are discussed in detail. Preparation of R e a g e n t Antibodies The choices to be made among adjuvants, routes of injection, dosage and immunization schedules, species of animal to be immunized, etc. can confound even a seasoned investigator. However, the fact that the literature on immunoassay techniques is growing so rapidly indicates that many people are succeeding in producing reagent antibodies and this should serve as a source of encouragement. With the practical informa29 J. C. Jaton and H. Ungar-Waron, this series, Vol. XVIIIA [93]. 30 j. C. Jaton and H. Ungar-Waron, this series, Vol. XVIIIB [189]. 3~ C. R. McBroom, C. H. Samanen, and I. J. Goldstein, this series, Vol. XXVIII [16]. 32 G. Ashwell, this series, Vol. XXVIII [17]. a3 K. Himmelspach and G. Kleinhammer, this series, Vol. XXVIII [18]. a4 G. R. Gray, this series, Vol. L [12]. 35 j. Lonngren and I. J. Goldstein, this series, Vol. L [13]. 36 D. A. Zopf, C. M. Tsai, and V. Ginsburg, this series, Vol. L [14]. 37 D. F. Smith, D. A. Zopf, and V. Ginsburg, this series, Vol. L [15]. 3s D. A. Zopf, D. F. Smith, Z. Drzeniek, C. M. Tsai, and V. Ginsburg, this series, Vol. L [16]. 39 A. L. Steiner, this series, Vol. XXXVIII [13]. ,0 R. M. Gutierrez-Cemosek, L. Levine, and H. Gjika, this series, Voi. XXXV [35].

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tion provided in the chapter by Hum and Chantler 4~ and a knowledge of the substances that have proven antigenic in the past, 3"12-14the chances of obtaining specific antibodies for a particular molecule can be assessed. Individual animals immunized with the same substance can produce antibodies that may differ in affinities, titer, and specificities. 3,4,H Such differences are apparent with antibodies studied by the more classical physical chemical procedures. 3 However, even among anti-enzyme sera harvested from individual rabbits that had been immunized with/3-1actamase, some were found to neutralize the activity of the enzyme, others to stimulate its activity, and still others were stimulatory and then inhibitory at higher concentrations.42 In radioimmunoassay, each antiserum from an individual animal must be characterized separately to select those that have the proper affinities and specificities. 3,4 The production of monoclonal antibodies by hybridoma t e c h n o l o g y 3'13'41'43 c a n yield molecules with defined specificities and affinities. As this technology becomes affordable for the average research laboratory, problems associated with antibody heterogeneity are due to diminish. Preparation of Radiolabeled Ligands Antigens can be labeled to high specific activity by a number of different techniques and with several different radioactive nucleides. The most commonly used isotopes are 3H (tl12 = 12.3 years, -~ 30 Ci/milliatom at 100% isotope enrichment) or 125I (tl/2 = 60 days, -------2200Ci/milliatom). Less often used are 131I(tl/2 = 8 days, --- 16,000 Ci/milliatom) and ~4C (--- 62 mCi/milliatom). The short half life of 1311and the low specific activity of 14C limit their use in radioimmunoassays. Tritiated products are usually prepared by custom synthesis or purchased from commercial laboratories. They can be relatively stable and are essentially identical to the molecule being measured. The introduction of 125I into molecules containing tyrosyl or histidyl moieties can be carried out chemically by the Chloramine T procedure ~ or enzymatically by lactoperoxidase. 45 The prelabeled Bolton-Hunter reagent [N-succinimidy] 3-(4-hydroxyl-5-[~sI]iodophenylpropionate] ~6 reacts under mild conditions with molecules containing amino groups (e.g., proteins and haptens) to introduce an iodinatcd propyltyrosyl moi41 B. A. L. H u m and S. M. Chantler, this volume [5]. 4~ M. H. Richmond and V. Betina, this series, Vol. XLIII [6]. 43 G. K6hler and C. Milstein, Nature (London) 256, 495 (1975). 44 p. j. McConahey and F. J. Dixon, this volume [11]. 45 M. Morrison, this volume [12]. 4e j. j. Langone, this volume [13].

[10]

RADIOIMMUNOASSAYS: AN OVERVIEW

207

ety. A table listing a large number of molecules labeled by this reagent is presented. 4e Cytosine residues can also be iodinated at the 5 position 4r and this labeling procedure has made feasible sensitive radioimmunoassays for nucleic acids. More complicated synthetic routes may be employed for specific compounds, e.g., the introduction of 125I at the 5' position of 11-nor-9-carboxy-8-9-THC. 4a In this study, the hapten was also attached to the carrier at this position to prepare the immunogen. In compounds lacking a group for iodination, it is possible to introduce tyrosyl, histidyl, or amino groups at the point of linkage for this purpose, e.g., the preparation of the tyrosyl methyl ester derivative of succinylated cAMP. aa It should be emphasized that if a group the size of 1251is introduced directly into a molecule it should be done at a position where it would not diminish the binding of the Ag* to the antibody. The relative merits of the individual labeling procedures are discussed within this volume. 45-49 Also described are radioiodination techniques suitable for the labeling of cell surface lipids and proteins for use in immunological studies .49 The theory and procedure for several immunoradiometric assays are given in the chapters by Hales and Woodhead s and by the Merretts. 5° 125Ilabeled protein A is rapidly becoming a valuable tracer in immunoassays. It can bind to the Fc region of IgG molecules without inhibiting the reaction between antigen and antibody. ~1 Procedures in which an antibody s,Sa or protein A 5~ are iodinated are particularly useful when the antigen cannot be labeled. Separation of Free Ag* from Antibody-bound Ag* To achieve separation between Ag* and Ag*.Ab procedures that utilize differences in properties between the two components and maintain the Ag*.Ab complex are used. I f a separation method is ineffective, and/or markedly dissociates the complex, the erroneous conclusion can be made that antibodies are unsuitable for use in radioimmunoassays. Problems relating to the efficiency of separation procedures and those arising from contributions made to the "blank" values arc discussed by Chard. 5~

4, S. L. Commerford, this volume [14]. 4s C. E. Cook, M. L. Hawkes, E. W. Amerson, C. G. Pitt, D. L. Williams, and R. (3. Willette, Pharmacologist 18, 291 (1976). 49 S. I. Schlager, this volume [15]. s o T. G. Merrett and J. Merrgtt, this volume [26]. 51 j. j. Langone, this volume [25]. s2 T. Chard, this volume [18].

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ASSAYS

[10]

Perhaps the most generally useful procedure (but not necessarily the most cost effective or rapid) is the immunological precipitation of the bound fraction by a second antibody (double-antibody method). 53 This mild procedure does not lead to significant dissociation of Ag*.Ab and is applicable to many different antigens. In our laboratory this procedure is used routinely to follow antibody production whether the antigen be a hapten (e.g., drugs, prostaglandins, insecticides, polycyclic hydrocarbons, various methylated nucleosides) or a macromolecule (e.g., protein or nucleic acid). Once specific antibodies are available and the assay is to be used routinely, more rapid, economical, and even automated separation methods can be adopted. Among reagents employed to separate Ag* from Ag.Ag* in radioimmunoassays are charcoal, 54 hydroxyapatite.55 ammonium sulfate and polyethylene glycol, 52 and zirconyl phosphate gels. 56 Two physical procedures are also described, gel centrifugation57 and microfiltration.5s Chard 52 lists other procedures used for this purpose and their bases for separation. Methods in which one component of the reaction mixture is immobilized are also becoming increasingly popular. Supports upon which antigen or antibody is immobilized include plastic-coated steel beads, 59 diazoand aminocellulose,8 polyacrylamide beads possessing either carboxyl or amino groups 51 on cyanogen bromide inactivated insoluble matrices, such as microcrystalline cellulose particles or paper discs, s and polymaleic anhydride particles.5° Smith and Gehle 59 refer to a number of other matrices that can serve as solid supports. With the plastic-coated iron beads, 5a magnetic devices are used for separation and this technique as well as others can be automated. Assay Conditions and Techniques The fundamental principles of assay design are discussed in the comprehensive review by Ekins.2 Suitable protocols can be found in the references already cited for other radioimmunoassays. Assay conditions should favor binding of the antigen to the antibody and assure stability of the reagents during incubation. The assays are usually carried out in protein-fortified isotonic buffers at a pH close to neutrality. While it is usual A. R. Midgeley, Jr. and M. P. Hepburn, this volume [16]. W. D. Odell, this volume [17]. s5 D. J. H. Trafford and H. L. J. Makin, this volume [19]. 5s j. W. Coffey, J. P. Vandevoorde, E. R. Sauerzopf, and H. J. Hansen, this volume [20]. 57 W. Fischer-Rasmussen and J. Larsen, this volume [22]. S. R. Chalkley and A. Renshaw, this volume [21]. s9 K. O. Smith and W. D. Gehle, this volume [27].

[10]

RADIOIMMUNOASSAYS; AN OVERVIEW

209

to carry out the assays in the absence of organic solvents, a recent assay for THC has been carded out in 25% methanol with gr6atly improved results, e° Once antibodies and labeled tracer are available, other assay conditions can be explored, e.g., the radioimmunoassay for cAMP is carded out at pH 5.5.39 Usually 10,000 cpm of Ag* is used per tube with an antibody dilution that binds approximately 50% of the counts. ~-4 As a rule, antisera to monovalent haptens cannot be diluted to the same extent as antisera directed against multivalent antigens. Even sera that can be diluted only 1/50 can be useful if it is specific. With multivalent antigens, the antisera can usually be diluted 104-105. The screening test for iodinated hormones utilizing the ability of some intact peptide hormones to be adsorbed to talc, excluded from resin and precipitated with TCA, offers a novel approach to checking the integrity of some radioiodinated hormones. 61 Rigorous methods for assessing immunological and biological properties of iodinated peptide hormones have been discussed. 4,62 The ability to ascertain the purity and stability of the compound used to construct the standard curve is as important in radioimmunoassay as it is to any other analytical technique. Because of the problem of cross reactivity, it is essential to compare results obtained by radioimmunoassay with those obtained by other reliable analytical techniques (e.g., MS-GC analysis for small molecules). The lack of correspondence between immunological and biological activities during analysis of physiological fluids by radioimmunoassay and bioassay led investigators to suspect that different forms of hormones existed. Fractionation of extracts after chromatography did reveal this to be true in a number of cases. Computation of Results Response variables can be plotted in a number of ways.~,4,5,e3 The statistical analysis of data derived from radioligand-antibody interactions and the added information that can be derived from logit-log plots has been considered in detail. 6

6o j. D. Teale, J. M. Ciough, L. J. King, V. Marks, P. L. Williams, and A. C. Moffat, J. Forens Sci. Soc. 17, 177 (1977). e~ B. B. Tower, M. B. Sigal, R. E. Poland, W. P. VanderLaan, and R. T. Rubin, this volume

[23]. J. Roth, this series, Vol. XXXVII [16]. ea F. W. Dahlquist, this series, Vol. XLVIII [13].

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[11] Radioiodination of P r o t e i n s b y t h e U s e o f t h e Chloramine-T By PATRICIA J. MCCONAHEY

Method and

FRANK J. DIXON

The incorporation of radioiodine into protein molecules provides increased dimensions to the study of their interactions and subsequent fates. This incorporation involves the covalent linkage of radioiodine in the tyrosyl and to a lesser degree histidine residues of the protein molecule. 1,2 Information gained through use of radioiodinated proteins can be used with confidence if it is established that minimal, if any, alteration occurs in the reactivity of the radioiodinated protein versus that observed in its native form. Radioiodination employing a modification 3 of the chloramine-T method originally described by Greenwood, Hunter, and Glover 4 meets this criterion, as has been demonstrated in many laboratories with a wide spectrum of proteins. In addition to the mild conditions employed in this modification, it has the following additional advantages: (a) inexpensive, uncomplicated, readily available reagents; (b) simple execution requiring no special skills or training; (c) quick completion of reaction; (d) minimal exposure of personnel to radiation. Below are listed the basic steps of the modified method of chloramineT radioiodination of proteins that have evolved in our laboratory over the past 15 years. We routinely radioiodinate 20-30 proteins at a time using this method. The entire radioiodination procedure is conducted in an ice bath and can be accomplished in about 60 min. 1. Dissolve protein in phosphate-buffered saline or any buffer at pH 7 taking care that no reducing reagents are present. Protein concentration ordinarily is in the range of 0.5-5 mg/ml, the total volume rarely exceeding 2 ml. 2. Add desired quantity of radioiodine to the protein solution. Just prior to addition, radioiodine is neutralized with 0.1 N HCI and diluted with 0.1 M phosphate buffer, pH 7. 3. Mix protein plus radioiodine. 4. Initiate radioiodination by adding freshly prepared chloramine-T. Chloramine-T is usually prepared at a concentration of 500/zg/ml in 0.1 M phosphate buffer, pH 7. The ratio of chloramine-T to protein most commonly used is 10/zg per milligram of protein. 1 W. L. H u g h e s , Jr., and R. Straessle, J. Am. Chem.Soc. 72, 452 (1950). G. L a m o u r e u x , P. R. Carnegie, and T. A. M c P h e r s o n , lmmunochemistry 4, 273 (1967). 3 p. j. M c C o n a h e y and F. J. Dixon, Int. Arch. Allergy 29, 185 (1966). F. C. G r e e n w o o d , W. M. H u n t e r , and J. S. Glover, J. Biochem. 89, 114 (1963). METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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PROTEIN RADIOIODINATION" CHLORAMINE-T METHOD

211

5. Mix protein plus radioiodine plus chloramine-T. 6. Permit radioiodination to proceed for 10 min. 7. Stop radioiodination by addition of freshly prepared Na~S2Os. Na~S205 is usually prepared at 500/.~g/ml in 0.1 M phosphate buffer, pH 7. The ratio of Na~S205 most commonly used is the same as that for chloramine-T: 10/.~g per milligram of protein. 8. Immediately remove unreacted radioiodine by dialysis. Where a large number of proteins are radioiodinated, it is convenient to transfer the protein mixtures to dialysis bags and dialyze away the unreacted radioiodine plus chloramine-T plus Na2S205 against a continuously changing volume of 0.1 M phosphate buffer, pH 7. This dialysis continues overnight at 4° against a total volume of 20 liters. However, there are many other suitable ways to separate free from protein-bound radioiodine, such as ion exchange chromatography. No laboratory procedure is so simple and direct that there are no areas of potential error. The following outlines the most common problems we have experienced with radioiodination by chloramine-T and some of the measures taken to increase the usefulness of radioiodinated proteins. 1. It is essential for successful radioiodination that no reducing substances be present. If protein solutions contain reducing substances including preservatives, they must be dialyzed away prior to radioiodination. 2. Carrier-free iodine should be employed to minimize the amount of iodine incorporated per molecule of protein which could lead to potential denaturation and alteration of the reactivity of the native molecule. 3. Radioiodine is usually supplied in 0.1 N NaOH. Direct addition to protein solutions is a potential cause of denaturation; it can be avoided by neutralization of the radioiodine solution with an equal volume of 0.1 N HC1 or one-tenth volume 1.0 N HC1 and further dilution with buffer at pH 7. Once radioiodine has been neutralized it must be used immediately to avoid loss by volatilization. 4. Efficiency of radioiodination is enhanced by minimizing the volume of reactants. In our laboratory the efficiency of radioiodination averages 50% when the following conditions are met: (a) protein at 0.5 mg in volumes from 0.5 to 2 ml; (b) chloramine-T at 100-500 /zg/ml, 10/xg of chloramine-T added per milligram of protein in volumes from 10 to 50/zl; (c) neutralized radioiodine from 50 to 5000/zCi in volumes less than 100/~1 for a final total volume of approximately 1-2 ml. 5. Chloramine-T radioiodination of proteins involves both oxidation of radioiodide to its reactive state, H2OI+, 5 and oxidation of sulfhydryl groups of the protein molecules. There must be sufficient chloramine-T 5 S. Sonoda and M. Schlamowitz, lrnmunochemistry 7, 885 (1970).

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PARTIAL LIST OF PROTEINS RADIOIODINATED BY THE CHLORAMINE-T METHOD

Albumins Bovine Horse Human Mouse Rabbit y-Globulins Bovine Burro Duck Goat Guinea pig Horse Human Mouse Mule Rabbit Rat Sheep Turkey IgA IgD IgE IgG IgM Aggregated IgG F(ab) F(ab')2 Fc Whole viruses and virus proteins AKR Moloney Rauscher Scripps leukemia Vaccinia plSE p30 gp70

Miscellaneous a-Crystallin ot-Fetoprotein ct2-Macroglobulin /3-Galactosidase fl~-Microglobulin Actin Aldolase Basic protein Catalase Cell membrane eluates Chymotrypsinogen Cobra factor Collagenase Concanavalin A Cytochrome c Endotoxins Fetuin Fibrin Fibrinogen Ferritin GBM eluates Hemoglobulin Hemopexin Horseradish peroxidase Immune complexes (preformed) Insulin Keyhole limpet hemocyanin Kidney eluates Lipoproteins Papain Phosphorylase A Phytohemagglutinin Plasmin Plasminogen Protamine Prothrombin Staphylococcus protein A Tetanus toxoid Thrombin Thyroglobulin Transferrin Trypsin Urokinase

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PROTEIN RADIOIODINATION: CHLORAMINE-T METHOD

213

available to complete both reactions. Therefore, proteins with high reducing capacity may be poorly radioiodinated under the standard conditions outlined above. For example, keyhole limpet hemocyanin under standard conditions is minimally radioiodinated. However, by increasing the chloramine-T to 100/zg per milligram of protein, an average 50% incorporation of radioiodine is achieved with no apparent denaturation. Therefore, proteins that cannot be radioiodinated successfully with standard conditions should be repeated at increased chloramine-T to protein ratios. 6. Denaturation of proteins may result from low concentrations and elevated storage temperatures. These conditions must also be controlled for radioiodinated proteins that are also subject to internal self-irradiation damage, which is more pronounced in TM I-labeled proteins than 125 I-labeled proteins owing to the beta radiation emission ofTM I. To minimize such problems, radioiodinated proteins are diluted in protective protein solutions that will not interfere in the subsequent studies to be made, such as 10% normal serum or BSA at 2 mg of protein per milliliter. Aliquots are kept frozen at - 2 0 ° to - 7 0 ° and thawed only as needed. 7. There are some proteins that can be radioiodinated successfully by chloramine-T but in the process lose some of their biological activity. The first five complement components fall in this category. Alternative methods of radioiodination must be used in such cases, as reported in this volume [12]. A partial list of proteins radioiodinated with chloramine-T without detectable loss of immunologic or enzymic activity can be found in the table. We have not been able to achieve greater than 10% incorporation of radioiodine into ovalbumin using chloramine-T at increased ratios of chloramine-T to ovalbumin. Some areas of investigation are restricted by the requirement for purified proteins because these may be limited in quantity of source material and their purification often involves tedious and inefficient procedures. Elder e t a l . e have reported a unique approach that bypasses some of these restrictions in their studies of viral and plasma membrane proteins. After separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they remove the gel slice containing the protein band of interest and radioiodinate it by chloramine-T while still in the gel. This application should prove to be useful in other systems. Acknowledgments This is publication No. 1889 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California. This work was supported by National Institutes of Health Grants AI-07007, CA-16600, and CP-71018 and by the Elsa U. Pardee Foundation. 8 j. H. Elder, R. A. Pickett, L Hampton, and R. A. Lerner, J. Biol. Chem. 252, 6510 (1977).

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RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[12] Lactoperoxidase-Catalyzed Iodination T o o l for I n v e s t i g a t i o n o f P r o t e i n s

[12]

as a

B y MARTIN MORRISON

The objectives of investigators using iodination of proteins have been (a) to study the structure function relationships in proteins, (b) to provide a label for proteins in order to investigate their metabolic fate, (c) to provide a method of increasing the sensitivity for assay procedures of proteins such as in radioimmunoassays, and (d) more recently, as a tool for the investigation of the arrangement of proteins in macromolecular structures such as membranes. A number of reviews dealing with various aspects of peroxidase-catalyzed iodination have appeared. 1-12 Peroxidase-catalyzed iodination has advantages over other methods of iodinating proteins. For one, the peroxidase halogenation has fewer side reactions than halogenation employing other reagents because the oxidant peroxide can be used in very low concentrations, a'13 It also has the advantage that, under the proper experimental conditions, the peroxidase-catalyzed halogenation can be very selective. Thus with lactoperoxidase M. Morrison, Gumrna Symp. Endocrinol. 5, 239 (1968). 2 M. Morrison, G. S. Bayse, and D. J. Danner, in "Biochemistry of the Phagocytic Process" (J. Schultz, ed.), pp. 51-66. North-Holland, Amsterdam, 1970. 3 M. Morrison, G. S. Bayse, and R. G. Webster, Irnmunochemistry 8, 289-297 (1971). 4 M. Morrison and R. Gates, in "The Molecular Basis for Electron Transport" (J. Schultz, ed.), pp. 327-345. Academic Press, New York, 1972. 5 M. Morrison and G. Bayse, in "The Second International Symposium on Oxidases and R e l a t e d Oxidation-Reduction Systems" (T. King, H. S. Mason, and M. Morrison, eds.), pp. 375-388. University Park Press, Baltimore, 1973. e M. Morrison, R. E. Gates, and C. T. Huber, in "Membrane Transformations in Neoplasia, Miami Winter Symposia" (J. Schultz and R. Block, eds.), Vol. 8, pp. 33-51. Academic Press, New York, 1974. 7 M. Morrison, this series, Vol. XXXII, p. 103. s M. Morrison, in "Cell Surfaces and Malignancy" (P. T. Mora, ed.), Proc. Fogarty Int. Center, No. 24, pp. 63-69. DHEW Publication No. (NIH) 75-6796, Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 1976. a M. Morrison and G. R. Schonbaum, in "Annual Reviews of Biochemistry" (E. E. Snell, P. D. Boyer, A. Meister, and C. C. Richardson, eds.), pp. 861-888. Annual Reviews, Palo Alto, CA, 1976. l0 E. S. Vitetto and J. W. Uhr, Science 189, 964 (1975). i1 A. L. Hubbard and Z. A. Cohn, in "Biochemical Analysis of Membranes" (A. H. Maddy, ed.), p. 427. Wiley, New York, 1976. ~2 S. G. Emerson, P. Reilly, and R. E. Cone, J. lmmunogenet. 6, 87 (1979). ~3 G. S. Bayse and M. Morrison, Arch. Biochem. Biophys. 145, 143 (1971).

METHODS IN ENZYMOLOGY,VOL. 70

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those tyrosine residues which can form an enzyme substrate complex with the peroxidase will be iodinated. In studies of isolated proteins, therefore, the peroxidase-catalyzed halogenation can be a very useful method of demonstrating that a particular tyrosine residue is on the exposed three-dimensional surface of the protein. 14 Because the tyrosine residues on the outside surface of the protein are usually not directly in the active center of enzymes, the lactoperoxidase procedure is especially good in preserving biological activity. Since iodination of external tyrosines does not readily perturb the three-dimensional structure of the protein, other biological activities such as binding activity in case of hormones immunoglobins etc. are also preserved. TM Peroxidase procedures can be easily controlled and readily followed during the course of the iodination reaction. Thus, it is a simple matter to add as few or as many atoms of iodine as desired. Procedures employing an iodide-sensitive electrode T M or spectrophotometric techniques 15,1~enable the extent of halogenation to be followed during the course of the reaction very readily. This is more difficult with other types of halogenation reactions. In attaining high specific activity in labeled proteins the peroxidase-catalyzed reaction has the advantage that iodination can be made to approach 100% of the iodide employed. Consequently, lower concentrations of radioactivity can be used. Only two amino acids, tyrosine and histidine, form stable derivatives as the result of peroxidase-catalyzed iodination. 9 All the tyrosine and histidine residues in a protein are not identical with respect to their reactivity or their geographic position. The residue which will be iodinated by lactoperoxidase must have the proper geometric position, while other methods of halogenation are influenced only by reactivity. The reactivity depends upon the microenvironment of the residue. There is an inverse relationship between the extent of tyrosine iodination and the dielectric constant of the environment of the tyrosine. Tyrosine iodination increases with decreasing dielectric constant. Steric factors also influence iodination since the relatively large iodine atom may be blocked in either the production of monoiodotyrosine or the formation of diiodotyrosine. Iodination of Proteins To prepare an iodinated protein with high specific activity, the following example is given for the iodination of immunoglobulin. ~4 N. Osheroff, B. A. Feinberg, E. Margoliash, and M. Morfison, J. Biol. Chem. 252, 7743 (1977). 15 M. Morrison and G. S. Bayse, Biochemistry 9, 2995 (1970). le M. Morrison, this series, Vol. XVII, p. 653.

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[12]

Reagents Lactoperoxidase was purified as previously described. TM A stock solution was prepared of 1 × 10-5 M. The concentration of the enzyme was determined by employing a millimolar extinction coefficient of 114 at 412 nm. Small aliquots of the enzyme were frozen at - 3 0 °. Samples were thawed just prior to use. A carder-free solution of Na 1251was obtained commercially. In these preparations, 1 mCi usually contained 2 × 10-6 mmol of iodide dissolved in 0.1 N NaOH. A stock solution is prepared by diluting 25 mCi to 0.1 ml with 0.05 M phosphate buffer, pH 7.4. Thus, each 0.004 ml contains 1 mCi. The stock solution of peroxide was freshly prepared for each experiment. Peroxide (30%) was diluted approximately 1 to 1000 in 0.05 M phosphate buffer, pH 7.4, to produce a 1 × 10-2 M solution. The concentration was established using a millimolar extinction coefficient of 72 at 230 nm.

Methods To each milliliter of a solution of 1 mg/ml of immunoglobulin, 0.015 ml of the stock solution of 1251 and 0.01 ml of the stock solution of lactoperoxidase was added. The stock peroxide 0.002 ml was then introduced with stirring. The peroxide addition was repeated four times at 1-min intervals. After the last addition, the iodinated immunoglobulin was freed of contaminating lactoperoxidase and excess iodide by passage through a gel filtration column. A 20 × 1 cm column of Sephadex G100 or BioGel 100 was prepared in 0.05 M phosphate buffer pH 7.4. The column eluate was monitored at 280 nm and the immunoglobulin which eluted in the void volume is collected free of the iodide and lactoperoxidase. R e c o m m e n d a t i o n s for Removing Free [125I]Iodide and Lactoperoxidase We have employed a variety of procedures for removing free iodide from the iodinated proteins. This can be accomplished by (a) gel filtration, (b) dialysis, and (c) use of ion exchange resin. Gel filtration is a very effective method for removal of both iodide and lactoperoxidase if the molecular weight of the iodinated protein and the molecular weight of the enzyme are sufficiently different as in the example given. Since the iodine derivatives are light sensitive all procedures should be carried out in dim light. Dialysis does not always remove excess free iodide readily since many proteins bind iodide. Therefore, it is best to dialyze against a dilute solution (0.01 M) of Nal in order to remove the 125I b y exchange.

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217

If the small amount of contaminating lactoperoxidase is not a problem, the free 1251 can be separated by passage through a small column of Dowex lXS. Dowex lX8 was converted to the acetate form and suspended in phosphate buffer (0.05 M, pH 7.4). A column of the resin, 3 cm in height, is prepared by plugging a Pasteur pipet from which the tip has been removed with a small wad of glass wool. The iodinated protein reaction mixture is allowed to percolate through the column and the eluate is collected immediately after the break through volume. The column is washed with twice the volume of the protein solution. The protein obtained in this way is freed of iodide since the resin has a high affinity and great capacity for the binding of iodide. With acidic proteins the pH of the buffer employed should be below their isoelectric point) 7 If gel filtration procedures cannot be employed for removal of lactoperoxidase, the enzyme can be removed by taking advantage of the fact that it is a basic protein. It is easily separated from most proteins which have lower isoelectric points by the use of ion exchange resin. The reaction mixture is passed through a small column of IRC 50 at a pH above the isoelectric point of iodinated protein but below pH 8.0. This will remove the lactoperoxidase from the mixture without adsorbing proteins of lower isoelectric points. Lactoperoxidase bound to an insoluble matrix TM has been employed. This has the advantage of providing a Simple way of removing the enzyme since it can be easily separated from the reaction mixture by filtration or centrifugation. With this procedure, a large percentage of the iodination activity takes place via iodine 02) generation probably because the bound enzyme has under these conditions a limited access to the protein substrate. However, it can be a very useful tool for high specific activity labeling. Separation of Iodinated Derivatives of Proteins It is frequently important to separate the different derivatives of the iodinated protein obtained upon iodination. In almost all cases the iodinated reaction mixture contains a mixture of uniodinated protein and different iodinated derivatives (mono-, diiodinated, etc.). It is usually not difficult to separate the purified protein on the basis of the number of iodine atoms incorporated. 14 The pH of the phenol group changes by approximately 2 pH units when the tyrosine phenol is converted to iodotyrosine and another 2 units when it is converted to the diiodotyrosine. Thus, 17G. Carpenter, K. J. Lembach, S. Cohen, and M. Morrison, J. Biol. Chem. 250, 4297 (1975). ~8G. S. David and R. A. Kersfeid,Biochemistry 13, 1014(1974).

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the iodinated protein derivatives can readily be separated by electrophoresis in the region of pH 7 to 8 or by ion exchange chromatography. 14

Macromolecular Probe The selectivity of the reaction also makes the peroxidase-catalyzed iodination a very good tool for the study of the position of proteins within macromolecular structure such as membranes, ribosomes, and micellular polypeptides. Its use in this way is based on the fact that it is a high molecular weight protein and therefore does not readily penetrate these macromolecular structures. ',~-12,1~,20 If the experimental conditions are correct, it catalyzes the halogenation selectively with those groups on the protein with which the enzyme has access. Thus, when the enzyme has access to proteins on only one side of the macromolecular structure such as the cell membrane, only the accessible proteins will be labeled with iodine. The labeled polypeptide of the macromolecular structure can then be isolated and identified. This provides a general method that can be applied to all macromolecular structures. As is the case with most methods, it is important to recognize the limitation of lactoperoxidase-catalyzed halogenation for macromolecular probe--that iodide can be oxidized by peroxidase to generate iodine (12) which is itself used as a iodinating agent. Unfortunately I2-catalyzed halogenation gives no information concerning the arrangement or geographical position of the tyrosine residue. Since it readily penetrates lipids 12 halogenation provides no information on the vectorial or geographic position of a protein. In order to minimize iodine generation iodide concentration should be kept very low. Further, it is desirable to have a high concentration of the iodinatable substrates, the proteins. Iodination may have an effect on the membrane. When the number of iodine atoms incorporated is very low there is little or no effect. However, as increasing quantities of iodine atoms are incorporated, iodination will disrupt membranes so that it is usually desirable to limit the extent of iodination to a very small percentage of the total protein of the membrane. In general, it has been observed that the iodination of proteins on macromolecular structures is random with respect to the accessible membrane protein. Increasing the extent of iodination does not alter the ratio of label incorporated into the different polypeptides of the membrane as long as the membrane peptides are in great excess.

1~ D. R. Phillips and M. Morrison, Biochem. Biophys. Res. Commun. 40, 284 (1970). so D. R. Phillips and M. Morrison, Biochemistry 10, 1766 (1971).

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219

Procedure for the Labeling of a Lymphocyte Plasma M e m b r a n e In order to illustratethe use of the lactopcroxidasc procedure as a vectorial probe, the example to bc given is the iodination of the plasma m e m brane of the human lymphocyte. All manipulations were carried out at 4 °. The lymphocytcs were isolated21 and washed frec of extraccllular protcin in isotonic phosphate buffer. The cells wcrc centrifuged at 700 g for I0 rain and the supernatant serum was removed. Cells are rcsuspended in 5 to 10 volumes of phosphate buffer and centrifuged frcc of the supernatant. This procedure was repeated four times or more until the supernatant gave a 280 n m reading below an optical density of 0. I absorbancc. The pellet of washed cells were then suspended in 3 volumes of isotonic phosphate buffer to produce 2 × 10s cclIs/ml. One miIlicuric of 1251 was added and 20 nmol of lactoperoxidase for each milliliterof cell suspension. Ten microlitcr portions of I m M peroxide was added at I rain intervals. The lymphocytcs were then spun down at 500 g for 5 rain and washcd three times with phosphate-buffered saline. The final pellet was diluted I :20 with water, mixed, and immediately heated at I00 ° for 3 rain. To I ml of this suspension was added 5/~I of bovine pancreas dcoxyribonuclcasc I (5 mg/ml Sigma). After a 30-rain incubation at 37 °, an equal volume of a solution of 2 % weight/volume sodium dodccyl sulfate and 2 % weight/volume/3-mercaptoethanol 2 m M E D T A 8 M urea was added. This mixture was immediately heated at I00 ° for 3 rain. The polypcptides in the sample wcrc separated by elcctrophoresis in S D S polyacrylamidc gels.~2

Recommendations for Vectorial Labeling of M e m b r a n e s Employing Lactoperoxidase I. Since lactopcroxidasc has a very high turnover, littleof the enzyme is required for labeling procedures. Concentrations of 10-7 M arc usually satisfactory for labeling cells. 2. In order to avoid generation of 12, low concentrations of iodide should bc used, preferably I × 10-e M or less. 3. Pcroxidase concentrations should be maintained at low levels since peroxide can inhibit the enzyme and will cause oxidation of the plasma membrane resulting in alterations and frequently breaking the membrane. Since the amount of peroxide added is the limiting step in iodination of membranes it should bc added in small multiple aliquots or by a continuous peffusion of low concentrations of peroxide. The solution should never obtain concentrations higher than I × I0-4 M. 4. The amount of membrane proteins that will bc iodinated should al21 A. Boyum, Scand. J. Immunol. $, 9 (1976). 22 U. K. Laemmli, Nature (London) 227, 680 (1970).

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ways be present in large excess and the amount of label incorporated should probably never exceed more than a few percent of the accessible tyrosine residues for best vectorial labeling. 5. Since cells, organelle, etc. are in a dynamic state during any interval, some of the membranes can be expected to break. For this reason it is preferable to iodinate first and then separate the membrane component by sedimentation to remove the broken cells, organelles, and debris from intact preparations. In this way, artifacts due to the broken membranes and dead cells are avoided. 6. Control experiments should be included in order to determine whether the cells under investigation contain endogenous systems capable of catalyzing iodination or iodide oxidation. These controls should include a sample in which the enzyme lactoperoxidase is omitted and one to which peroxide is not added. 7. Inhibitors of peroxidase-catalyzed iodination, such as azide and sulfhydryl compounds, should always be removed prior to attempts at membrane labeling. 8. Soluble proteins in the supernatant should also be removed, since the soluble proteins probably represent better substrates for iodination than the plasma membrane and would then be iodinated in preference to the plasma membrane. This would result in low label of the membrane. 9. When employing high specific activity radionuclides, care should be taken to be sure that the iodine atom is in the form of iodide (I-). Oxidized forms of iodine, such as periodate or iodate which commonly occur in commercial preparations, are not incorporated into proteins by the procedures outlined here. 10. When investigating the proteins or polypeptides subsequent to iodination, care should be taken to inhibit endogenous cellular proteases and other hydrolases. This can be achieved by using any of the wide variety of serine protease inhibitors or other hydrolase inhibitors. A general method which has been described above is to inactivate all the enzymes by heating in order to prevent the degradation of the proteins. This method, of course, allows the separation and identification of the polypeptides by SDS polyacrylamide gels, but does not provide for the isolation and purification of the biologically active proteins.

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RADIOIODINATION

WITH

BOLTON--HUNTER

REAGENT

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[13] R a d i o i o d i n a t i o n b y U s e o f t h e B o l t o n - H u n t e r and Related Reagents B y J O H N J. L A N G O N E

Antigens, haptens, and antibodies radiolabeled with 12~I or 1311 are commonly used as tracers in immunoassay. These nuclides can be introduced directly into functional groups normally present in proteins and other macromolecules or into suitable derivatives that can be synthesized by a variety of chemical procedures. The most widely used iodination methods have been direct chemical or enzymic substitution of hydrogen in tyrosine or related groups using chloramine-T or lactoperoxidase, respectively. These methods are described in separate chapters in this volume. More recently, techniques have been developed in which a highly reactive precursor is labeled with radioiodine by one of the standard methods, separated from the iodination mixture, and allowed to react under relatively mild conditions with the target molecule. Thus, the antigen or hapten never comes in contact with potentially damaging oxidizing or reducing agents or with high concentrations of radioactive iodine. Iodinated N-succinimidyl 3-(4-hydroxyphenyl)propionate (BoltonHunter reagent, Fig. 1) is the first conjugation reagent that has been widely used to prepare tracers for immunoassay. 1 Similar reagents derived from methyl p-hydroxybenzimidate2,3 and diazotized aniline 4 (Fig. 1) have been synthesized and used to iodinate proteins, but not specifically for use in immunoassay. However, since potentially they could be used for this purpose, these reagents also will be discussed. Bolton-Hunter Reagent General Considerations

The Bolton-Hunter reagent is an 12SI-labeled acylating agent prepared by the chloramine-T method that reacts spontaneously and under mild conditions primarily with lysine residues. 1 Use of this reagent supplei A. 2 F. a F. 4 C.

E. T. T. E.

Bolton and W. M. H u n t e r , Biochern. J. 133, 529 (1973). W o o d , M. M. W u , and J. C. Gerhart, Anal. Biochem. 69, 339 (1975). W o o d , J. Dent. Res. 54, C86 (1975). H a y e s and I. J. Goldstein, Anal. Biochem. 67, 580 (1975).

METHODS IN ENZYMOLOGY. VOL. 70

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I o

I

c--o

I

®®

CH 2

I CH 2

H3CO\C~, NH2Cl

11251)~I~12.51 1251"~ 1251(1251)/~ 1251 OH OH N2Cl ®®

(1:)

('rr)

(:Ill:)

FIG. 1. Chemical structures: (I), 125I-iabeled N-succinimidyl 3-(4-hydroxyphenyl)propionate (Bolton-Hunter reagent); (II), di-'~I-labeled methyl p-hydroxybenzimidate; (III), nSI-labeled diazotized aniline.

ments the chloramine-T5 and lactoperoxidase methods 6 in which radioactive iodide is introduced into the phenol ring of tyrosine or related moieties and to some extent into histidine residues. When labeling tyrosine can lead to loss of immunoactivity- or biological activity, or when the substrate lacks the appropriate group, the Bolton-Hunter reagent is an excellent alternative. Specificity. The Bolton-Hunter reagent reacts primarily with free amino groups. Knight and Welch 7 reported careful studies in which human serum albumin was iodinated by several methods including the Bolton-Hunter technique. The protein was degraded enzymically, and labeled amino acids were analyzed by high performance liquid chromatography. Lysine residues were labeled predominantly, but iodinated histidine and tyrosine also were produced. For example, when the molar ratio of Bolton-Hunter reagent to albumin was 150: 1, 12 lysines, 1-2 histidines, and 0.3 tyrosine groups were labeled per albumin molecule. The ratio shifted slightly when less reagent was used. Reactivity with tyrosine and histidine was confirmed using phenol and imidazole as model compounds. Others 4 have suggested that the reagent should react with sulfhy5 p. McConahey and F. Dixon, this volume [11]. e M. Morrison, this volume [12]. T L. C. Knight and M. J. Welch, Biochim. Biophys. Acta 534, 185 (1978).

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dryl groups and therefore would not be suitable for labeling enzymes or other proteins in which cysteine was necessary to maintain structural integrity or biological activity. Although this reaction is likely, it has not been shown experimentally. Availability. Mono- and di-nSI-labeled Bolton-Hunter reagent (sp. act. 1500 and 4000 Ci/mmol, respectively) are available from Amersham/Searle, Arlington Heights, Illinois, and New England Nuclear (NEN), Boston, Massachusetts. The NEN product is packaged in benzene, the Amersham product in benzene containing 0.2% dimethylformamide (DMF). If aliquots of the reagent are to be used, samples of the Amersham reagent can be withdrawn directly (by syringe) from the vial. However, dry DMF (approximately 0.5% of the sample volume) must be added to the NEN vial to ensure solubilization of the reagent, which otherwise sticks tenaciously to the glass.

N-Succinimidyl-3- (4-hydroxyphenyl)propionate 1 Reagents 3-(4-Hydroxyphenyl)propionic acid (HPPA): 1.661 g (10 mmol) N-Hydroxysuccinimide (NHS): 1.151 g (10 mmol) Dicyclohexylcarbodiimide (DDC): 2.47 g (12 mmol) Tetrahydrofuran (THF) Acetic acid Ethyl acetate 2-Propanol Petroleum ether Procedure. Prepare a solution of HPPA and NHS in 7 ml of THF at - 1 8 °. Add the DCC and allow the solution to stir at -18 ° (magnetic stirrer) for 2 hr, then at room temperature for 10 hr. Add the acetic acid (0.12 ml) to destroy excess DCC, then after 1 hr dilute the reaction mixture with ethyl acetate (10 ml). Precipitated dicyclohexylurea is removed by filtration, washed with a few milliliters of ethyl acetate, and discarded. The combined filtrates are evaporated to dryness under reduced pressure on a Btichi rotary evaporator or similar device, and the residue is recrystallized from ethyl acetate (20 ml) by addition of low boiling petroleum ether (10 ml). The product (2.51 g; 95% yield) has a melting point (m.p.) of 120-122 °. Although this material reportedly is suitable for iodination and coupling reactions, it contains a small amount of unreacted HPPA. Pure product (0.91 g; 36% yield; m.p. 129°) is obtained by recrystallization from 2-propanol (20 ml) by addition of water (60 ml) at 0° followed by drying under reduced pressure. The activated ester must be kept dry to prevent hydrolysis.

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lz~I-Labeled N-Succinimidyl 3-(4-hydroxyphenyl)propionate (Bolton-Hunter Reagent) 1 Reagents N-Succinimidyl 3-(4-hydroxyphenyl)propionate (SHPP), 0.2-0.25 /zg Naa25I, 2-5 mCi/10/zl Chloramine-T, 50/zg per 10-20 ~1 of 0.25 M phosphate buffer, pH 7.5 Sodium metabisulfite, 120/~g per 10/zl of 50 mM phosphate buffer, pH 7.5 KI (carrier), 200/zg per 10/zl of 50 mM phosphate, pH 7.5 Dimethylformamide (DMF) Benzene Procedure. In a small glass vial, add the Na125I solution at room temperature to crystalline SHPP. Immediately add sodium metabisulfite to reduce the oxidizing agent and iodide, stopping the reaction. Carder KI is added and the iodinated ester (12hi-labeled Bolton-Hunter reagent) isolated by addition of DMF (5/zl) followed by extraction with two portions of reagent grade benzene. To prevent hydrolysis (i.e., inactivation) of the labeled ester, the entire procedure must be carded out as quickly as possible (< 1 min). In an alternative procedure, the SHPP is added to the vial at the appropriate concentration in benzene (10-20/zl), which is then evaporated to dryness under a stream of dry nitrogen. Na125I is added, followed by metabisulfite then KI. The product is isolated as described above. Analogous acylating agents labeled with 131F-a or 7rBr9 also have been synthesized and used to radiolabel proteins. Protein Labeling The following procedure, taken from the original paper by Bolton and Hunter, x describes the labeling of human growth hormone (HGH). Reagents HGH, 5 p,g in 10 p,l of 0.1 M borate buffer, pH 8.5 x~5I-labeled Bolton-Hunter reagent, 0.2/zg in benzene-DMF equivalent to 3.2 mol of ester per mole of protein Glycine, 0.5 ml of 0.2 M solution in 0.1 M borate buffer, pH 8.5 Procedure. Evaporate the b e n z e n e - D M F to dryness under a stream of dry nitrogen. Cool the vial to 0° and add the solution of protein (0°). s R. J. Schneider, R. L. Burger, C. S. Mehlman, and R. H. Allen, J. Clin. Invest. 57, 27 (1976). 9 L. C. Knight, S. S. L. Harwig, and M. J. Welch, J. Nucl. Med. 18, 282 (1977).

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225

Agitate the vial gently and allow the reaction to proceed for 15 min. To destroy unreacted ester, add the glycine and agitate the mixture for 5 min. The iodinated product is collected by gel filtration on Sephadex G-50 (fine), wet-packed, and eluted with 50 mM phosphate buffer, pH 7.5, containing 0.25% gelatin. Since the low molecular weight compounds will bind to serum albumin and other serum components, these proteins should not be added to the elution buffer. If desired, serum protein can be added after the iodinated product has been isolated. Specific activities of HGH, thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) up to 170, 120, and 55/xCi/gg were achieved. Results

Since the Bolton-Hunter reagent was introduced, it has been used to label a wide variety of targets including viruses and cell lysates as well as pure compounds. Undoubtedly one of the most important applications has been the preparation of iodinated antigens and hapten derivatives of high specific activity for use in radioimmunoassay. Because the original procedure is straightforward, there has been little room (or need) for major modification. Among the numerous applications that have appeared, the changes usually involve running the reaction at different temperatures or for longer than the 5 min originally proposed. In some cases, compounds first have been acylated with SHPP then labeled with 1251 by the chloramine-T method) °-13 Generally there appears to be no advantage compared to using the prelabeled BoltonHunter reagent. However, gentamycin and other aminoglycoside antibiotics may be exceptions. When gentamycin was acylated then iodinated, up to 70% of the available radioactivity was incorporated and the specific activity ranged up to 1200/zCi//~g. When prelabeled Bolton-Hunter reagent was used, no more than 5% of the label was incorporated and the specific activity was only 73/~Ci//~g. a° The basis for this difference is not clear, but the two-step procedure yielded labeled gentamycin, toramycin, and amikacin suitable for use in radioimmunoassay) 1 The table lists the substances that have been radiolabeled using the Bolton-Hunter reagent along with the specific activity of the product, literature references, and informative comments. An attempt has been made to cover the literature into the first quarter of 1979. to D. J. Casley, Clin. Exp. Pharmacol. Physiol. 4, 525 (1977). tt C. D. Ashby, J. L. Lewis, and J. C. Nelson, Clin. Chem. 24, 1734 (1978). ~2 j. p. McMurtry, S. C. M. Kwok, and G. D. Bryant-Greenwood, J. Reprod. Feral. 53, 209 (1978). ta G. B. Tener, A. B. Delaney, T. A. Grigliotti, G. J. Cowling, and I. J. Gillam, Biochemistry 17, 741 (1978).

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R A D I O I M M U N O A S S A Y S A N D I M M U N O R A D I O M E T R I C ASSAYS

[13]

Other Iodination Reagents General Considerations Other prelabeled small molecules have been synthesized and used to prepare radioiodinated protein derivatives under conditions that circumvent exposure of the protein to oxidizing conditions. One of these compounds, x25I-labeled methyl p-hydroxybenzimidate, reportedly is more selective for amino groups than the Bolton-Hunter reagent 2,3 (Fig. 1). In the other method, 125I-labeled aniline was prepared by the chloramine-T method, diazotized, and allowed to react with the protein under basic conditions 4 (Fig. 1). Presumably it couples to the phenol moiety of tyrosine residues. If this is the case, the product is analogous to that obtained by direct iodination by the chloramine-T or lactoperoxidase procedures. Papain and certain plant lectins in which cysteine residues are part of the active site were labeled without loss of activity. When variations of the chloramine-T or lactoperoxidase methods were used, 64-83% of the activity was lost.

12~I-Labeled Methyl p-Hydroxybenzimidate Methyl p-Hydroxybenzimidate z,z Reagents p-Hydroxybenzonitrile, 1.2 g; 10 mmol Methanol, 16 ml; 0.4 tool HCI gas, dry Diethyl ether Molecular sieve, a drying agent commercially available from several sources Procedure. Mix p-hydroxybenzonitrile, methanol, ether (10 ml), and 5 pellets of molecular sieve in a 50-ml round-bottom flask equipped with a drying tube containing anhydrous calcium sulfate or similar desiccant. Cool the mixture to - 2 0 ° (ice-salt bath) and bubble HCI gas through an inlet tube reaching nearly to the bottom of the solution. After the solution is saturated with HCI, allow the temperature to warm to 4 °. After 30 min collect the orange crystals by filtration and wash them quickly with cold methanol-ether (1:2) followed by cold ether. The product (90% yield, m.p. 171-172 °) can be stored at 0° in a vacuum desiccator for at least 6 months without deterioration.

[13]

RADIOIODINATION W I T H B O L T O N - - H U N T E R REAGENT

245

Di-125I-Labeled Methyl p-Hydroxybenzimidate 213 Reagents Methyl p-hydroxybenzimidate, 3.7 mg in 1 ml of 50 mM sodium borate buffer, pH 8.5 NaI, 1.0 ml of 40 mM solution Nal~5I, 2 mCi in 10/~1 Chloramine-T, 1.0 ml of 40 mM solution /3-mercaptoethanol, 0.1 ml of 1 M solution Acetic acid, 2/zl of 1 M solution Procedure. Add the NaI and Nal~5I to the solution of benzimidate followed by the chloramine-T solution. After 15 rain at room temperature, add/3-mercaptoethanol to stop the reaction. Add acetic acid to precipitate the iodinated benzimidate, which is collected by centrifugation at 10,000 rpm for 5 min, dissolved at 37° in 2 ml of 50 mM sodium borate buffer, pH 8.5, and stored at 0° or frozen at - 2 0 °. The frozen product was stable for at least 7 days. Protein Labeling 2"z Reagents Bovine plasma albumin, 20 mg Di-125I-labeled methyl p-hydroxybenzimidate; 4 /xM containing 5.6 × 10n cpm of ~2sI Buffer: 50 mM sodium borate, pH 9.5 Procedure. Add the albumin to the ~5I-labeled benzimidate in a total volume of 1.0 ml of buffer maintained at 37°. After 24 hr the product is isolated by dialysis against 0.15 M NaCI containing 5 mM sodium phosphate, pH 7.4. Presumably, separation could be effected be gel filtration as described above for isolation of the product labeled with the BoltonHunter reagent. Under these conditions (14:1 molar ratio of nSI-benzimidate to albumin), maximum incorporation of 1251was 30%. Results Bovine plasma albumin and dog serum albumin have been iodinated with di-125I-labeled methyl p-hydroxybenzimidate. The authors did not attempt to obtain maximum specific activity, but rather examined the effect of reaction conditions on the rate of amidination and yield of iodinated product. The set of conditions given above is only one of the many tested. It was chosen because it gave the highest incorporation of 1251.Overall the results showed that the iodination reaction will proceed at pH 7.5-9.5 and

246

R A D I O I M M U N O A S S A Y S A N D I M M U N O R A D I O M E T R I C ASSAYS

[13]

between 21 ° and 37° with a yield of 20-30% incorporation after 24 hr. Specific activities up to 104 cpm per microgram of protein were obtained, but the authors suggested that modifications of the method would permit the use of lesser amounts of reagents and lead to at least a 10-fold increase in the specific activity. These improvements would be necessary to adapt the procedure to the preparation of microgram amounts of high specific activity antigen or antibody required for immunoassay. Diazotized x25I-Aniline

125I-Aniline Reagents Aniline, 5 nmol; 50/zl of a solution prepared by dissolving 10/.d of aniline in 100 ml of 0.1 M HCI Na125I, 2/zl; 1.0 mCi; 0.5 nmol Chloramine-T, 10/~1, 1 mM, 10 nmol NaOH, 1 M Chloroform Procedure. To the solution of aniline in a 3-ml tapered glass tube add the NaX25I followed by chloramine-T. Agitate the tube for 1 min then adjust the pH to 11 with NaOH (indicator paper). Extract the labeled aniline with three 1-ml portions of chloroform and evaporate the combined chloroform extracts to dryness under a stream of dry nitrogen. The residual product is dissolved in 100/zlof 0.1 M HCI.

Diazotization of [1251]Aniline and Protein Labeling Reagents Sodium nitrite, 10/zl; 10 mM; 1 nmol [125I]Aniline, 5.0 nmol in 0.1 M HCI Protein, 10 mg; 10 ml of 1 mg/ml solution at pH 10 Procedure. Add the sodium nitrite to the [~25I]aniline. The solution of diazonium salt is added dropwise to the stirred solution of protein, and the, pH is maintained at 9-10 for 1 hr. Isolate the a25I-labeled product either by dialysis against 10 mM phosphate-buffered saline, pH 7.2, or by chromatography on a BioGel P column (2.5 × 30 cm) wet-packed and eluted with this same buffer.

Results Papain, Bandeiraea sirnplicifolia lectin, and lima bean lectin have been labeled with diazotized [125I]aniline according to the procedure described

[14]

I O D I N A T I O N OF N U C L E I C ACIDS

247

above.4 The specific activity ranged between 760 and 1085 cpm per microgram of protein. The effect of varying the ratio of [nSI]aniline to protein was tested using B. simplicifolia lectin. As the amount of lectin was decreased from 10 to 0.5 mg and the diazonium coupling reaction volume decreased correspondingly from 10 to 0.5 ml, the specific activity of labeled lectin increased from 1085 to 7109 cpm per microgram of protein. Conclusion Conjugation labeling has become an important addition to the available methods of introducing 1251or 1311into antigens, haptens, and antibodies for use in immunoasssay and other immunochemical studies. The Bolton-Hunter reagent is already widely used, especially to label compounds that lack tyrosine groups or are labile to the reaction conditions encountered in the chloramine-T and lactoperoxidase methods. Prelabeled derivatives of methyl p-hydroxybenzimidate and diazotized aniline are potentially useful for preparing tracers for immunoassay, although they have not been developed specifically for this purpose. Synthesis of new reagents and adaptation of existing techniques are sure to enhance the value of this mehodology.

[14] I n V i t r o I o d i n a t i o n

of Nucleic Acids 1

By S. L. COMMERFORD Nucleic acids can be labeled with a radioactive isotope in vitro by heating them in a mixture containing radioactive iodine and thallic trichloride (TICI3). 2 The reaction is rapid and simple. It results in the formation of a stable covalent bond between the radioactive iodine atom and carbon atom 5 of cytosine in the nucleic acid. The biological properties of the nucleic acid are not significantly affected by this procedure. The reaction requires single-stranded nucleic acid (RNA or DNA), an acid pH, and temperatures of 40 ° or higher. Other conditions, such as time of reaction, ionic strength, volume, concentration of iodine, TICIz, and nucleic acid, can be varied over a wide range to accommodate the specific purposes for which labeling is being done.

i

Research was supported by the U.S. Environmental Protection Agency S. L. Commefford, Biochemistry 10, 1993 (1971).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1960by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

[14]

I O D I N A T I O N OF N U C L E I C ACIDS

247

above.4 The specific activity ranged between 760 and 1085 cpm per microgram of protein. The effect of varying the ratio of [nSI]aniline to protein was tested using B. simplicifolia lectin. As the amount of lectin was decreased from 10 to 0.5 mg and the diazonium coupling reaction volume decreased correspondingly from 10 to 0.5 ml, the specific activity of labeled lectin increased from 1085 to 7109 cpm per microgram of protein. Conclusion Conjugation labeling has become an important addition to the available methods of introducing 1251or 1311into antigens, haptens, and antibodies for use in immunoasssay and other immunochemical studies. The Bolton-Hunter reagent is already widely used, especially to label compounds that lack tyrosine groups or are labile to the reaction conditions encountered in the chloramine-T and lactoperoxidase methods. Prelabeled derivatives of methyl p-hydroxybenzimidate and diazotized aniline are potentially useful for preparing tracers for immunoassay, although they have not been developed specifically for this purpose. Synthesis of new reagents and adaptation of existing techniques are sure to enhance the value of this mehodology.

[14] I n V i t r o I o d i n a t i o n

of Nucleic Acids 1

By S. L. COMMERFORD Nucleic acids can be labeled with a radioactive isotope in vitro by heating them in a mixture containing radioactive iodine and thallic trichloride (TICI3). 2 The reaction is rapid and simple. It results in the formation of a stable covalent bond between the radioactive iodine atom and carbon atom 5 of cytosine in the nucleic acid. The biological properties of the nucleic acid are not significantly affected by this procedure. The reaction requires single-stranded nucleic acid (RNA or DNA), an acid pH, and temperatures of 40 ° or higher. Other conditions, such as time of reaction, ionic strength, volume, concentration of iodine, TICIz, and nucleic acid, can be varied over a wide range to accommodate the specific purposes for which labeling is being done.

i

Research was supported by the U.S. Environmental Protection Agency S. L. Commefford, Biochemistry 10, 1993 (1971).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1960by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

248

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[14]

T h e Effect of Reaction Conditions

Tempeature, pH, and Time o f Reaction. The rate of reaction is sensitive to temperature and pH. It proceeds very slowly or not at all at pH values greater than 5.5 or at temperatures below 40°. The rate increases rapidly as temperature increases and pH decreases. However, temperatures greater than 80° and pH values less than 4.5 should ordinarily be avoided because of the risk of depurinating the nucleic acid. Good yields are obtained by heating to 70° at pH 4.7 for 30 rain. This produces no significant adverse effects on the labeled nucleic acid. 3 Ionic Strength. The iodination reaction is not greatly affected by ionic strength, but very high ionic strength will decrease yield. TICl3 Concentration. The stoichiometry of the iodination requires a concentration of TIC13 at least six times as great as the iodide concentration for maximum yield. Higher levels do no apparent harm. Iodide Concentration. The iodide concentration has a major influence on the reaction. This is shown by the data in Table I. 4 Maximum labeling of DNA is obtained when the iodide concentration is 10-5 M whereas maximum specific activity is obtained when the iodide concentration is 8 x 10-5 M. Nucleic Acid Concentration. As the nucleic acid concentration increases, the fraction of 125I covalently bound increases whereas the specific activity of the labeled nucleic acid decreases. Typical data for DNA are shown in Table II. 4 Volume. As the reaction volume decreases, the amount of water and acetic acid lost to the gas phase in the scaled container during heating becomes more significant. For any given container a point will be reached where these losses raise the pH enough to affect yield. With 12 x 125 mm test tubes this point is reached somewhere between 50 ~1, which gives normal yield, and 20 tzl, where the yield is about 70% of normal. This effect can be avoided by using a smaller container. Another factor that becomes more important as volume decreases is the presence of protein or detergent residues in the reaction container or the pipettes used to transfer components of the reaction mixture. In addition, this apparatus becomes more difficult to clean as the size is reduced. Iodine Isotope. There are many radioactive isotopes of iodine. Four are commercially available. For most purposes, one of these, 125I, is the most useful because it has a reasonable half-life (60 days), it decays to a stable isotope, and it decays by an electron capture process that results in a cascade of low energy electrons and the emission of X-rays and 3,-rays. a j. M. Orasz and J. G. Wetmur, Biochemistry 13, 5467 (1974). 4 S. L. Commerford, unpublished results, 1979.

[14]

249

I O D I N A T I O N OF N U C L E I C ACIDS TABLE I EFFECT OF IODIDE CONCENTRATION ON THE EXTENT OF D N A IODINATIONa DNA-bound iodine

Iodide concentration M

mCi/mP

5 x 10-tl

--

1 x

10 - 7

--

1 ×

10 - 6

2

53

6

3 x 10-e

6 22 43 87 130 173 217

73 78 77 71 65 57 43

24 85 166 310 423 499 467

l x

l0 -5

2 4 6 8 1

10-5 l0 -5 10-5 10-5 10-4

x x x × ×

% 2 20

/zCi/~g b ---

a Reaction conditions: 200 p,g/ml denatured calf t h y m u s DNA, 200 ~ g / m l TICis and iodide, as listed, in 0.2 M acetate buffer, pH 4.7 were heated at 70 ° for 30 min. The unstable addition product was removed by heating at 70 ° for 30 min at pH 6.7. b Levels were calculated for carrier-free 125I.

T A B L E II EFFECT OF D N A CONCENTRATION ON THE EXTENT OF D N A IODINATIONa DNA-bound iodide D N A concentration (/zg/ml)

%

/~Ci/mi b

10 20 50 100 200

18 30 57 73 80

397 331 246 158 86

a The reaction mixture contained 10 /~M iodide and denatured D N A , as listed. Other conditions were the same as listed in Table I. b Levels were calculated on the basis of carrierfree t25I. Theoretical maximum is 1500/~Ci//~g.

250

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[14]

As a result the isotope gives high resolution autoradiographs, can be detected by liquid scintillation or, with no sample preparation, by y-ray counting. In addition the X-rays and y-rays emitted are low energy, so that little shielding is required for safe handling. Less than 0.1% of the X-rays and y-rays penetrate 0.5 mm of lead. Principles Governing Experimental Procedure During iodination of nucleic acids some of the radioactive iodine isotope becomes volatile. Radioactive contamination of the laboratory can be minimized by placing a gastight seal on the reaction container, by adding lZSIto the mixture through the seal by means of a hypodermic syringe, and by withdrawing air from the sealed container by means of a syringe so that the inner pressure remains less than atmospheric even when the container is heated to 70°. Serum bottle stoppers, 13 x 20 mm, are suitable for 12 x 125 mm Pyrex glass test tubes, and Burrell silicone seals are suitable for 4 × 50 mm Pyrex tubes. In addition, iodination involving substantial quantities of isotope should be done in a hood. A number of other precautions that can be taken when handling large amounts of isotope are discussed in some detail by Prensky. 5 After terminating the iodination reaction by chilling in ice, two further steps must be taken. First, an unstable addition product formed between iodine and nucleic acid must be eliminated. This is accomplished by adding phosphate buffer (via hypodermic syringe through the rubber seal) to raise the pH to between 6.5 and 7.0 then heating at 70° for 30 min. Finally, TICI3 and unbound radioactive iodine are separated from the labeled nucleic acid. This is most conveniently done by chromatography on Sephadex G-50, since this method is rapid, simple, and uses disposable apparatus.

Example Solutions Acetate buffer: 0.2 M acetic acid, 0.2 M ammonium acetate DNA, 1 mg/ml in H20, denatured by heating 10 min at 70° Potassium iodide (KI), 0.1 mM Thallic trichloride (TICI3), 2 mg/ml in 0.1 M acetic acid, 0.1 M ammonium acetate 1251, 1 mCi/ml, carrier free, in dilute NaOH, pH 8-11 Potassium monohydrogen phosphate (K2HPO4), 1 M Phosphate buffer: 50 mM K2HPO4, 50 mM KH2PO4, 0.2 mM EDTA, pH 6.7 5 W. Prensky, Methods Cell Biol. 13, 121 (1976).

[14]

IODINATION OF NUCLEIC ACIDS

251

Sephadex G-50 column. Add 20 ml of phosphate buffer to 1 g of Sephadex G-50 Fine. Let stand at least 3 hr at room temperature, then pour the slurry into a 10-ml disposable glass serological pipette containing a small amount of glass wool at the tip. The bed volume should be about 10 ml. Procedure. Acetate buffer (450/xl), DNA (100/xl), KI (100/~1), and HzO (150/xl), are added to a 12 x 125 mm Pyrex glass test tube, which is then sealed with a 13 x 20 mm serum bottle stopper. All further additions and withdrawals are made through this stopper by means of hypodermic syringes equipped with a 25-gauge needle; 5 ml of air are withdrawn, and 1251 (I00/zl) and finally TICI3 (100 ~1) are added. After stirring, the test tube is heated for 30 min at 70° then chilled in ice. K2HPO4 (200 txl) is added, the test tube is stirred then heated again for 30 min at 70 ° and chilled on ice. The reaction mixture is loaded onto the Sephadex column and eluted by gravity with phosphate buffer at 2030 ml/hr. The labeled nucleic acid elutes first and is well resolved from TICI3 and the unbound 1251, which appear later. Collection of fluid from the column is begun when the reaction mixture is added. Labeled nucleic acid first appears when a volume equivalent to 0.4 the bed volume of the column has passed through, and over 90% has appeared when 0.6 volume has been collected. Unbound 1251 does not appear until 1.0 volume has passed through.

Optimum Composition of the Reaction Mixture The optimum composition depends on the amount and specific activity of labeled nucleic acid required. If high specific activity is not important, then, for economy and to minimize exposure to radioactive isotope, the iodide concentration should be brought to 10 p ~ / b y adding carrier iodide, if necessary, as in the example given above. Such a reaction mixture will give about 70 ~Ci of labeled DNA with a specific activity of 0.7 ~Ci per microgram of DNA. Higher specific activity can be achieved by reducing the amount of DNA and carrier iodide added, decreasing the total volume and increasing the concentration of 1~5I. For example, 70/zCi of labeled DNA with a specific activity of over 100 ~Ci/~g should be obtained by allowing 6 pA of a mixture containing 100 I.~Ci of ~25I, 0.6 ~g of DNA, and no carrier iodide to react in a suitable small container.

Storage of Labeled Nucleic Acid Labeled nucleic acids can be stored frozen if their specific activity and concentration are low. However, as 125I concentration (~Ci/ml) and spe-

252

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[15]

cific activity (~Ci per microgram of nucleic acid) increase, radiation damage during storage may cause significant alterations in the properties of the labeled nucleic acid, especially in its molecular weight. These changes can be minimized by keeping the 1251concentration as low as possible and by storing at 0 ° to 4° in 20% (by volume) ethanol.

[15] R a d i o i o d i n a t i o n o f C e l l S u r f a c e L i p i d s a n d Proteins for Use in Immunological Studies B y SEYMOUR I. SCHLAGER

The importance of the plasma membrane as the site of action of immunologically mediated cytotoxicity reactions involving humoral or cellular factors has recently become evident. In this regard, several studies have shown that humoral immune killing reactions involve a complex series of biochemical interactions between the attacker moieties and the cell surface membrane. 1-4 One approach in studying and elucidating such interactions could therefore focus on the effects of the immune attack processes on the synthesis and/or turnover of cell surface macromolecules known to be structural and functional components of the plasma membrane (e.g., proteins and lipids). There are presently available several reagents, including diazotized iodosulfanilic acid (ISA) and a NaI-lactoperoxidase (LPO) complex, that have been used to label cell membranes radioisotopically, presumably by labeling exposed tyrosine and/or histidine residues on plasma membrane proteins, s-13 Recently, the lactoperoxidase-catalyzed NaI reaction has i M. M. Mayer, Harvey Lect. 72, 139 (1978). 2 M. M. Mayer, in "The Nature and Significance of Complement Activation" (W. Pollack, ed.), p. 29. Ortho Research Inst. of Med. Sci., Raritan, New Jersey, 1977. 3 S. H. Ohanian, S. I. Schlager, and T. Borsos, in "Contemporary Topics in Molecular Immunology" (R. Reisfeld and F. P. Inman, eds.), p. 153. Plenum, New York, 1978. 4 S. H. Ohanian and S. I. Schlager, in "CRC Critical Reviews in Immunology" (M, Z. Atassi, ed.). CRC Press, Palm Beach, Florida, 1980. In press. 5 A. L. Hubbard and Z. A. Cohn, J. Cell Biol. SS, 390 (1972). 8 A. L. Hubbard and Z. A. Cohn, J. Cell Biol. 64, 438 (1975). 7 j. j. Marchalonis, R. E. Cone, and V. Santer, Biochem. J. 124, 921 (1971). s M. Morrison, G. S. Bayse, and R. G. Webster, lmmunochemistry 8, 289 (1971). g H. C. Berg, Biochim. Biophys. Acta 183, 65 (1969). 1o H. C. Berg and D. Hirsch, Anal. Biochem. 66, 629 (1975). 1~ j. N. George, P. C. Lewis, and D. A. Sears, J. Lab. Clin. Med. 88, 247 (1976). ~z J. N. George, R. D. Potted', P. C. Lewis, and D. A. Sears, J. Lab. Clin. Med. 88, 232 (1976). la D. A. Sears, C. F. Reed, and R. W. Helmkamp, Biochim. Biophys. Acta 233, 716 (1971).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright ~) 19~0 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181970-1

252

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[15]

cific activity (~Ci per microgram of nucleic acid) increase, radiation damage during storage may cause significant alterations in the properties of the labeled nucleic acid, especially in its molecular weight. These changes can be minimized by keeping the 1251concentration as low as possible and by storing at 0 ° to 4° in 20% (by volume) ethanol.

[15] R a d i o i o d i n a t i o n o f C e l l S u r f a c e L i p i d s a n d Proteins for Use in Immunological Studies B y SEYMOUR I. SCHLAGER

The importance of the plasma membrane as the site of action of immunologically mediated cytotoxicity reactions involving humoral or cellular factors has recently become evident. In this regard, several studies have shown that humoral immune killing reactions involve a complex series of biochemical interactions between the attacker moieties and the cell surface membrane. 1-4 One approach in studying and elucidating such interactions could therefore focus on the effects of the immune attack processes on the synthesis and/or turnover of cell surface macromolecules known to be structural and functional components of the plasma membrane (e.g., proteins and lipids). There are presently available several reagents, including diazotized iodosulfanilic acid (ISA) and a NaI-lactoperoxidase (LPO) complex, that have been used to label cell membranes radioisotopically, presumably by labeling exposed tyrosine and/or histidine residues on plasma membrane proteins, s-13 Recently, the lactoperoxidase-catalyzed NaI reaction has i M. M. Mayer, Harvey Lect. 72, 139 (1978). 2 M. M. Mayer, in "The Nature and Significance of Complement Activation" (W. Pollack, ed.), p. 29. Ortho Research Inst. of Med. Sci., Raritan, New Jersey, 1977. 3 S. H. Ohanian, S. I. Schlager, and T. Borsos, in "Contemporary Topics in Molecular Immunology" (R. Reisfeld and F. P. Inman, eds.), p. 153. Plenum, New York, 1978. 4 S. H. Ohanian and S. I. Schlager, in "CRC Critical Reviews in Immunology" (M, Z. Atassi, ed.). CRC Press, Palm Beach, Florida, 1980. In press. 5 A. L. Hubbard and Z. A. Cohn, J. Cell Biol. SS, 390 (1972). 8 A. L. Hubbard and Z. A. Cohn, J. Cell Biol. 64, 438 (1975). 7 j. j. Marchalonis, R. E. Cone, and V. Santer, Biochem. J. 124, 921 (1971). s M. Morrison, G. S. Bayse, and R. G. Webster, lmmunochemistry 8, 289 (1971). g H. C. Berg, Biochim. Biophys. Acta 183, 65 (1969). 1o H. C. Berg and D. Hirsch, Anal. Biochem. 66, 629 (1975). 1~ j. N. George, P. C. Lewis, and D. A. Sears, J. Lab. Clin. Med. 88, 247 (1976). ~z J. N. George, R. D. Potted', P. C. Lewis, and D. A. Sears, J. Lab. Clin. Med. 88, 232 (1976). la D. A. Sears, C. F. Reed, and R. W. Helmkamp, Biochim. Biophys. Acta 233, 716 (1971).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright ~) 19~0 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181970-1

[15]

RADIOIODINATION OF CELL SURFACE MACROMOLECULES

253

also been shown to label a variety of cell surface lipids, including phospholipids, triacylglyceddes, free fatty acids, and lysophosphatides. 14,'5 Although the exact mechanism of the labeling reaction is not fully understood, it is enzyme-dependent in its initial steps, in which an oxidizing agent (HzO2) is generated [Eq. (1)] with the subsequent oxidation of I(from NaI) to I2 [Eq. (2)].5"e-2° The final interaction of Iz with lipid molecules [Eq. (3)] apparently does not involve a simple addition reaction of I, to unsaturated fatty acid constituents of lipids, since fully saturated fatty acids or saturated fatty acid-containing neutral lipids and phospholipids can also be labeled by this reaction./5 This suggests that I~ substitution or exchange reactions can also occur in this system./5 Glucose

glucose oxidase ~--- H20~ H20, 02

a~I-

;

a ~ s e

Protein or lipid

12~I-Protein or lipid

*2sI2

/

(1)

(2)

(3)

In the present report, the use of radioiodinated diazotized iodosulfanilic acid and a lactoperoxidase-catalyzed iodination method for the labeling and examination of plasma membrane proteins and lipids in cells under humoral immune attack is described. P r e p a r a t i o n of 125I-Diazotized I S A 125I-labeled ISA is available from New England Nuclear, Boston, Massachusetts, including all reagents required for carrying out the diazotiza14M. Mersel, A. Beneson, and F. Doljanski, Biochem Biophys. Res. Commun. 70, 1166 (1976). is S. I. Schlager,J. Immunol. 123, 2108 (1979). le j. C. Marshall and N. D. Odell, Proc. Soc. Exp. Biol. Med. 149, 351 (1975). 17y. Miyachi, A. Crambach, R. Mecklenburg, and M. B. Lipsett, Endocrinology 92, 1725 (1973).la M. J. Murphy, Biochem. J. 159, 287 (1976). lg B. B. Tower, B. R. Clark, and R. T. Rubin, Life Sci. 21, 959 (1977). 20I. Schenkein, M. Levy, and J. W. Uhr, J. Lab. Clin. Med. 76, 46 (1970).

254

R A D I O I M M U N O A S S A Y S A N D I M M U N O R A D I O M E T R I C ASSAYS

[15]

tion reaction, fhe iodinated product offers several distinct advantages over analogous reagents labeled with all, ~4C, or 35S in that it can be used under conditions resulting in minimal damage to membrane components, it can be used to label whole viable cells or subcellular fractions, and it can be used in studies designed to follow the fate of labeled cells in vivo. 11-13

Diazotization Procedure Reagents [125I]Iodosulfanilic acid, 1 mCi, > 1000 Ci/mmol Distilled water NaNO2, 50 mM HCI, 0.1 M NaOH, 0.1 M Phosphate buffer, 0.1 M, pH 7.5 Procedure The [125I]ISA, supplied as an n-propanol : H20 (1 : 1) solution, is first dried by blowing off the solvent in a gentle stream of N2; the dried sample is dissolved immediately in 10/.el of distilled HzO (dry ISA will undergo autoradiolysis within minutes). The solution is cooled to 0 5° in an ice bath; 5/zl of NaNO2 and 5 tzl of HCI are added, and the mixture is allowed to react for 5 min at 0 - 5 °. The reaction is stopped by neutralizing the mixture with 5 tzl of NaOH or diluting with 0.5 ml of phosphate buffer. The diazotized product should be used immediately or may be stored frozen for up to 24 hr. M e m b r a n e Labeling In the labeling of intact cells or subcellular fractions, it is important to have the cell suspension washed thoroughly and free from extraneous proteins or protein-containing macromolecules that could couple with the diazotized ISA. Procedure. An aliquot of the neutralized diazotized [125I]ISA is added to a washed, buffered suspension of cells (pH 7.5) at 5°. The reaction goes to completion within 15-30 rain depending on the cells being labeled? 3 The reaction is stopped by washing the cell suspension 3 or 4 times with a 10× volume of 0.15 M NaC1 containing 1% bovine serum albumin or serum-containing tissue culture medium (e.g., RPMI 1640 with 10% fetal calf serum).

Distribution of [125/]/SA The distribution of diazotized [~2nI]ISA into subcellular fractions of nucleated cells (i.e., line-10 guinea pig hepatoma cells) that were first labeled

[15]

RADIOIODINATION OF CELL SURFACE MACROMOLECULES

255

TABLE I UPTAKE AND DISTRIBUTION OF DIAZOTIZED [1251]IoDoSULFANILIC ACID INTO CELL SURFACE AND INTRACELLULAR MEMBRANES OF LINE-10 TUMOR CELLS

Activity (cpm) Total activity added Activity taken up per 10s cells Activity recovered from subcellular fractions

31,000,000

Rough endoplasmic

(100)

807,978 759,500

2.6 2.5

Percentage of total activity in fractions

Subcellular fraction Plasma membrane Mitochondria Ribonucleoprotein/smooth endoplasmic reticulum Nuclear membrane

Percentage of total

592,400 60,760 5,924

78 8 1

53,165 60,760

7 8

reticulum/ribosomes

and then fractionated into plasma membrane and intracellular fractions 21 is shown in Table I. Approximately 2.6% of the 4.4 × 107 dpm (3.1 x 107 cpm) added to the cell suspension was taken up by 10a line-10 cells when the reaction was carded out in a 0.5 ml total volume; in addition, 94% of the activity taken up by the cells was recovered in the subcellular membrane fractions (Table I). Approximately 78% of the total membrane activity in the cell was in the plasma membrane; all intracellular membranes had taken up -< 8% of the total activity (Table I). Quantitation of the Release of Cell Surface Proteins and Lipoproteins from Cells under Humoral Immune Attack

General Considerations The ability to label specifically plasma membrane proteins with ['25I]ISA makes it possible to analyze the effects of an immune attack system (e.g., antibody-complement) on cell surface proteins and proteincontaining macromolecules (e.g., lipoproteins). In this regard, cells can be labeled with [125I]ISA, sensitized with antibody, and treated with corn2~ S. I. Schlager and S. H. Ohanian, Cancer Res. 39, 13"69(1979).

256

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[15]

plement, and the release of radioisotopically labeled plasma membrane macromolecules can be monitored. In the methods being described herein, care has been taken to absorb the antisera and complement sources with the appropriate cells (i.e., rabbit anti-line-10 antiserum has been absorbed with sheep erythrocytes to remove Forssman specificities and with line-1 guinea pig hepatoma cells to remove xenoantibody and non-tumor-specific antibody specificities~2'za; GPC and HuC were both absorbed with sheep erythrocytes, line-I and line 10 tumor cells to remove naturally occurring anti-Forssman and anti-tumor-specific antibody activities).

Reagents [125I]ISA-labeled line-10 tumor cells; 5 × 105 cells/ml in Hanks' balanced salt solution (HBSS) Rabbit anti-Forssman IgM antibody, diluted 1:8 Rabbit anti-line-10 antibody, diluted 1:20 Guinea pig serum (GPC), diluted 1 : 8 Normal human serum (HuC), diluted 1:6 Dextran sulfate, 10% MgCI2 1 M Procedure. The labeled tumor cells (0.5 ml of 5 × 105 cells/ml) and 0.5 ml of the appropriate dilution of antibody are mixed and incubated for 30 min at 0°. The cells are washed twice with 3 ml of RPMI 1640; 0.5 ml of the appropriate dilution of GPC or HuC is added to the cell pellet, and the mixture is incubated at 37 °. The reaction is stopped by addition of 2 ml of HBSS, the cells are pelleted (5 min at 500 g), and the activity in the cell pellet and supernatant is quantified. Figure 1 shows the kinetics of release of radioiodinated protein from antibody-complement-treated tumor cells. Guinea pig serum caused the maximum enhanced release compared to untreated cells of lZSI-labeled protein from anti-Forssman antibody-sensitized cells within 10 min of incubation; there was no enhanced release of ~25I-labeled protein compared to controls from anti-line-10 antibody-sensitized cells treated with GPC (Fig. 1A). Similarly, HuC caused maximal enhanced release of cell surface protein from cells sensitized with either antibody within 10 min (Fig. 1B). No enhanced release of membrane protein was observed from cells treated with antibody alone or complement alone (Fig. 1). The values in Fig. 1 represent 23-40% of the total cell-bound activity associated with [125I]ISA. ~2 H. J. Rapp and T. Borsos, "Molecular Basis of Complement Action," p. 75. Appleton, New York, 1970. 23 T. Borsos, A. K. Richardson, S. H. Ohanian, and E. J. Leonard, J. Natl. Cancerlnst. 51, 1955 (1973).

[15]

RADIOIODINATION OF CELL SURFACE MACROMOLECULES

GUINEA PIG COMPLEMENT

B.

257

HUMAN COMPLEMENT

x P, /,~..

~

s~

Q <

--

t

~D._

_

_

~ .E}- -- - - D

_.E]

uJ ._J uJ ¢Z:

>_ t~

<

I 2

I 5

I 10

I 1,5

1 2

I 5

I 10

I

15

MIN AFTER ADDITION OF COMPLEMENT

FIG. 1. Release of [mI]iodosulfanilic acid-labeled plasma membrane proteins from line10 tumor cells treated with antibody plus complement. * - - * , Tumor cells alone (T); []---D T sensitized with anti-Forssman IgM antibody diluted 1:8 (TAr); A---A, T sensitized with specific anti-line-10 antibody diluted 1:20 (TATu); V---V, T treated with guinea pig serum (GPC) diluted 1:8 or human serum (HuC) diluted 1:6 (TC~ or TC,u); 1 - - 1 1 , TAvCcp or TArCnu; & - - & , TATuCGp or TATvC.u.

The correlation between the release of cell surface proteins and the killing of the cells by antibody plus HuC is shown in Fig. 2. The amount of plasma membrane protein released and the percentage of antibody-sensitized cells killed by HuC are both dependent upon the concentration of antibody used to sensitize the cells (Fig. 2). To determine what percentage of the activity released from antibodycomplement-treated cells is associated with protein-containing macromolecules (e.g., lipoprotein), the supernatant from the treated cells is collected as described above, 0.7 ml of dextran sulfate and 2.0 ml of the MgCl= are added to the supernatant, and the mixture is centrifuged at 4 ° for 15 min at 5000 g. Depending upon the lipid content of the macromolecules released from the cells, the fl-lipoprotein-dextran sulfate insoluble complexes thus formed may either float or pellet. 24 The results in Table II 24 D. G. Cornwell and F. A. Kruger, J. Lipid Res. 2, 110 (1961).

258

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[15]

A

I R"
IO0

x

-rt

,A

O.

/

--80

/" /

_x

/ W --I LM

m

/ /

T

/ I.-

rm '-

/

r-,

>_

a~ o

2-

-40

/ .jR . . . . . . . /

N

-n

/

-20

,It // 0

I-L

l

l

1

2

I 4

l 8

RELATIVE ANTIBODYCONCENTRATION FIG. 2. Effect of antibody plus human serum (Hue) on the release of [l=SI]iodosulfanilic acid-labeled proteins from line-10 tumor cells as compared to cell killing, e, tumor cells alone (T); m, T sensitized with anti-Forssman IgM antibody (1 = 1:32 dilution) and treated with HuC diluted 1:6 (TAr(2); A, T sensitized with specific anti-line-10 antibody (1 = 1 : 160 dilution) and treated with HuC (1 : 6) (TATuC). TAF, TATu, TC, and TAFACor TATuAC(TAF or TATu treated with heat-inactivated HuC) released < 4430 cpm and were killed < 10% (SchlagerlS). s h o w that a p p r o x i m a t e l y 14% o f the proteins shed f r o m the surface o f unt r e a t e d line-10 t u m o r cells are in the f o r m o f fl-lipoproteins. Sensitization o f the cells with a n t i b o d y or t r e a t m e n t o f cells with G P C o r H u C did not m a r k e d l y affect the a m o u n t o f protein o r lipoprotein released f r o m the cells. Cells t r e a t e d with a n t i b o d y plus G P C o r H u C w e r e e n h a n c e d in their release o f cell surface p r o t e i n ; in addition, the p e r c e n t a g e o f these m o l e c u l e s in the f o r m o f fl-lipoprotein rose to 23 to 33% (Table II). Lactoperoxidase-Catalyzed

Iodination of Plasma Membrane

Lipids

General Considerations A l t h o u g h the L P O - c a t a l y z e d iodination o f p l a s m a m e m b r a n e proteins has b e e n in use for s o m e time, the d i s c o v e r y that cell surface lipids c a n be

[15]

RADIOIODINATION OF CELL SURFACE MACROMOLECULES

259

TABLE II [I~5I]IoDoSULFANILIC ACID-LABELED PROTEINS AND LIPOPROTEINS RELEASED FROM LINE-10 TUMOR CELLS TREATED WITH ANTIBODY PLUS COMPLEMENT Activity released (cpm/2.5 × 105 cells) Treatment a

Total

Dextran-precipitable b

T TAr TATu TCGp TCxu TA~CGp TAruCGp TAFCNv TATuCrlv

3895 3997 4083 3837 4178 4952 4617 5218 5513

545 (14)c 520 (13) 367 (9) 345 (9) 418 (10) 1139 (23) 1339 (29) 1565 (30) 1819 (33)

T, tumor cells alone; TAr or TArv, cells sensitized with anti-Forssman IgM or antitumor antibody, respectively; TCcp or TCnu, cells treated with guinea pig or human serum (GPC or HuC), respectively; TAC, cells sensitized with antibody and treated with complement. b Dextran sulfate-precipitated proteins are /3-1ipoprotein in nature (Cornwell and Kruger2~). c Values in parentheses indicate percentage of total.

labeled simultaneously in this reaction has only recently been observed. 14,15 The hallmarks of the reaction are that it apparently labels all lipids regardless of class (i.e., phospholipids, neutral lipids, and free fatty acids), it can bind 1251to saturated as well as unsaturated fatty acid-containing lipids, and it can be used to label whole cells or artificially constructed liposomes. 14"15In addition, the lipid-125I bond is stable in face of lytic or cytotoxic stimuli to the cell surface) 5

Reagents Carrier-free Na-125I (New England Nuclear, Boston, Massachusetts, 17 Ci/mg) Lactoperoxidase (EC 1.11.1.7) Glucose oxidase (EC 1.1.3.4) Glucose

260

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[15]

T A B L E III CHROMATOGRAPHIC MIGRATION OF LIPIDS IODINATED IN A LACTOPEROXIDASE-CATA LYZED REACTION

R t Value"

Lipid Phosphatidylcholine b Tripalmitin Palmitic acid (16:0) Linoleic acid (18: 2)

Iodinated compound 0.17 0.97 0.78 0.73

-+ 0.05 -+ 0.05 -+ 0.03 _+ 0.04

Unlabeled parent 0.22 0.94 0.75 0.73

_+ 0.07 -+ 0.06 --_ 0.02 --- 0.01

a The solvent system used for separating the lipid compounds was CHCIa: C H a O H : H~O (65 : 25 : 4).~8 todinated lipids were detected by assaying 5-mm sections of the chromatography plates from the origin to the solvent front for radioactivity; unlabeled lipids were detected in an iodine vapor chamber. b Naturally occuring bovine phosphatidylcholine is a mixture of saturated and unsaturated fatty acid-containing molecules.

Buffer: Dulbecco's Ca2+-Mg~+-free phosphate-buffered saline 25 (Grand Island Biological Co., Grand Island, New York) (PBS) Standard lipids: chromatographically pure phosphatidylcholine, tripalmitin, palmitic acid, linoleic acid Silica gel chromatography plates, 0.25 mm (EM Laboratories, Elmsford, New York) CHCla : CHsOH (2 : 1, v : v) Procedure

Liposomes. The chromatographic purity of the lipids is tested by high pressure liquid chromatography.~6a7 A 500-/~g sample of lipid is dried and sonicated in 0.4 ml of PBS. The resulting liposomes are incubated with 10/~g of lactoperoxidase, 3 mU of glucose oxidase, 2.5 p,M glucose (0.23/xg), and 100/zCi of Na125I in a total volume of 0.5 ml for 15 min at ambient temperature. The liposomes are washed 3 times with 10 ml of PBS and dissolved in 1 ml of CHCIs:CH3OH. Each lipid is chromatographed on thin layer silica gel chromatography plates2S; the Rf values of the iodinated lipids obtained are comparable to the Re values of the unlabeled parent compounds (Table III). This indicates that the iodination 25 R. Dulbecco and M. Vogt, J. Exp. Med. 99, 167 (1954). z6 W. S. M. Geurts V a n K e s s e l , W. M. A. H a x , R. A. I)emel, and J. Degier, Biochim. Biophys. Acta 486, 524 (1977). 27 S. I. Schlager and S. H. Ohanian, J. lmmunol.i124, 626 (1980). S. I. Schlager, S. H. Ohanian, and T. Borsos, J. Immunol. 120, 472 (1978).

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reaction does not change the chromatographic migration of neutral lipids, phospholipids, or free fatty acids. Cell Membrane Labeling. Five million line-10 tumor cells are washed 3 times with 25 ml of PBS, resuspended in 0.4 ml of PBS, and incubated with 10 ~g of lactoperoxidase, 3 mU of glucose oxidase, 2.5/zM glucose (0.23/xg), and 100/~Ci of Na12~I in a total volume of 0.5 ml for 15 min at ambient temperature. The cells are washed 5 times with 10 ml of PBS, and the lipid and nonlipid fractions of the cells are obtained through a Folch extraction. 29 The cellular lipids are separated by thin layer chromatography.2a As shown in Table IVA, the cells bind approximately 5% of the total radioactivity added; 32% of this activity is associated with the cellular lipid fraction and 64% with the nonlipid (presumably protein) fraction, is Of the lipid-associated activity, the distribution of lz~I into cellular phospholipids, lysophosphatides, triacylglycerides, and free fatty acids is shown in Table IVB. The iodination reaction labels a variety of lipids of all classes without preference to charge, acid-base properties, or number of fatty acyl constituents, appears not to label intracellular lipids (e.g., cardiolipin, a major mitochondrial component), and labels lipids on both the exterior and interior hydrophilic surfaces of the plasma membrane (i.e., phosphatidylcholine and sphingomyelin as well as phosphatidylethanolamine and phosphatidylserine)(Table IVB). is Release of Cell Surface Lipids during Humorai Immune Attack. Lactoperoxidase-iodinated line-10 tumor cells (0.25 ml of 106 cells/ml) suspended in RPMI 1640 are incubated with 0.25 ml of the appropriate dilution (in RPMI 1640) of rabbit anti-Forssman IgM antibody or specific antiline-10 antibody for 30 min at 0°.The cells are washed twice with 4 ml RPMI 1640 and incubated for 15 min at 37° with 0.5 ml of the appropriate dilution (in RPMI 1640) of GPC or HuC. One-half milliliter PBS is added to the cells, the cells are centrifuged for 5 min at 500 g at 4°, and the radioactivity in the supernatants is quantified. This is a measure of total lipidand protein-containing macromolecules released from the cells. Controls include tumor cells alone (T), tumor cells treated with antibody alone (TA), tumor cells treated with complement alone (TC), and tumor cells sensitized with antibody and treated with heat-inactivated (30 min at 56°) complement

(TAAC). The lipid fraction of the supernatants is obtained through a Folch extraction, 29 and the lipid classes are separated by analytical thin layer chromatography.2S The amount of activity associated with each separated lipid moiety is then quantified. 2s Treatment of line-10 cells with antibody plus zs j. Folch, M. Lees, and G. H. Sioan-Stanley, J. Biol. Chem. 226, 497 (1957).

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T A B L E IV DISTRIBUTION OF 1~51 IN LINE-10 TUMOR CELLS IODINATED IN A LACTOPEROXIDASE (LPO)-CATALYZED REACTION a A. Distribution in Cellular Pools b

Pool

Activity (cpm)

Percentage o f total

Cells, 5 x l0 s Extracted pellet A q u e o u s (nonlipid) fraction Organic (lipid) fraction

7,987,800* 286,277 5,102,910 2,518,506

(100) 4 64 32

B. Distribution in Cellular Lipids d Lipid Lysophosphatidylserine Sphingomyelin Phosphatidylserine Phosphatidylcholine Phosphatidylinositol Phosphatidylglycerol Cardiolipin Phosphatidylethanolamine Free fatty acids Cholesterol Triglycerides Free 1251

Activity (% o f total - SD) 2 4 6 14 II 6 0.3 13 4 7 13 16

+- 0.5 -+ 0.5 -+ 1.5 -+ 0.5 -+ 4 -+ 1 +- 0 -+ 0.5 -+ 1 -+ 0.5 -+ 2 -+ 4

a From S. I. Schlager. 15 b Distribution of 125I-associated activity in 5 × 106 LPOcatalyzed iodinated line-10 t u m o r cells that were subjected to Folch lipid extraction. c Total activity taken up by 5 × i06 line-10 t u m o r cells. a Approximately 63,000 c p m (25 ttl) of the lipid fraction extracted from 5 x i0 s LPO-iodinated line-10 t u m o r cells were c h r o m a t o g r a p h e d , and the activity associated with each separated lipid moiety was quantified.

GPC (TAC) increased the amount of radioisotopically labeled macromolecules that was released from the cells from 30-32% up to 55%, and increased the release of labeled lipids from 9-12% up to 19% (Table VA). The indentity of the 125I-labeled lipid molecules released from the cells after antibody-GPC treatment is shown in Table VB; TAC released a significantly higher proportion of sphingomyelin, phosphatidylserine, and

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TABLE V ANTIBODY-GUINEA PIG SERUM (GPC)-MEDIATED RELEASE OF x25I-LABELED MEMBRANE LIPIDS FROM LINE-]0 TUMOR CELLSa A. Activity Distribution

Treatment b

Total activity bound (cpm/5 x 105 cells)

Percentage of activity released

Percentage of lipid extractable activity released

310,339 306,189 305,489 308,304 309,154

31 30 30 32 55

12 11 9 11 19

T TA TC TAAC TAC

B. Distribution of Activity in Lipids Released Percentage of total extracted activity c Lipid

T

TA

TC

TAAC

Ly sophosphatidylserine Sphingomyelin Phosphatidylserine Phosphatidylcholine Phosphatidylinositol Phosphatidylglycerol Cardiolipin Phosphatidylethanolamine Free fatty acid Cholesterol Triglyceride Free x25I

5 7 4 15 6 14 1 10 7 4 17 9

3 4 5 16 6 16 2 19 2 4 18 10

4 10 4 16 6 13 1 17 3 2 14 10

4 9 4 14 6 16 1 13 3 4 17 8

TAC 3 18 a 18 27

6 1 0 4 3 3 8 8

a From S. I. Schlager. 15 b T, tumor cells alone; TA, antibody-sensitized T; TC, T treated with GPC; TAAC, T sensitized with antibody and treated with heat-inactivated GPC (AGPC); TAC, T sensitized with antibody and treated with GPC. Antibody used was anti-Forssman IgM diluted 1:2; AGPC and GPC were diluted 1:8. Cells remained >90% viable after all treatments. c Total activity chromatographed was 15,887 cpm, 15,431 cpm, 15,240 cpm, 15,951 cpm, and 15,310 cpm for T, TA, TC, TAAC, and TAC, respectively. a Italicized numbers indicate a significant difference compared to controls.

phosphatidylcholine than untreated control cells; TAC released significantly less phosphatidylglycerol, phosphatidylethanolamine, and triglyceride than T. Neither TA, TC, nor TAAC showed these differences in membrane lipid release (Table VB). The effects of the specificity of the antibody used to sensitize the cells

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and of the C species used to treat the cells on release of membrane lipids can also be examined in this manner. Line-10 cells sensitized with antiForssman or anti-line-10 antibody and treated with GPC (TAFCGp or TATuCGp) demonstrated enhanced release of sphingomyelin and phosphatidylcholine and depressed release of phosphatidylethanolamine from the plasma membrane as compared to T, TAF, TAro, or TCGp (Table VI). In addition, TAFC showed enhanced release of phosphatidylserine, not seen in TATuC. Similarly, cells sensitized with either antibody and treated with HuC showed enhanced release of sphingomyelin, phosphatidylserine, and phosphatidylcholine and depressed release of phosphatidylethanolamine compared to T (Table VI). Treatment of cells with HuC alone (TCHu) TABLE VI COMPARISON OF THE EFFECTS OF GUINEA PIG AND HUMAN SERUM (GPC AND H u C ) ON THE RELEASE OF IZSI-LABELED MEMBRANE LIPIDS FROM LINE-TUMOR CELLS a

Distribution (%) of total extracted activityb Treatment c GPC (1 : 8) Lipid

T

TAr

TAro

TC

TAFC

Ly sophosphatidylserine Sphingomyelin Phosphatidylserine Phosphatidylcholine Phosphatidylinositol Phosphatidylglyceroi Cardiolipin Phosphatidylethanolamine Free fatty acid Cholesterol Triglyceride Free 1~5I

6 5 8 15 11 9 I 10 7 9 12 8

6 7 6 15 8 8 0 17 5 5 10 12

9 7 6 16 6 8 1 10 6 3 15 12

4 10 3 12 6 10 0 14 4 10 16 10

4 20 a 15 26 5 5 1 0 4 5 10 10

HuC (1 : 8)

TATuC TC 4 26 5 27 5 4 0 3 3 10 15 9

4 11 3 12 5 5 1 32 2 7 8 12

TAgC

TAroC

3 24 19 27 5 4 0 0 5 6 9 7

3 34 13 21 5 3 0 1 4 5 5 8

From S. I. Schlager. 15 b Total activity chromatographed was: 11,656 cpm T, 12,040 cpm TAr, 13,146 cpm TAro, 10,010 cpm TCc~, 12,760 cpm TCHv, 11,509 cpm TArCap, 10,679 cpm TAroCae, 12,030 cpm TAFCno , and 12,275 cpm TArvCHv. c Antibody dilutions used: anti-Forssman IgM was diluted 1 : 2 for TAr and TAFCGp, and 1 : 32 for TAFCnu ; anti-line- 10 antibody was diluted 1 : 40 for TAro and TAa~CGp, and 1 : 160 for TAroCao. Cells remained >90% viable after any of the treatments. a Italicized numbers indicate a significant difference compared to controls.

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caused an enhancement in their phosphatidylethanolamine release compared to all other treatments (Table VI). The effects of cytotoxic and noncytotoxic combinations of antibody and complement on the release of cell surface lipids can also be studied; approximately 40% of a line-10 cell population sensitized with a near excess (1 !4 dilution) concentration of anti-Forssman antibody instead of a limiting (1:32 dilution) concentration (as used in the experiments shown in Table VI) will be killed by excess HuC3°; the identity and amounts of lipids released from the TAxsC population were observed to be equivalent to that released from T A u t o G . 15 Rema~s

Since[125I]ISA and Na125I can be used to label cell surface proteins, lipids, and protein- and/or lipid-containing macromolecules (e.g., lipoproteins, glycolipids, glycoproteins), the assays described here may be useful in studying the effects of immunologically mediated cytotoxic stimuli (e.g., antibody-complement-mediated attack, T cell-mediated attack, antibody-dependent cell-mediated attack, activated macrophage killing) on a variety of cell surface macromolecules. Since most immune attack systems are thought to involve an interaction between an immunological eflector molecule(s) and a cell surface receptor, these techniques may be used to ascertain the role that plasma membrane macromolecules play in influencing the susceptibility of nucleated cells to these immune attack processes. In addition, these methods incorporate a great deal of flexibility in application to various cell systems since the precise technique used to isolate the macromolecule of interest from the supernatants of cells under immune attack can be varied. For example, the assay of lipoproteins released from the cell surface can be accomplished as easily using molecules isolated with a KBr flotation method 3L32 or a n ( N H 4 ) ~ S O 4 precipitation method 24 as with the dextran sulfate method outlined here. Also, the ability to specifically label and identify cell surface macromolecules may make it possible to study other types of surface membrane receptor-ligand interactions (e.g., cellular interactions with hormones, lectins, pharmacological agents, or drugs).

30 S. H. Ohanian, T. Borsos, and H. J. Rapp, J. Natl. Cancer Inst. 50, 1313 (1973). 3~ R. J. Havel, H. A. Eder, and J. H. Bragdon, J. Clin. Invest. 34, 1345 (1955). 32 C. M. Radding and D. Steinberg, J. Clin. Invest. 39, 1560 (1960).

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[16] U s e o f t h e D o u b l e - A n t i b o d y Method to Separate Antibody Bound from Free Ligand in Radioimmunoassay By

A.

REES MIDGLEY,

JR.

and

MARJOR1E R.

HEPBURN

Since first described by Morgan and Lazarow 1 and Utiger et al.,Z most radioimmunoassays have utilized double-antibody methods to separate the fraction of ligand that is bound to antibody from that which is free. The reasons for this are numerous. Double-antibody methods have applicability to more types of radioimmunoassay than any other separation procedures devised to date: they can be used with any ligand able to be bound by an antibody. Double-antibody methods are generally associated with low nonspecific binding, in part because the only required additional component is another immunoglobulin. Thus, the conditions selected to minimize nonspecific binding to the ligand-specific antibody usually minimize nonspecific binding to the second antibody as well. Double-antibody methods also have the advantage of being able to be carried out with no substantive compositional differences in the incubation mixture. Thus, separations can be completed with only minimal effects on the primary binding reaction, and, as a consequence, with minimal effects on the portions ofligand that are either bound or free. Double-antibody methods can be conducted under conditions that can provide very rapid separations. Finally, double antibody methods are easy to run and can be used to obtain reliable results of unsurpassed specificity, sensitivity, precision, and reproducibility. The various methods utilizing a second antibody for the purpose of separation can be divided into four different types. These are" post-precipitation (the most common method), primary antibody pre-precipitation (formation of a precipitate of first and second antibody before adding to the assay vessel), solid phase (second antibody conjugated to an inert solid matrix), and nonimmune globulin pre-precipitation (a new method to be described here). Post-Precipitation Methods The concentrations of ligand and antibody generally employed in a radioimmunoassay are too low to permit formation of an immunologic preC. R. Morgan and A. Lazarow, Proc. Soc. Exp. Biol. Med. 110, 29 (1962). 2 R. D. Utiger, M. L. Parker, and W. H. D a u g h a d a y , J. Clin. Invest. 41, 254 (1962).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright ~) 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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cipitate. The latter requires concentrations of reactants sufficient to form a lattice. Most double antibody procedures achieve this by including nonimmune immunoglobulin of the same species as the primary ligand-specific antibody, and in concentrations sufficient to form a precipitate. A second antibody to T-globulin of the species forming the primary ligandspecific antibody is prepared in an unrelated species and added to the reaction mix in a concentration sufficient to form an optimal precipitate with the nonimmune y-globulin. The primary antibody, present in minor concentrations relative to nonimmune y-globulin, becomes included in the immunoprecipitate. Since the sites on the primary antibody molecule that bind with the second antibody are unrelated to the sites on the primary antibody that bind the ligand, any ligand bound to the primary antibody is also included in the immunoprecipate. Optimal precipitation requires application of the well known principles of quantitative precipitin tests. 3 In general these involve use of an ionic strength approximating 0.15, pH between 6.5 and 8.5, temperature between 0° and 37°, and sufficient time to ensure that the reaction reaches equilibrium. In addition, the second antibody must be present in sufficient but not excessive amounts in order to ensure maximal precipitation of the nonimmune y-globulin (and thereby the primary antibody). The major difficulties with post-precipitation methods result from effects of compositional differences in samples on the rate or maximal extent of precipitate formation. Thus, if a sample contains serum, these parameters can be independently affected, either positively or negatively. The complement system is one of the components affecting the reaction.. Accordingly, complement should be inactivated by heating or, more easily, by including a chelating agent in the assay buffer. Additionally, one may add ligand-free serum to provide constant total amounts of serum in all assay tubes. The greatest problem with this latter approach is the difficulty in locating sufficient volumes of truly ligand-free serum. However, this approach can substantially improve assay reliability, especially parallelism between standards and unknowns. We have attempted to select conditions that minimize the effects of sample compositional differences. The efforts have included using EDTA as part of the assay buffer, using sufficient reaction time, optimizing for maximum sensitivity, which permits dilution of the sample, elevating the concentrations of nonimmune y-globulin and second antibody, and, in some cases, rejecting a specific lot of second antibody. The time required for completion of immunoprecipitation using low concentrations of reactants can require up to a week at 4 °. However, use of sufficient concentra3 E. A. Kabat, this volume [i].

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tions of nonimmune y-globulin can effectively reduce this to a few hours or less. Increased utilization of second antibody is the trade-off for shorter reactions. The following describes the assay conditions we have used for a large number of different ligands. For the reasons mentioned above, the conditions can be varied to meet individual requirements, but will be used here to simplify presentation. We usually employ, first, antibodies generated in rabbits and, second, antibodies generated in sheep (sheep anti-rabbit yglobulin = anti-RGG). Our standard sample volume has been set at 0.5 ml mainly because this permits a 5000-fold range in amount of added sample (0.1 tzl to 500/xl) without requiring a secondary dilution step. This is desirable because differential adsorptive losses to pipettes and containers can generate serious errors when sequential dilution procedures are used. The volumes for labeled ligand (0.1 ml) and first and second antibody (0.2 ml) were originally set because these volumes can be dispensed with high accuracy using a variety of pipetting instruments. The carrier nonimmune y-globulin is provided with the first antibody as 0.1 ml of 1 : 100 nonimmune rabbit serum. In some cases it is desirable to fractionate the serum to purify the y-globulin component. 4 However, this is rarely helpful, adds additional work, and removes inert proteins that are helpful in reducing nonspecific adsorption of some labeled ligands to vessel surfaces. The assay sample buffer we have used is 0.14 M sodium chloride with 10 mM phosphate, pH 7.0, phenol red at 10 mg/liter, and an inert protein, usually 0.1% gelatin. The protein is included in all samples to minimize adsorptive losses. Gelatin has proved to be most useful because it is free of most potentially cross-reactive proteins that occasionally contaminate some preparations (e.g., luteinizing hormone in crystalline bovine serum albumin); it is free of most small, nonproteinaceous molecules that occasionally contaminate other preparations (e.g., steroids in ovalbumin); it effectively reduces nonspecific adsorption; it is inexpensive; and it does not cause foaming or create problems with valves on some automatic pipetting equipment. The concentration of phosphate is low and could be increased or supplemented. In effect we accomplish this by including 50 mM EDTA in the buffer used for the first antibody. The phenol red is included to serve as an indicator of dangerous pH shifts upon addition of sample.

4 H. F. Deutsch, in "Methods in Immunology and Immunochemistry" (C. A. Williams and M. W. Chase, eds.), Vol. 1, p. 315. Academic Press, New York, 1967.

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Reaction Components, Order of Addition for Post-Precipitation 1. Sample buffer (0.14 M NaCI, 10 mM phosphate, pH 7.0, 10 mg of phenol red per liter, 0.1% w/v gelatin, 0.02% Merthiolate or 25 mM sodium azide) in volume such that sample addition gives 0.5 ml 2. Labeled ligand in sample buffer, 0.1 ml 3. Sample in sample buffer, 0-0.5 ml 4. Rabbit antiserum 1 : 100, diluted further in 1 : 100 nonimmune rabbit serum to give desired working concentration of antiserum from nothing to maximum of 1:100 in 0.2 ml volume. The nonimmune serum is diluted in sample buffer supplemented with 50 mM EDTA, pH 7.0. 5. Sheep anti-RGG, 0.2 ml, diluted to give an optimal concentration for maximal immunoprecipitation of the rabbit y-globulin. The anti-RGG is diluted in sample buffer without gelatin and phenol red but supplemented with bromophenol blue at 15 mg/liter. The latter serves solely as a visual aid to ensure that the second antibody has been added.

Reaction Conditions For most ligands, it is possible to work rapidly at room temperature and add the first four components in sequence keeping racks of tubes at 4 ° between additions. The assay tubes are vortexed before and after addition of the rabbit antiserum. The tubes are then covered and the reaction is allowed to proceed (usually at 4 °) for the desired time (5 min to several days). The second antibody is then added, and the tubes are again vortexed and incubated for an additional 4-24 hr at 4°. This reaction slows but does not fully terminate the primary ligand-antibody reaction. At the completion of the immunoprecipitation step, the reaction is terminated by addition of 3 ml of sample buffer at 4 ° and the tubes are centrifuged at 3000 g for 20-30 min. The supernatant is then removed by aspiration (a glass syringe and long needle work well) or, more conveniently, by decantation. Decantation is facilitated by using tubes with a diameter greater than 10 mm (we use 12 × 75 mm disposable glass or plastic tubes) and some device to hold the tubes in the centrifuge carrier. For some carriers, e.g., the International Equipment Corporation 26-tube carrier No. 1021, this can be accomplished by cementing a rubber gasket to the top surface and cutting holes in the gasket through which the tubes may be pushed. In most cases the 3 ml of buffer substitutes for a separate washing step. However, if nonspecific binding needs to be reduced, an additional 3 ml of cold buffer may be added, and the tube centrifuged and decanted (aspirated) again.

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Titration of Second Antibody By adding a series of increasing concentrations of second antibody to a set of tubes containing completed primary ligand reactions involving only labeled ligand, primary antibody, and carrier globulin, a concentration of second antibody suitable for the amount of included carder globulin can be determined. The minimal amount would be given by the lowest concentration of second antibody that results in precipitation of a maximal percentage of the labeled ligand. However, because of potential effects of variation in sample composition, it is better to use larger amounts. A safe amount may be calculated as three times the amount necessary to precipitate one-half the maximally precipitable radioactivity. Since the same concentration and lot of nonimmune y-globulin are used in most assays, the determined concentration of second antibody usually suffices for all assay systems using primary antibodies obtained from the same species. This is generally true except for assays in which the concentration of y-globulin in the primary antibody approaches that in the added carder globulin. This is particularly true when the primary antibody and the added globulin are provided as unfractionated sera, and the concentration of y-globulin in each is not known. In this case the hypergammaglobulinemia induced by immunization may require that greater amounts of second antibody be used. In all cases in which a high concentration of primary antibody is used, one may need to run an additional second antibody titration using the intended concentration of primary antibody.

Primary Antibody Pre-Precipitation Method The primary antibody pre-precipitation method entails precipitating the primary ligand specific antibody with second antibody prior to the addition of sample and labeled ligand. Thus, since the precipitation step occurs in the absence of sample, it can bc brought to completion without interference. After the immunoprccipitate has fully formed, the sample and then the labeled ligand may bc added. This method suffers from the effects of pre-prccipitation on the availability of reactive sites on the primary antibodies. Steric hindrance results in many sites becoming unavailable. Pre-precipitation thus lowers the apparent affinity and capacity of the primary antibody. We find that pre-precipitation techniques require from 5 to 10 times more antibody to bind the same amount of labeled ligand. As expected, the resulting sensitivity is also lower. The procedure also requires that the tubes be set up some time before the samples are added. This adds the problem of needing to know the

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number of samples to be assayed in advance. To avoid this problem, the pre-precipitation could be done in bulk, and the precipitate washed and stored for latter use. Although this can be done, difficulties in dispensing exact amounts of a suspension of primary antibody lead to relatively poor precision. The approach has little to commend it. Solid Phase Methods

In 1966 Wide and Porath 5 and Catt e t a l . 6 independently described the use of primary antibodies covalently coupled to a solid matrix. Although this general approach is still being used in many forms, there are a number of limitations. These include reduced assay sensitivity secondary to reduced affinity of the coupled antibody, reduced precision, and increased nonspecific effects of variation in sample composition. In 1969 Midgley e t a l . 7 noted that most of these problems and others associated with doubleantibody post-precipitation methods could be eliminated by covalently linking the second antibody to a solid matrix. The feasibility of this approach, sometimes termed double-antibody solid phase (DASP), was demonstrated by Karlberg e t a l . 8 and den Hollander and Schuurs 9 in 1971. With this approach compositional variation cannot affect the formation of a precipitate since the second antibody is already insoluble. Because carrier nonimmune y-globulin is not needed, less second antibody is required. Further, reactions may be stopped more rapidly because time does not have to be allowed for precipitate formation. The solid phase reagent can be added in excess, and little care in dispensing is required. In theory and in practice the procedure can result in a higher fraction of the labeled ligand being bound. Thus, since a l l the added second antibody is insoluble, the chance for loss of soluble complexes of primary and secondary antibody is less. A related derived advantage is that greater variation of sample composition can be tolerated. Thus, larger volumes of serum can be used with an effective increase in the ability to detect low concentrations of ligand. Among the relatively few limitations to the approach are the problems that relate to the properties of the chosen matrix. This matrix introduces another variable into the reaction: a surface that may bind substantial 5 L. Wide and J. Porath, Biochim. Biophys. Acta 130, 257 (1966). 8 K. J. Catt, H. D. NiaU, and G. W. Tregear, Biochem. J. 100, 316 (1966). 7 A. R. Midgley, Jr., R. W. Rebar, and G. D. Niswender, Acta Endocrinol. (Copenhagen) Suppl. 142, 247 (1969). a B. Karlberg, S. Almqvist, and S. Werner, Acta Endocrinol. (Copenhagen) 67,288 (1971). 9 F. C. den Hollander and A. H. W. M. Schuurs, in "Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 419. Livingstone, Edinburgh, 1971.

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quantities of some labeled ligands nonspecifically. Further, the chemical linkage of the second antibody to the solid matrix presents a potential new site of attack by a hydrolytic enzyme. Another consideration is the effort and chemical expertise required in preparing the coupled solid phase reagent. Although the procedures are not difficult, most published methods have involved use of cyanogen bromide, a hazardous compound that must be handled with considerable care. Once large batches are prepared, however, it is possible to store the suspension in a frozen state for at least a year with little loss in activity. TM Obtaining an appropriate particle size and density can also be a problem. Ideally one desires a solid matrix that can be easily dispensed, remain evenly in suspension throughout the pipetting and incubation procedures, and yet be easily separated by centrifugation or filtration. The particle size of a commercially available cyanogen bromide-activated support (Sepharose 4B; Pharmacia, Uppsala, Sweden), was found to be too large to be satisfactory, 11"~2but it may be possible to fragment the beads to obtain a more usable product. Thus, after coupling to second antibody, Wang et al. 12fragmented Sepharose 4B beads by vigorously stirring them for 24 hr at 4° at high speed with a magnetic stirrer. The resulting fragmented beads could be harvested and washed more efficiently than intact beads. Microcrystalline cellulose (Merck) x3 appears to have been used most successfully,9"1°'13but even this support matrix should be used with continuous mixing to maintain adequate suspension. The inclusion of a nonionic detergent in the incubation mixture (0.5% v/v Tween-201°'14; 0.1% v/v Brij-35 TM) helps to keep the particles dispersed and to reduce nonspecific binding. Other aproaches and matrices used with variable success have included conjugation to iron oxide particles coated with polymerized m-diaminobenzene, is adsorption to individual polystyrene balls 6.4 mm in diameter, TM and adsorption to polystyrene plastic tubes. 17

10 p. Koninckx, R. Bouillon, and P. De Moor, Acta Endocrinol. (Copenhagen) 81, 43 (1976). 11 j. M. Chesworth, Anal. Biochem. 80, 31 (1977). ~2 R. Wang, E. D. Sevier, R. A. Reisfeld, and G. S. David, J. lmmunol. Methods 18, 157 (1977). 13 p. O. Lundberg, J. Walinder, I. Werner, and L. Wide, Eur. J. Clin. Invest. 2, 150 (1972). 14 L. Wide, Acta Endocrinol. (Copenhagen) Suppl. 142, 207 (1969). 15 L. Nye, G. C. Forrest, H. Greenwood, J. S. Gardner, R. Jay, J. R. Roberts, and J. Landon, Clin. Chim. Acta 69, 387 (1976). le B. R. Ziola, M.-T. Matikainen, and A. Salmi, J. Immunol. Methods 17, 309 (1977). lr F. Cocola, A. R. Genazzani, and P. Neri, J. Nucl. Biol. Med. 17, 14 (1973).

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N o n i m m u n e Globulin Pre-Precipitation Method Nonimmune globulin pre-precipitation combines the advantages of post-precipitation and DASP and appears to be the procedure of choice. The approach involves simply mixing a large batch of unfractionated nonimmune serum (rabbit in our illustration) with a suitable amount of unfractionated second antibody (sheep anti-RGG in our illustration). After incubation the immunoprecipitate, composed solely of rabbit nonimmune 7-globulin and sheep anti-RGG ~/-globulin, is washed, and stored for later use. Although many of the reactive sites on the sheep anti-RGG molecules are lost in forming the precipitate, enough are still available that the suspension may be added directly to any primary reaction to separate labeled ligand bound to antibody from that which is flee. Since the primary reaction can be conducted with all soluble reagents, and the suspension can be added in excess, high sensitivity, precision, and accuracy of the assay are maintained. By adding sufficiently large amounts of the RGG-anti-RGG precipitate, an assay may be terminated in a few minutes. The method is associated with low nonspecific binding. As with DASP procedures, it gives higher specific binding and, as a consequence, shows sensitivity equal to or greater than that given by postprecipitationmethods. Because carrier globulin does not need to be added to the primary antibody, the method consumes less second antibody and is consequently more economical. The washed RGG-anti-RGG precipitate stays in suspension with minimal agitation, can be separated easily by centrifugation or filtration, and is free of cross-reacting contaminants that might otherwise be included in serum. The washed precipitate may be stored frozen with little loss of activity for at least several months. The method has few disadvantages other than that it cannot be used easily with high concentrations or amounts of primary antibody. As with all double-antibody methods, it is species specific.

Preparation of RGG-anti-RGG Precipitate All steps are at 4 °. To 10 ml of normal rabbit serum in 1 liter of sample buffer minus phenol red and gelatin, add the amount of undiluted sheep anti-RGG that is necessary to precipitate maximally the RGG in the rabbit serum (this will depend on the titer of anti-RGG and often consists of 75150 ml). Mix and incubate overnight. Centrifuge at 1000 g for 20 min, and wash once by resuspending in 1 liter of the same buffer followed by recentrifugation. Resuspend the precipitate in 200 ml of the same buffer supplemented with 50 mM EDTA and 10 mg of bromophenol blue per liter. Allow the suspension to sit at 4 ° overnight (this facilitates final disper-

274

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[17]

sion). The suspension is then dispersed thoroughly by sonication (less than 2 min) or by repeated pipetting.The suspension can, as desired, be diluted further with the EDTA-bromophenol blue buffer, divided into aliquots, rapidly frozen in a bath of solid carbon dioxide and ethanol, and stored at or below - 2 0 ° . For use, snap thaw by agitation in luke warm water. The thawed suspension may be stored at 4° for several weeks, but should not be refrozen. Utilization of R G G - a n t i - R G G Precipitate The primary antibody should be diluted with 0.1% w / v gelatin substituted for carder globulin. Otherwise the assay is conducted as described above for post-precipitation procedures, steps 1-4. To terminate the assay, add suitable aliquots of the RGG-anti-RGG suspension, taking care that the suspension is thoroughly mixed and remains so while aliquots are removed. This may be accomplished by keeping a rotating magnetic stirrer bar in the suspension. The time for the subsequent incubation will depend upon the amount of added suspension relative to the concentration of the T-globulin in the primary antibody and may vary from a few minutes to overnight.

[17] U s e o f C h a r c o a l t o S e p a r a t e A n t i b o d y C o m p l e x e s from Free Ligand in Radioimmunoassay By WILLIAM D. ODELL

Charcoal is widely used to separate antibody-bound ligand (bound) from non-antibody-bound ligand (free) in competitive protein binding assays. Its use was originally described by Miller for vitamin Blz assay 1 and later by Herbert et al. for B12.2 In 1965, Herbert suggested its use to separate bound and free in the radioimmunoassay of insulin. 3 Since then it has been used for a large number of radioimmunoassays and radioreceptor assays. Use of charcoal to separate antibody complexes from free ligand is dependent on the existence of rather large size-charge differences in the two moieties. Charcoal is an absorbent, without specific recognition sites (such as antibodies have). However, it has irregular surfaces, which adi O. N. Miller, Arch. Biochem. Biophys. 68, 255 (1957). 2 V. Herbert, C. Gottlieb, K. S. Lau, and L. R. Wasserman, Lancet 2, 1017 (1964). a V. Herbert, K. L. Lau, C. W. Gottlieb, and S. J. Bleieher, J. Clin. Endocrinol. Metab. 25, 1375 (1965).

METHODS IN ENZYMOLOOY, VOL. 70

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[17]

sion). The suspension is then dispersed thoroughly by sonication (less than 2 min) or by repeated pipetting.The suspension can, as desired, be diluted further with the EDTA-bromophenol blue buffer, divided into aliquots, rapidly frozen in a bath of solid carbon dioxide and ethanol, and stored at or below - 2 0 ° . For use, snap thaw by agitation in luke warm water. The thawed suspension may be stored at 4° for several weeks, but should not be refrozen. Utilization of R G G - a n t i - R G G Precipitate The primary antibody should be diluted with 0.1% w / v gelatin substituted for carder globulin. Otherwise the assay is conducted as described above for post-precipitation procedures, steps 1-4. To terminate the assay, add suitable aliquots of the RGG-anti-RGG suspension, taking care that the suspension is thoroughly mixed and remains so while aliquots are removed. This may be accomplished by keeping a rotating magnetic stirrer bar in the suspension. The time for the subsequent incubation will depend upon the amount of added suspension relative to the concentration of the T-globulin in the primary antibody and may vary from a few minutes to overnight.

[17] U s e o f C h a r c o a l t o S e p a r a t e A n t i b o d y C o m p l e x e s from Free Ligand in Radioimmunoassay By WILLIAM D. ODELL

Charcoal is widely used to separate antibody-bound ligand (bound) from non-antibody-bound ligand (free) in competitive protein binding assays. Its use was originally described by Miller for vitamin Blz assay 1 and later by Herbert et al. for B12.2 In 1965, Herbert suggested its use to separate bound and free in the radioimmunoassay of insulin. 3 Since then it has been used for a large number of radioimmunoassays and radioreceptor assays. Use of charcoal to separate antibody complexes from free ligand is dependent on the existence of rather large size-charge differences in the two moieties. Charcoal is an absorbent, without specific recognition sites (such as antibodies have). However, it has irregular surfaces, which adi O. N. Miller, Arch. Biochem. Biophys. 68, 255 (1957). 2 V. Herbert, C. Gottlieb, K. S. Lau, and L. R. Wasserman, Lancet 2, 1017 (1964). a V. Herbert, K. L. Lau, C. W. Gottlieb, and S. J. Bleieher, J. Clin. Endocrinol. Metab. 25, 1375 (1965).

METHODS IN ENZYMOLOOY, VOL. 70

Copyright © 1960by Academic Press, Inc. All rightsof reproduction in any formreserved. ISBN 0-12-181970-1

[17]

CHARCOAL SEPARATION OF ANTIBODY COMPLEXES

275

sorb charged molecules. Since the sizes of the in-pocketings of charcoal (or any adsorbent) vary, the amount of surface area accessible to a given protein varies depending on the size, shape, and charge of that protein. Charcoal can be used effectively if the ligand is small relative to the antibody-ligand complex. For large ligands equaling or exceeding immunoglobulins in size, the size-charges differences between antibody-ligand complexes and the ligand are too small to permit charcoal to be used, As extreme examples, charcoal is useful for separation of free steroid or small peptides from antibody-steroid or antibody-peptide complexes, but it is not useful for separating free virus from antibody-virus complexes. In addition, use of charcoal depends on recognizing that sufficient charcoal will bind antibody-ligand complexes as well as ligand; i.e., that dose-response relations exist for dose (or amount) of charcoal versus percentage of both antibody-bound and free ligand complexes. One must always, and for each ligand-antibody system of interest, study the full dose-response relations prior to selecting an amount of charcoal. Variation of protein content in the medium bathing the antibody complexes and ligand results in varying possibilities of competition for the charcoal surface. Thus, in general, with the amount of charcoal held constant, increasing protein content decreases the charcoal binding of both free ligand and antibody-ligand complexes. Last, it is important to dispense with the concept of molecular sieving by dextran coating of charcoal. Gottlieb e t al. 4 suggested that by selecting the appropriate dextran (Sephadex), to coat the charcoal one could produce a reagent that selectively bound small molecules and failed to bind larger ones; it was hypothesized that a Sephadex molecular sieve had been produced. In extensive studies by Binoux and Odell in 1973,5 it was shown that Sephadex coating does not produce such a selective reagent. Sephadex does shift charcoal dose-response curves to the right (see Fig. 1); it also makes charcoal stickier, permitting easier centrifugation into a pellet, but it does not limit access to charcoal based on molecular size. Estimating Charcoal Dose-l~esponse Curves The amount of charcoal required is different for separating different ligand- and antibody-ligand complexes. The amount of charcoal required also varies with protein content of the solutions being separated. For these reasons it is important to develop full charcoal dose-response 4 C. Gottlieb, K. S. Lau, L. R. Wasserman, and V. Herbert, Blood 25, 875 (1965). 5 M. A. Binoux and W. D. Ode[l, J. Clin. Endocrinol. Metab. 36, 303 (1973).

276

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[17]

I00

~

...J < 8o o o T 0

i/,

FREELIGAND/

/we

60

0 1--

iiii

o40 z o m20

,,

/ AJ~NTIBODYBOUNDLIGAND :._----

'

+ +

'++'

,"

. . . . .

'"o4

'+,4+'%+

CHARCOAL CONCENTRATION ~ g / t u b e ( Log scole ) FIG. 1. Dose-response curve for amount of dextran-coated charcoal added versus percentage free and bound, adsorbed. These are theoretical curves drawn from generalizations

derived from several assays. - - , Curve showing binding of free ligand; ---, curve showing binding of antibody bound ligand. The exact amounts of charcoal giving a particular bound value vary with each assay (e.g., growth hormone assay differs from insulin assay). For significance of arrows A and B, see the section on selection of charcoal dose.

curves for the particular assay and conditions selected prior to choosing the amount of charcoal to be used. Once selected, conditions of assay, including protein content, must be kept constant. For example, immunoassay tubes containing reference preparations or standards must contain an identical kind and amount of protein as unknown samples. "Charcoal stripped" plasma or serum is not identical to plasma or serum being assayed, for charcoal stripping removes many highly charged small molecules that effectively influence subsequent charcoal-binding of antibodyligand complexes and of free ligand. However, for some assays if "carefully" assessed, it may be satisfactory. Better choices included plasma or serum from patients or animals rendered free of the hormone in question or plasma or serum freed of hormone by immunoadsorption. To develop charcoal dose-response curves, we suggest routinely using dextran-coated charcoal because it is stickier than uncoated charcoal, thus forming a distinct and firm pellet on centrifugation. Which size dextran is used to coat is unimportant, but once selected, continue to use Sephadex of that size. The charcoal is placed in buffer (10: 1, v/w) (10 mM

[17]

CHARCOAL SEPARATION OF ANTIBODY COMPLEXES

277

PO4, 0.15 M NaCI, pH 7.4) and stirred for 1-2 hr. This suspension is centrifuged at a low speed (500 g) for 10 min and the supernatant buffer is decanted to remove fine particles of charcoal. The charcoal is resuspended in a second aliquot of buffer, stirred again for 1-2 hr, and recentrifuged; the supernatant is discarded again. Next, the charcoal is mixed with an equal amount of dextran (e.g., Sephadex G-10) and suspended in buffer to form a final concentration of 10% charcoal. Prior to addition, the dextran is swollen in buffer. This mixture is placed on an automatic magnetic stirrer, when pipetting and dispensing is to be done. It may be prepared in large quantities and stored in sealed bottles until used. A series of tubes containing radiolabeled ligand (without antibody present) are prepared in assay buffer containing all reagents to be used in the final assay except the antiserum. Thus for steroid assays, which generally employ extracts of serum or plasma dissolved in buffer, labeled ligand in buffer suffices. For peptide or protein radioimmunoassays, tubes containing labeled hormone in ligand-free plasma or serum are prepared. If the ligand is species specific (e.g., thyrotropin, growth hormone), serum or plasma from a different species may be used as a source of ligand free material. If the ligand is not completely species specific (e.g., thyroxine or insulin), serum or plasma from thyroidectomized or triiodothyronine-suppressed animals or depancreatectomized animals could be used. For ligand that are ubiquitous, and if no source of ligand-free serum or plasma is known, samples rendered ligand-free by purification on immunoabsorbent columns might also be used. A second series of tubes are prepared containing labeled ligand and excess antibody. 6 As for the tubes not containing antibody, the plasma, serum, or buffer content must be identical to the content that would be present an assay containing unknown samples--i.e., samples to be quantified. The charcoal-dextran suspension is pipetted into the two series of tubes in increasing amounts (volumes) to give a final charcoal amount in each assay tube ranging from 1 /~g per tube (0.001%) to 50 mg per tube (5%). The tubes are allowed to stand for 15 min then centrifuged at about 500 g for 10 min. The supernatants are decanted into separate tubes for counting (if desired), or the tubes containing the charcoal pellets are counted. Since all tubes contain the same total counts, counting of either fraction is satisfactory. Generally, counting the charcoal pellets is easiest; if this is done, the supernatant can be removed either by decanting or aspiration. If decanting is performed, one ensures that the mouth of each 6 Excess antibody is that amount that will bind all (>90%) of an immunocompetent (nondamaged) labeled ligand.

278

[17]

RADIOIMMUNOASSAYS AND IMMUNORAD1OMETRIC ASSAYS

FREE L I G A N D IO0-

/

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NOSERUM--/" ##

.J

o 80-

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/

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/

/

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I

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i

)!;/,.,"_/ • ,! l

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/

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"/ # / .os,.o~/ / II I,o.,.sE,u,4--i

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ANTIBODYBOUND LIGANO

i

/

,

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. . . . . . . . L i

i i

i i

i i

i i

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, i

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CHARCOAL CONCENTRATION Ng/tube (Log scale) FiG. 2. Effects of adding varying amounts of serum (as protein source) in the charcoal separation of bound and free.

tube is touched to absorbent paper to remove as much supernatant as possible. If aspiration is performed, a continual suction device with a fine glass tip (e.g., a Pasteur pipette) may be used, and care is used not to aspirate any charcoal. Data are calculated and plotted as in Fig. 1. One finds that (a) increasing amounts of charcoal bind increasing amounts of both free ligand and antibody-bound ligand; (b) an amount of charcoal (indicated by arrow) can be selected that binds all free ligand and little of the antibody-bound ligand. This would be the amount of charcoal selected for assay purposes if all assay tubes were to contain buffer only (e.g., many steroid assays). Figure 2 shows the effect of adding protein to assay tubes (in the form of ligand-free serum in this example). Each dose-response curve is shifted to the right. The amount of charcoal selected for assay purposes in protein-poor buffer (Fig. 2) is indicated again, to show that this amount of charcoal is incorrect for the same immunoassay when protein content is changed. For several specific examples see our previous studies. 5,7. 7 W. D. Odell, C. Silver, and P. K. Grover, in "Steroid Immunoassay" (E. H. D. Cameron, S. G. Hillus, and K. Griffiths, eds.), p. 207. Alpha Omega Publ., Cardiff, Wales, 1975. a p. K. Grover and W. D. Odell, J. Steroid Biochem. 6, 1373 (1975).

[17]

CHARCOAL SEPARATION O F A N T I B O D Y COMPLEXES

279

Selection of Charcoal Dose and Method of Use Once the detailed data illustrated schematically in Figs. 1 and 2 are collected, an amount of dextran-coated charcoal is selected that binds all of the free ligand and little of the antibody-bound ligand. This amount is selected, if possible, to lie on plateaus of the charcoal dose-response curves, so that errors in pipetting charcoal will produce little effect on the amount of labeled ligand bound, i.e., arrow (A) in Fig. 1. The amount of charcoal indicated by arrow (B) is less satisfactory, since an error in pipetting on the low side would decrease bound ligand and be erroneously " r e a d " or interpreted as hormone or ligand in the assay tube. Once this amount of charcoal is selected, then time-response curves are constructed. To do this, a series of assay tubes containing all ingredients except the antibody are prepared as before. A second series is prepared containing excess antiserum as before. After incubation to permit antibody binding of ligand, charcoal is added at the following time intervals to centrifugation in a refrigerated centrifuge: 120 min, 60 min, 45 min, 30 min, 20 min, 5 min, and 0 rain. All tubes are then centrifuged for 10 min at 500 g, the supernatants are aspirated, and counts bound to charcoal are plotted against time. For assay conditions the time charcoal is permitted to be in the tube is determined by selecting the time interval when variations in time would produce the least error--that is, the time that is on a plateau of a time-response curve. This is done because for large assays the time charcoal is present in the first and last tubes (e.g., tubes 1 and 500) will vary greatly. It will be possible to add charcoal to all the tubes of an asay at " o n e " time only, if the time of addition to all tubes is short, relative to consideration of the " t i m e " plateau on a graphic plot of time versus amount bound. Thus, if the amount of ligand bound increases between 0 and l0 min and is constant thereafter for 90 min, charcoal would be left in tubes for 15-75 min prior to centrifugation. Once conditions for assay and amount of charcoal are selected, they must be kept constant. If conditions are changed (e.g., larger volumes of serum assayed) the charcoal dose must be reselected. Last, as a precaution, we recommend recharacterizationof these data at least once each 6 months to assure proper use. It is important to note that errors in separation of antibody-bound from free ligand are interpreted in the assay as hormone being measured in the assay tube. Such errors in separation can even show parallel dose-response curves between unknown sample and reference preparation.

280

R A D I O I M M U N O A S S A Y S A N D I M M U N O R A D I O M E T R I C ASSAYS

[18]

[18] A m m o n i u m Sulfate and Polyethylene Glycol as Reagents to Separate Antigen from Antigen-Antibody Complexes

By T. CHARD Separation of free antigen from antigen-antibody complexes is a universal procedure in radioimmunoassay and related techniques and is necessary to determine the distribution of the antigen between the bound and the free forms. It requires that the free fraction be physically separated from the bound fraction, and a variety of techniques have been developed for this purpose, all of which depend on physicochemical differences between the two forms. For example, both ammonium sulfate ~ and polyethylene glycol z'a can be used at concentrations that will precipitate the antibody molecules and thus the antigen-antibody complex, but will not precipitate the free antigen. Before considering the details of the use of these materials, it is worthwhile to review briefly the two most important operational criteria for a separation procedure: efficiency and practicality. Operational Criteria

Efficiency of a Separation Method The efficiency of a separation procedure in radioimmunoassay can be defined as the completeness with which the free and bound fractions are separated. This concept is illustrated diagrammatically in Fig. 1.4 A perfect separation system would totally divide the two phases. In reality this is never achieved because of deficiencies in the separation system, on the one hand, and of the primary reagents, on the other hand. Invariably, some part of the free antigen behaves identically with the bound fraction, even when there is no antibody present. This misclassification of free as bound is usually referred to as the "diluent" or "assay T. Chard, M. J. Martin, and J. Landon, in " R a d i o i m m u n o a s s a y M e t h o d s " (K. E. Kirkham and W. M. Hunter, eds.), p. 257. Churchill-Livingstone, Edinburgh, 1971. 2 B. Desbuquois and G. D. Aurbach, J. Clin. Endocrinol. Metab. 33, 732 (1971). 3 A. E. Leek, C. F. Ruoss, M. J. Kitau, and T. Chard, Br. J. Obstet. Gynaecol. 82, 669 (1975). 4 T. Chard, " A n Introduction to Radioimmunoassay and Related Techniques.'" North-Holland Publ., Amsterdam, 1978.

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181970-1

[18]

SEPARATION OF BOUND AND FREE ANTIGEN

10,~

281

t Nonreacti ve tracer ligand

6~

Counts

Tracer ligand reacting in the assay

20~

Assay blank

0~.~ FIG. 1. Definitionof the efficiency of a separation procedure in a radioimmunoassy. In a perfect assay all the labeled antigen would distribute between the free and bound fractions. In practice, some of the free is classified as bound (assay blank, nonspecific binding), and some of the free will be nonimmunoreactive. The greater the total amount of tracer ligand that reacts in the system, the better the assay. From Chard.' b l a n k " or as "nonspecific binding." For example, chemical precipitation o f the type described here will inevitably precipitate some of the free antigen in the absence of antibody, despite the fact that the antigen should be fully soluble under the chosen conditions. There are a number of possible explanations for the existence of this " b l a n k " value: (a) physical entrapment of the free antigen in the interstices of the p r e c i p i t a t e - - t h i s can be quantitated and sometimes eliminated by repeated washing of the precipitate or by observing the distribution of an isotope (such as 2ZNa) that appears only in the liquid phase; (b) the existence in the labeled antigen o f contaminants whose chemical behavior is similar to that of the a n t i g e n - a n t i b o d y complex: blank values are often reduced by careful attention to the purification of the tracer (Fig. 2); (c) absorption of the free antigen to the walls of the tube in which the assay is performed; and (d) failure to achieve complete separation of the bound and free phases because the characteristics of the free antigen are similar but not identical to those of the a n t i g e n - a n t i b o d y complex (see Fig. 7). In practice it may be difficult to determine which of these processes is responsible for the occurrence o f the assay blank. More important, the actual value o f the blank may vary according to the materials present in the incubation medium, and the presence or absence of serum can have a substantial effect. The type of problem that can arise because o f differences between blank values in samples and standards is illustrated in Fig. 3.

282

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[18]

8000

7000 6000

5000

lOO

e'o, Counts per

4000

8o

3000

60

second

% of counts in precipitate

",/"5 ',,

< \':

2000

~0

2O

1000

8

10

12

14

16

18

Eluate volume (ml)

FIG. 2. Chromatography of iodinated human pregnancy-specific/31-glycoprotein(PS/31G) on a 40 x 1 cm Sephadex G-100 column eluted with 50 mM phosphate buffer, pH 7.5. Binding of the tracer in the presence and in the absence of an antiserum to PSflIG is shown by the dashed lines, the separation procedure being addition of polyethylene glycol as described in Table II. Earlier fractions, containing aggregated ~=H-labeledPS/3tG, give an unacceptably high assay blank value. Selection of the later fractions yields material with an acceptable blank and also shows the best distinction between blank and zero standard. From data kindly supplied by A. T. Al-Ani. T h e second p r o b l e m is failure to achieve c o m p l e t e precipitation of the b o u n d complex. In theory, 100% o f the tracer antigen should be bound in the p r e s e n c e o f an excess o f antibody, but this o p t i m u m is rarely achieved and it is not u n c o m m o n to find values o f 70% or less. There are a n u m b e r of possible explanations of this p h e n o m e n o n . 1. A proportion o f the antibody, and therefore o f the a n t i g e n - a n t i b o d y c o m p l e x , m a y b e h a v e similarly to the free antigen: in other words, precipitation is incomplete. 2. Impurities m a y exist in the tracer antigen that fail to react with the antibody and thus a p p e a r in the free fraction (for e x a m p l e , free 12H would b e h a v e in this way). 3. The separation p r o c e d u r e m a y lead to dissociation o f the a n t i g e n antibody complex. A good e x a m p l e o f this is seen with separation s y s t e m s in which the free antigen is a b s o r b e d onto charcoal; the

[18]

SEPARATION OF BOUND AND FREE ANTIGEN

283

100

80 anctmdreadingof sample (2) true readingof sample(4)

60 % tracer bound

40

20 blamkvadueof ml~lLdO blank vaduoof mmdards 1

2

4

8

16

32

Concentration of Standard

FIG. 3. A diagram to show the effect of a variable blank value on a radioimmunoassay. If the blank for the sample is higher than that of the standard (which might be the case if the former is serum and the latter is in aqueous buffer), the result will be artifactually low (2 instead of 4). This type of problem does not occur if the composition of sample and standard is identical. From Chard. 4

charcoal can "compete" with the antibody and effectively "strip" antigen from the bound complex (Fig. 4). In terms of overall efficiency, chemical precipitation of the bound fraction with ammonium sulfate or polyethylene glycol is often less satisfactory than systems such as second antibody. In particular, they tend to yield high assay blank values. However, for many routine assays this defect is compensated by their high level of practicality.

Practicality of a Separation Method Five characteristics of a separation method determine its practicality: speed, simplicity, applicability, reproducibility, and cost. Speed is important because, with assays intended for clinical use, a result available to the clinician on the day that the sample is taken is of greater value than the same result reported the following day; estimation of a cardioactive drug

284

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[18]

70 60 50

'.'\

%'km ~ \.

40 z

30 20

'%.

\\..

~\'b.x \ ~ , "°.....°

10 n

~

n

"'°~''o~

~.,~ ~ m °u

|

1:100 1:400 1:1600 1:6400 1:25600 ANTIBODYDILUTION FIG. 4. The effect of different separation procedures on the apparent titer of two different antisera to oxytocin (A and B), using mI-labeled oxytocin. A - - & antiserum A, ammonium sulfate; A ...... A antiserum B, ammonium sulfate; • ...... • antiserum A, dextrancoated charcoal; [] ..... [] antiserum B, dextran-coated charcoal. From Chard?

such as digoxin is a good example. However, most common separation procedures, including precipitation with ammonium sulfate or polyethylene glycol, take much less time than the incubation of the primary reagents and add little to the time of the assay as a whole. Simplicity is a key factor because it may determine the total number of samples that a technician can process in a single day. For example, a separation requiring detailed handling of every tube, such as application to a chromatographic column or an electrophoretic strip, will severely limit sample throughput. In addition, there is a direct relation between the complexity of a technique and its reproducibility. Chemical precipitation scores high on simplicity, since it involves the simple addition of one reagent, followed by mixing, centrifugation, and counting of the precipitate or supernatant. Applicability--the ability to apply a single procedure to a wide variety of different assays--is desirable but not essential. Some procedures, such as second antibody and solid-phase antibody, are universal. Precipitation with ammonium sulfate or polyethylene glycol is not, because in some cases the properties of the antigen in the bound and free phases are not sufficiently distinct. Reproducibility is highly important, and two aspects should be considered. The first is batch-to-batch variation of the separating agent used, which may be very poor with some systems (e.g., charcoal, coated tubes),

[18]

S E P A R A T I O NOF BOUND AND FREE ANTIGEN

285

8o

60 % tracer bound 40

20

I

I

i

i

i

1

2

3

4

5

Amount of separating agent added

FIG. 5. Diagram to show how precision can be affected by a separation procedure. In system~B!variationsin the amount of separating agent added will have a considerable effect on the observed distribution of free and bound antigen. In system A, there is a long plateau at which the amount of separating agent is noncritical, and even large errors will not affect the result. Systems of type A are much to be preferred. See Chard. 4 but in general is excellent with both a m m o n i u m sulfate and polyethylene glycol. The second is the potential for e r r o r in the technical execution of the procedure; methods in which the exact amount of separating agent is highly critical are obviously less satisfactory than those in which it is not (Fig. 5). The costs of any r a d i o i m m u n o a s s a y system lie largely in labor; reagents and capital equipment m a k e only a small contribution. H o w e v e r , costs o f reagents should always be taken into account, and in the case of the separation s y s t e m can vary widely. Chemical precipitations are the c h e a p e s t of a l l - - a fraction o f a cent per tube. The m o s t e x p e n s i v e syst e m s are those that are labor intensive (e.g., electrophoresis) or that require considerable effort for the original preparation of reagents (e.g., solid-phase systems). C u r r e n t S e p a r a t i o n M e t h o d s U s e d in R a d i o i m m u n o a s s a y The principal methods are listed in Table I.

286

RADIO1MMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[18]

TABLE I COMMON SEPARATION PROCEDURES USED IN RADIOIMMUNOASSAY a

Electrophoresis (starch gel, cellulose acetate, polyacrylamide gel) Gel filtration (column or batch) Adsorption (charcoal, silicates, hydroxyapatite) Fractional precipitation (ethanol, dioxane, polyethylene glycol, sodium sulfite, ammonium sulfate, tfichloroacetic acid) Second-antibody precipitation (soluble and solid phase) -Solid-phase antibody (particles, disks, tubes, gel entrapment, polymerized antibody) a From Chard.*

Fractional Precipitation Methods Precipitation of proteins with organic solvents or salts was one of the earliest methods for the separation of free and bound antigen in radioimmunoassays and related systems. The use of water-soluble nonionic polymers in protein fractionation has been reviewed by Fried and Chun. ~ The likely mechanism by which fractional precipitation serves to separate protein molecules is reduction of the amount of " f r e e " water in the system--in other words, the water that normally forms a shell around a dissolved molecule and thus keeps it in solution (Fig. 6). Ammonium sulfate, for example, can bind water, which then is not available to form a hydration shell for other molecules. The solubility of a protein at any given concentration of the separating agent is then dependent on its own ability to attract water molecules. With most proteins and other biological materials this is determined by electrostatic charge and, in turn, depends on the isoelectric point (pI) of the material and the pH of the medium. The greater the difference between pl and pH, the greater is the net charge on the molecule and the greater its ability to form a water shell. Radioimmunoassays are usually conducted at or around neutral pH. When a hydrophilic material such as ammonium sulfate or polyethylene glycol is added it will first precipitate substances with a pl near neutrality. As the concentration is increased it will progressively precipitate other materials in the order of their pls. The result is a fractionation very much like that achieved by electrophoresis at the same pH; the slowest running molecules (those with little net charge) are precipitated first. Relatively hydrophobic molecules, such as unconjugated steroid hormones and certain drugs, remain soluble even at very high concentrations of a separating agent. However, the fractionation will never be quite identical to that of electrophoresis because it depends on the total number of charged groups, as opposed to the balance between charged groups, and may also 5 M. Fried and P. W. Chun, this series, Vol. 22, p. 238.

[18]

SEPARATION OF BOUND AND FREE ANTIGEN

287

0 o ~ O

Biological molecule

~(~ 0

Water molecules

00@~ O

)

Molecule of separatingagent

u

FIG. 6. The probable mechanism by which ammonium sulfate or polyethylene glycol precipitates an antigen-antibody complex. The salts take up water molecules, which are then not available to provide a hydration shell for the protein. See Chard. 4

~

BO 70 60

of °

~' Insulin

50 %BOUND

l

i

Blank

/

40

l

;

i//

30

i ACTHI / B,ank/

20

il.~ I

/ '~ ~

] /

/21/ . E A m , tI

10

Oxymcin Blank

,. f .

.

I 0.5

.

.

I 1.0

I 1.5

I 2.0

FINALCONCENTRATION OFAMMONIUMSULFATE( M )

I 2.5

FIG. 7. An experiment showing the efficiency of fractional precipitation by ammonium sulfate in assays for oxytocin ( 0 - - ( 3 , A - - A ) , ACTH ((3---(3, A___A) or insulin ((3---(3, A---&). With oxytocin there is a substantial gap between the concentration of ammonium sulfate required to precipitate bound antigen and the concentration that will precipitate free antigen. By contrast, with insulin there is virtually no gap and the procedure would not be suitable for an assay. From Chard. 4

288

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[18]

depend to some extent on nonelectrostatic interactions. Contrary to the views of some workers, the fractionation is not closely dependent on the molecular weight of the antigen; polythylene glycol, for example, is highly efficient in the separation of antibody-bound and free a-fetoprotein, which has a molecular weight of 60,000-70,000. Antibody molecules are y-globulins that have only a small charge at neutral pH and therefore can be precipitated by relatively low concentrations of reagents, such as sodium sulfate (the first material of this type to be used in a radioimmunoassay system6), ammonium sulfate, ethanol, dioxane, and polyethylene glycol. The mechanisms described above seem to apply to most of the salts used in protein fractionation. Nevertheless, there are discrepancies, and whether or not a given reagent can be used for separation has to be determined by experiment. The approach to this is illustrated in Fig. 7, and the primary criterion is that the bound complex should be precipitated at concentrations that do not cause significant precipitation of the free antigen. Experience with a wide variety of antigens has now shown that the use of high molecular weight polyethylene glycol (MW 6000) is almost invariably superior to the use of ammonium sulfate. Thus, to take the example shown in Fig. 7, polyethylene glycol can be used very effectively in the separation of free and bound ACTH, whereas with ammonium sulfate there is significant precipitation of the free fraction at the concentrations required to render the bound fraction insoluble. A feature of some chemical precipitation of the bound fraction (including ammonium sulfate and polyethylene glycol, but not ethanol) is that the antigen-antibody reaction will continue in the presence of the reagent-that is to say, with the antibody in the precipitated form. This phenomenon can be used to design a system in which both the antibody and the separating agent are added together, thus eliminating one pipetting step. A protocol for the use of either ammonium sulfate or polyethylene glycol in the separation of free and bound antigen in a radioimmunoassay is given in Table II. This protocol can be considered as virtually universal for all those antigens for which the method has been shown to be appropriate, i.e., if it satisfies the criteria of the type of experiment shown in Fig. 7.

Chemical Precipitation Combined with Second Antibody It has been shown by several workers e.g.,7,8 that the incorporation of a small amount of ammonium sulfate or polyethylene glycol has many ad6 G. M. Grodsky and P. H. Forsham, J. Clin. Invest. 39, 1070 (1960). 7 M. J. Martin and J. Landon, in "'Radioimmunoassay in Clinical Biochemistry" (C. A. Pasternak, ed.), p. 269. Heyden, London, 1975. a M. A. Peterson and R. S. Swerdloff, Clin. Chem. 25, 1239 (1979).

TABLE II SEPARATION OF ANTIBODY AND FREE ANTIGEN USING POLYETHYLENE GLYCOL OR AMMONIUM SULFATE Polyethylene glycol solution": 20% (w/v) solution of polyethylene gylcol 6000 in phosphate buffer (0.05 M, pH 7.5) Ammonium sulfate solutiona: 4 M solution in distilled water, adjusted to pH 7.5 with NaOH 1. Prepare separating agent by mixing on a magnetic stirrer. Up to 30 min may be required for complete solution to occur. 2. Add 2 volumes of polyethylene glycol or 1 volume of ammonium sulfate to each incubation tube (a repeating syringe is particularly convenient for this), b 3. Mix carefully on a vortex mixer to yield a homogeneous solution. 4. Centrifuge for 30 min at 1500 g or greater. The temperature control is not essential, though results may be marginally improved at 4°. 5. Decant or aspirate the supernatant. Aspiration, using a Pasteur pipette attached to a simple Venturi pump, is normally preferable unless very wide-base tubes are used. 6. Count the precipitate by placing the tube in the well crystal of a gamma counter. a Both polyethylene glycol and ammonium sulfate can be obtained through the catalog of any supplier of general laboratory chemicals. b Note that precipitation will occur only if there are y-globulins present in the incubation mixture (e.g., with serum samples or standards prepared in serum). If there is no y-globulin present (e.g., urine samples, standards prepared in buffer alone), then a separate source should be added such as 10% (v/v) serum or purified y-globulins to yield a final concentration of 1 mg/ml. Some workers have.used a premixed ~uspension of y-globulin in polyethylene glycol. 2

60 50 40

30 (..)

20

v~

10

0

assay diluent

3.1% Dextran 40

1.2M (NH4)2SO4

FIG. 8. The acceleration of second antibody separation in an ACTH assay using ammonium sulfate (1.2 M) or Dextran 40 (3.1%). Bound ACTH was separated after 15 rain of incubation at 4 ° with a donkey anti-rabbit serum at a dilution of 1 : 200 and carrier rabbit serum. From Martin and Landon. r

290

RAD101MMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[18]

60 ~...-~_

~

:

b I

4O

I

l

~" 30 .¢:

l l

L/_

l l l l

k\ , 1:100

1:400

• ~°"'~"o'-~--o-T--o 1:?00

final dilution of second antibody

,

1:101]0 (K8135)

Fl6. 9. The effect of ammonium sulfate (1.2 M) on second antibody separation of the bound fraction in a radioimmunoassay for the fibrinogen degradation product, fragment E (FgE). In the presence of ammonium sulfate, complete precipitation of the bound fraction occurs at considerably reduced concentrations of second antibody despite a much shorter incubation period O - - O , 30 min, 1.2 M (NI-h)2SO4; O---O, 24 hr, no (NI-I~)sSO4. From Martin and Landon. 7

vantages in a second antibody separation. The incubation time can be reduced (Fig. 8); the concentration of second antibody can be reduced (with considerable saving in costs) (Fig. 9); and antibodies previously rejected because of their poor precipitation characteristics can be used successfully. Presumably all of these are due to enhancement of micelle formation in the presence of even low concentrations of the salt. Conclusions There is no such thing as a perfect separation method for use in a radioimmunoassay. All techniques represent some sort of compromise between efficiency and practicality. The major problem of fractional precipitation methods, including the use of ammonium sulfate and polyethylene glycol, is that they tend to yield high assay blank values, in the range 5-15%. The lower values are found with haptens such as thyroxine; the higher values, with some of the protein hormones. Whether or not this is important depends very much on the overall design of the assay: for those assays in which the binding of tracer in the "0" standard is 50% or greater, a blank value of 10% is im-

[19]

HYDROXYAPATITE IN RADIOIMMUNOASSAY

291

material because variation or "noise" in the blank will have little influence on the overall precision; however, for assays with a low " 0 " standard, blank variation can have a substantial effect. Under these circumstances, every effort should be made to reduce the blank, and almost invariably this will mean choosing an alternative system, such as second antibody or solid phase. In terms of practicality the fractional precipitation methods are difficult to equal, being simple, fast, and cheap. The only systems offering greater convenience to the user are the antibody-coated tube methods, but these suffer from major problems of reproducibility, particularly at the manufacturing stage, and are totally unsuited to the laboratory wishing to set up its own procedures. In setting up a new radioimmunoassay, and on the assumption that the primary reagents are known to be appropriate, the worker would be well advised to examine the use of a simple precipitation system such as polyethylene glycol. If this does not prove to be optimal, for the reasons set out above, then it is worthwhile to explore the use of a second-antibody system.

[-19] U s e o f H y d r o x y a p a t i t e

in R a d i o i m m u n o a s s a y

B y D. J. H. TRAFFORD and H. L. J. MAKIN

Any radioimmunoassay analysis relies upon the efficiency of the separation of the unbound (free) antigen or hapten from that bound to antibody. 1 When using immunoassay to estimate low molecular weight compounds such as steroids, the problem is perhaps somewhat simpler than in protein immunoassay. Because of the large difference in molecular weight between steroid haptens and their appropriate antibodies, relatively simple methods of separation, such as dialysis,z can be used. Although nonspecific precipitation of the antibody-bound steroid by ammonium sulfatea and polyethylene glycol4 are widely used, the utilization of second-antibody immunoprecipitation of the steroid-antibody complex 5 is not corni W. H. Daughaday and L. S. Jacobs, in "Principles of Competitive Protein-Binding Assays" (W. D. Odell and W. H. Daughaday, eds.), p. 303. Lippincott, Philadelphia, Pennsylvania, 1971. 2 F. F. G. Rommerts, W. F. Clotscher, and H. J. van der Molen, Anal. Biochem. 82, 505

(1977). 4 R. J. Liedtke,J. P. Greaves, J. D. Batjer, and B. Busby,Clin. Chem. 24, 1100(1978). 5 A. R. Midgelyand G. D. Niswender,in "Steroid Assay by Protein Binding"(E. Diczfalusy, ed.), p. 320.2nd KarolinskaSymposiumon Research Methodsin ReproductiveEndocrinology. Stockholm, 1970. METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-71

[19]

HYDROXYAPATITE IN RADIOIMMUNOASSAY

291

material because variation or "noise" in the blank will have little influence on the overall precision; however, for assays with a low " 0 " standard, blank variation can have a substantial effect. Under these circumstances, every effort should be made to reduce the blank, and almost invariably this will mean choosing an alternative system, such as second antibody or solid phase. In terms of practicality the fractional precipitation methods are difficult to equal, being simple, fast, and cheap. The only systems offering greater convenience to the user are the antibody-coated tube methods, but these suffer from major problems of reproducibility, particularly at the manufacturing stage, and are totally unsuited to the laboratory wishing to set up its own procedures. In setting up a new radioimmunoassay, and on the assumption that the primary reagents are known to be appropriate, the worker would be well advised to examine the use of a simple precipitation system such as polyethylene glycol. If this does not prove to be optimal, for the reasons set out above, then it is worthwhile to explore the use of a second-antibody system.

[-19] U s e o f H y d r o x y a p a t i t e

in R a d i o i m m u n o a s s a y

B y D. J. H. TRAFFORD and H. L. J. MAKIN

Any radioimmunoassay analysis relies upon the efficiency of the separation of the unbound (free) antigen or hapten from that bound to antibody. 1 When using immunoassay to estimate low molecular weight compounds such as steroids, the problem is perhaps somewhat simpler than in protein immunoassay. Because of the large difference in molecular weight between steroid haptens and their appropriate antibodies, relatively simple methods of separation, such as dialysis,z can be used. Although nonspecific precipitation of the antibody-bound steroid by ammonium sulfatea and polyethylene glycol4 are widely used, the utilization of second-antibody immunoprecipitation of the steroid-antibody complex 5 is not corni W. H. Daughaday and L. S. Jacobs, in "Principles of Competitive Protein-Binding Assays" (W. D. Odell and W. H. Daughaday, eds.), p. 303. Lippincott, Philadelphia, Pennsylvania, 1971. 2 F. F. G. Rommerts, W. F. Clotscher, and H. J. van der Molen, Anal. Biochem. 82, 505

(1977). 4 R. J. Liedtke,J. P. Greaves, J. D. Batjer, and B. Busby,Clin. Chem. 24, 1100(1978). 5 A. R. Midgelyand G. D. Niswender,in "Steroid Assay by Protein Binding"(E. Diczfalusy, ed.), p. 320.2nd KarolinskaSymposiumon Research Methodsin ReproductiveEndocrinology. Stockholm, 1970. METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-71

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mon. The alternative approach of nonspecific adsorption of the unbound hapten, using such materials as Florisiln and dextran-coated charcoal, 7 has gained wide acceptance. More recently, solid-phase immunoassays using antibody-coated tubes, s antibody bound to magnetic particles, 9 and microparticulate glass TM have been described. To the best of our knowledge, there has been no description of the use and evaluation of a nonspecific absorbent for antibody-steroid other than hydroxyapatite. H This article describes our experience in the radioimmunoassay of steroids using hydroxyapatite as an adsorbent for the steroid-antibody complex. Materials and Methods

Hydroxyapatite. This can be obtained from many sources (hydroxylapatite, BDH Chemicals Ltd., Poole, Dorset, U. K.; BioGel HT, BioRad Laboratories, Richmond, California), or it can be prepared in the laboratory. TM Our experience is limited to "hydroxylapatite" from BDH Chemicals, which is supplied in 50 mM phosphate buffer, pH 7.0. Before use, it was resuspended in 5 mM phosphate buffer, pH 7.0, or in Tris(hydroxymethyl)methylamine-hydrochloricacid (Tris-HCl) buffer, pH 7.5, at a concentration of approximately 1 g/ml. It can also be used as an acetone-dried powder la in a similar fashion to that described for Florisil. 6 Antibody-bound steroid was adsorbed to hydroxyapatite after addition to the incubation medium as a suspension or a solid powder. Free and antibody-bound steroid can also be separated in small columns of hydroxyapaptite, eluting antibody-bound steroid with a continuous, increasing concentration of phosphate buffer, pH 7.0 (see Fig. 1). Radioactive Steroids. All were 3H-labeled and were obtained from the Radiochemical Centre, Amersham, Bucks., U. K.; they were purified, if necessary, to >95% before use. Antisera. These were kindly provided by many colleagues H and were purchased from commercial sources (rabbit antitestosterone serum, Searle Diagnostics, High Wycombe, Bucks., U. K.). 6 B. E. P. Murphy, J. Crn. Endocrinol. Metab. 27, 973 (1976). 7 M. A. Binoux and W. D. Odell, J. Clin. Endocrinol. Metab. 36, 303 (1973). s S. L. Jeffcoate and J. E. Searle, Steroids 19, 181 (1972). 9 G. C. Forrest, Ann. Clin. Bioehem. 14, 1 (1977). ~0 Immo P h a s e - - a commercial immunoassay kit for plasma cortisol (Corning Medical, Halstead, Essex, CO9 2DX United Kingdom). 11 D. J. H. Trafford, P R. Ward, A. Y. Foo, and H. L. J. Makin, Steroids 27, 405 (1976). lz C. J. O. R. Morris and P. Morris, eds. "Separation Methods in Biochemistry," 2nd ed., p. 179. Pitman, London, 1976. 13 A. F. Hofman, in "Biochemical Separation Techniques" (A. T. James and L. T. Morris, eds.), p. 283. Van Nostrand-Reinhold, Princeton, New Jersey, 1964.

[19]

HYDROXYAPATITE Radioactive

293

IN R A D I O I M M U N O A S S A Y

counts

(cpm)xlO 3 15 Antibody -bound

14

fraction

13

12

11

10

9

8

7

6

5

4 ~ Free.

l

t

3

2

1

0

I

I

I

I

I

I

I

10

20

30

40

50

60

70

Fraction

number

FlG. 1. Separation of free and antibody-bound [3H]testosterone on small columns (0.4 cm i.d. x 8 cm) of hydroxyapatite. Jail]Testosterone was incubated with antibody at 37 ° for 1 hr and applied to the column in 5 mM phosphate buffer. Radioactivity was eluted from the column by slowly increasing the concentration of eluting phosphate buffer. The concentration (C, in moles per liter) o f the phosphate eluent was related to time (t, in minutes) by the equation: C × 0.5 - 0.445e -t~4°. The flow rate was 0.1 ml/min, and 0.2 ml fractions were collected.

294

RADIOIMMUNOASSAYS

AND

IMMUNORADIOMETRIC

ASSAYS

[19]

I m m u n o a s s a y . This assay was carried out as described by Trafford et al. 11 unless otherwise specified. Antiserum [200/xl appropriately di-

luted with 0.1% bovine serum albumin (BSA) in phosphate buffer, pH 7.0, so as to give the maximum binding at zero together with the maximum displacement of labeled tracer over the range of hapten concentration being examined] was added to a dried extract of plasma, or standard steroid solution, to which 3H-labeled steroid had been added. After incubation at 37 ° for 1 hr, 50/.d of hydroxyapatite suspension were added, and tubes were gently shaken and centrifuged. Supernatant (100/.d) was removed for liquid scintillation counting, at least 1000 counts being collected for each pot. Efficiencies were approximately 25% with a background count rate of 40 cpm. It has been found possible to carry out the

%Unbound 100

1: 200 diln. present throughoutincubation)

.~..f.O(HA

8O e/

o..~-'~" I"

60

o 1:600 diln. (Normal, HA added after incubation)

f~

f

.,//

'°,7 20

1:600diln. (HA presentthroughout incubation)

= = y O "Oj o j -

0

I

I

40

i

I

80

i

I

120

I

I

160

I

i

200

Testosterone (pg) F I G . 2. Standard curves for the immunoassay of testosterone either adding hydroxyapa-

tite (HA) after incubation or with hydroxyapatite present during incubation.

[19]

HYDROXYAPATITE IN RADIOIMMUNOASSAY

295

EFFECT OF HYDROXYAPATITE ON VARIOUS LOW MOLECULAR WEIGHT COMPOUNDS

Labeled compounds [l~q]Triiodothyronine [l~q]Thyroxine [aH]Guanosine 3',Y-cyclic phosphate 3-O-Succinyldigitoxigenin-L-tyrosine, IZq-labeled [3H]Estradiol-17/3 [3H]Testosterone [all] 17a-Hydroxyprogesterone

Percentage of added radioactivity recovered in supernatant after addition of hydroxyapatite 91.8 -+ 1.7 a,o 99.7 -+ 3.8a 98.8 -+ 2.4a 105.3 -+ 3.8a 97.2 - 9.7c 97.2 _+ 5.2c 96.3 -+ 11.6c

a In 50 mM Tris-HCl buffer, pH 7.5. 0 Value +- standard deviation. c In 5 mM phosphate buffer, pH 7.0. immunoassay with hydroxyapatite present throughout the incubation with the antiserum, although the concentration of antiserum used has to be increased. At the end of the incubation, the tubes are simply centrifuged and the supernatant is sampled as before. Figure 2 shows standard curves for testosterone immunoassay obtained by adding hydroxyapatite after the incubation and also by having hydroxyapatite present throughout the incubation. E v a l u a t i o n of H y d r o x y a p a t i t e as a n A d s o r b e n t of A n t i b o d y - B o u n d Steroid. 11 Investigations have shown that the use o f hydroxyapatite appears to be a very satisfactory procedure for the separation o f free and antibodybound steroids and other low molecular weight compounds (see the table). The adsorption of antibody-bound steroid appears to be independent of time in contact with hydroxyapatite (up to 1 hr), temperature (4-37°), and amount of hydroxyapatite added. Buffers of p H > 7.5 appear to prevent adsorption to varying degrees, and phosphate buffers elute antibody-bound steroid from hydroxyapatite (see Fig. 3). H o w e v e r , TrisHC1 buffers up to molarities of 0.1 M have no appreciable effect on the adsorption o f antibody-bound steroid. No significant effects on the crossreactivity o f the various antisera used has been noted. E f f e c t of P r o t e i n Because of the relative lack of specificity of many steroid antisera, it has been the custom to extract the hydrophobic steroids from plasma or

296

[19]

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS %Unbound

80

60

40

20 10', 0 0"001

I 0"01

I 0-1

I 0-2

Concentration of phosphate ( m o l e s / I ) FIG. 3. Effect of increasing concentrations of phosphate buffer, pH 7.0, on the adsorption of antibody-bound steroid. [all]Testosterone was incubated with antibody without added unlabeled testosterone. Hydroxyapatite suspensions in phosphate buffers of different molarities were added; tubes were shaken and centrifuged, and the supernatants were counted. Phosphate buffer strengths recorded are the final concentrations after addition of the hydroxyapatite suspension.

urine prior to immunoassay. The presence of plasma protein has therefore been largely irrelevant. With the increasing specificity of modem antisera, it has become more common to dispense with the organic extraction step, and many steroids are now being assayed in unextracted plasma. ~4 Studies on the effect of protein on hydroxyapatite adsorption of antibody-bound steroid have been inconclusive. Hydroxyapatite adsorbs protein, and the presence of increasing concentrations of added BSA or gelatin (up to 2% protein) in the incubation medium merely requires the addition of extra hydroxyapatite in order to achieve the same adsorption of antibody-bound steroid. The addition of plasma or serum is more com14 E. A. S. AI-Dujaili and C. R. W. Edwards, J. Clin. Endocrinol. Metab. 46, 105 (1978).

[19]

297

HYDROXYAPATITE lN RADIOIMMUNOASSAY

plex, however, and there appears to be some component present in plasma that prevents the adsorption of antibody-bound steroid by hydroxyapatite. This effect was first noted during attempts to utilize hydroxyapatite in an immunoassay for insulin that is assayed directly in plasma: good standard curves were obtained in 1% human serum albumin but after the addition of plasma samples, hydroxyapatite was ineffective. Hydroxyapatite, when added to solutions of BSA in sufficient amounts, reduced the extinction of the protein solution at 280 nm to background, indicating total adsorption of protein. A similar experiment using diluted plasma gave no reduction in the extinction at 280 nm. It has been found, therefore, that hydroxyapatite cannot be used in the presence of significant concentrations of plasma or serum. Figure 4 shows the effect

•Unbound

1°°f , ,,e Undiluted 80 i

•

I :,,lO,oOOO

/ ~

~ • 1 in 10

•

I in 1,000

4°If/~~ ot

I

ill

100

I

200

Testosterone (pg)

FIG. 4. The effect of charcoal-stripped plasma of various dilutions on the standard immunoassay curve for testosterone, l m m u n o a s s a y s were carded out as described in the text in plasma diluted with 0.1% bovine serum albumin.

298

R A D I O I M M U N O A S S A Y S A N D I M M U N O R A D I O M E T R I C ASSAYS

[19]

of various dilutions of charcoal-stripped plasma on the immunoassay of testosterone. Plasma has been treated in various ways in attempts to identify and remove the interfering component. Dialysis, heat treatment (80° for 20 min), and oxidation with periodate have had no effect. Precipitation of plasma proteins with trichloroacetic acid or perchlorate, and subsequent dialysis, produced a dialyzate that had no appreciable effect on hydroxyapatite. This approach is impractical, however, because it cannot easily be applied to plasma immediately prior to assay. So far, we have not managed to find a simple solution to this problem that will enable hydroxyapatite to be used to separate free and antibody-bound steroid in the presence of significant concentrations of nonextracted plasma. Comparison of a Radioimmunoassay for Plasma Testosterone Using Hydroxyapatite with Other I m m u n o a s s a y s Methods The use of hydroxyapatite as a means of separating free and antibodybound steroid in a routine immunoassay for plasma testosterone n was evaluated by comparing the results obtained using hydroxyapatite with the mean values obtained on the same plasma samples estimated by a number of other laboratories taking part in the U. K. Supraregional Assay Service Quality Control scheme (by courtesy of Dr. M. Wheeler). Four female and six male plasma samples were provided in the Quality Control scheme, and each was analyzed a number of times on different occasions. A total of 56 assays were compared. The correlatin coefficient was 0.9650 and the equation of the regression line was: y(SAS) = 0.84(HA) + 0.28. The correlation coefficient was not significantly different from 1.00, and the intercept was not significantly different from zero. The slope was, however, highly significantly different from 1.00 (p < 0.001). Summary Hydroxapatite has proved to be very useful for the separation of free and antibody-bound steroid in the immunoassay of various steroids. Successful immunoassays have been set up, using hydroxyapatite, for the estimation of plasma testosterone, estradiol-17fl, 17a-hydroxyprogesterone, and progesterone. No steroid so far examined has been found to be bound to hydroxyapatite. Some other low molecular weight compounds, for which immunoassays are available, have also been found not to be bound to hydroxyapatite (see the table), and it may therefore be possible to use hydroxyapatite in immunoassays for compounds other than steroids.

[20]

ZIRCONYL P H O S P H A T E GEL IN RAD1OIMMUNOASSAY

299

[20] U s e o f Z i r c o n y l P h o s p h a t e G e l for t h e S e p a r a t i o n of Antigens from Antigen-Antibody Complexes in Radioimmunoassays: Assays for Carcinoembryonic Antigen, ot-Fetoprotein, and Insulin

By J. W.

COFFEY,

J. P. V A N D E V O O R D E , E. R. and H. J. HANSEN

SAUERZOPF,

During the last two decades, radioimmunoassay has become one of the most widely used techniques for the quantitation in both clinical and experimental situations of proteins, hormones, drugs, and many other physiologically important molecules that are present at very low levels in biological fluids. As a result, many methods have been developed for the separation of free antigen from the antigen-antibody complexes in incubation mixtures (see Felber 1 for review). Zirconyl phosphate (Z-gel) is a convenient and inexpensive reagent for the separation of free antigen from antigen-antibody complexes in certain radioimmunoassays. Examples of radioimmunoassays in which Z-gel has been used successfully, such as assays for carcinoembryonic antigen2 (CEA), a-fetoprotein (AFP), and insulin,3 are described in this report. Stragand and Hagemann4 have also used Z-gel in their assay for cyclic AMP. Webb and NowowiejskP have described the use of Z-gel in a radioimmunoassay for prostaglandins. Methods

Preparation of Zirconyl Phosphate Gel Zirconyl chloride.8H20 (Matheson-Coleman and Bell), 100 g, is dissolved at room temperature in 15 liters of 0.1 M HCI; 200 ml of 14.6 M HsPO4 are added slowly as the solution is stirred vigorously. The resultant precipitate is allowed to settle for 2 days, and then the supernatant is removed by aspiration. The settled gel is brought to a final volume of 15 liters with distilled H20, resuspended by stirring, allowed to settle as 1 j. p. Felber, Adv. Clin. Chem. 20, 129 (1978). z H. J. Hansen, L. Hainsselin, D. Donohue, R. Davis, O. N. Miller, and J. P. Vandevoorde, Methods Cancer Res. 11, 355 (1975). 3 j. W. Coffey, C. F. Nagy, R. Lenusky, and H. J. Hansen, Biochem. Med. 9, 54 (1974). 4 j. j. Stragand and R. F. Hagemann, Experientia 31, 1375 (1975). 5 D. R. Webb and I. Nowowiejski, Cell. lmmunol. 41, 72 (1978). METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181970-1

300

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

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above; then the supernatant is aspirated as before. Washing of the gel with distilled H20 at room temperature is repeated five times; the gel is then brought to a volume of 15 liters with distilled H20. Concentrated acetic acid (17.6 M) is added with stirring to a final concentration of 0.1 M. The gel is allowed to settle for 2 days in this solution of 0.1 M acetic acid; then the supernatant is removed completely by aspiration. The pH of the resultant slurry of Z-gel is adjusted to 6.25 by the addition of concentrated NH4OH (15.1 M); the slurry is stored at room temperature after the addition of 0.01% (w/v) thimerosal as a preservative. The percentage of solids should be 37-45% when centifuged at 1000 g for 10 min in a hematocrit tube.

Preparation of Antigen, 125I-Labeled Antigen and Antisera Since the purpose of this report is to illustrate the usefulness of Z-gel as a reagent for the separation of antigens from antigen-antibody complexes, the methods 6,7 used for the purification of CEA and AFP will not be described. Bovine insulin was purchased from Schwarz-Mann, and anti-bovine insulin serum was purchased from Miles Laboratories. Antisera to CEA and AFP were prepared in goats and rabbits, respectively. Standard solutions of nonradioactive CEA (125 ng/ml) and AFP (180 ng/ml) are prepared in 50 mM borate buffer (pH 8.4) containing 10% (v/v) group A human plasma for CEA or 0.25% human albumin for AFP and 0.01% thimerosal. The bovine insulin standard (100/~IU/ml) is prepared in 40 mM phosphate buffer, pH 7.4, containing 0.15 M NaCI, 0.5% bovine serum albumin (BSA), and 0.01% thimerosal (phosphate buffer 1).

Iodination of CEA and AFP Proteins were iodinated by the method of Hunter and Greenwood. s Briefly, CEA and AFP are dissolved in 50 mM borate buffer (pH 8.4) at a concentration of 1 mg/ml. Fifty microliters of the protein solution are added to a vented V vial containing 5 mCi of carrier-free Na125I (pH 8-10) from Amersham Searle (IMS-30). Twenty microliters of freshly prepared chloramine-T (5 mg/ml in borate buffer) are added to the vial with a Ham-" ilton syringe, and the contents are mixed immediately. After 1 rain at room temperature, 20 t~l of Na~S205 (10 mg/ml in borate buffer) are added to stop the iodination reaction. The reaction mixture is combined with e j. Krupey, P. Gold, and S. O. Freedman, J. Exp. Med. 128, 387 (1968). 7 N. G. Anderson, D. W. Holladay, J. W. Caton, E. L. Candler, P. J. Dierlan, J. W. Eveieigh, F. L. Ball, J. W. Holleman, J. P. Breillatt, and H. J. Coggin, Jr., Cancer Res. 34, 2066, (1974). 8 W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1964).

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1.0 ml of 5% human albumin in the case of AFP, or of group A human plasma in the case of CEA. The nSI-labeled proteins are separated from free 1251 by filtration through a Sephadex G-100 column (2.5 × 90 cm) equilibrated with 0.1 M Tris-HCl (pH 7.0) containing 0.15 M NaCI and 0.02% (w/v) NAN3. The column at 4° is eluted at a flow rate of 0.5 ml/min, and 5-ml fractions are collected into tubes containing 0.5 ml of 5% human albumin (AFP) or 0.5 ml of group A human plasma (CEA). The amount of 1251 in a 25-/~1 aliquot of each fraction is determined in a gamma scintillation spectrometer. The tubes containing the '25I-labeled protein are pooled, and the pool is diluted in 50 mM borate buffer (pH 8.4) supplemented with 0.25% human albumin (AFP) or with 10% group A plasma (CEA) and 0.01% thimerosal to yield a working solution of 125I-labeled CEA, which contains 200,000 dpm per 25 /~1, and of [125I]AFP, which contains 120,000-160,000 dpm per 50t~l.

Iodination of Insulin Twenty-five micrograms of insulin in 25 ~1 of 40 mM phosphate buffer (pH 7.4) are added to a vial containing 5 mCi of Na125I, and the iodination reaction is started by the addition of 10 t~l of chloramine-T (10 mg/ml in 40 mM phosphate buffer, pH 7.4). The reaction is allowed to proceed at room temperature for 2 min and then is stopped by the addition of 21~ tzl of Na~S205 (10 mg/ml in phosphate buffer). The contents of the iodination vial are combined with 0.2 ml of 1% BSA, and the p25I]insulin is separated from free 1251on a 2.5 × 20 cm column of Sephadex G-25 (4°) equilibrated with 0.1 M Tris-HCl (pH 7.4). The column is eluted at a flow rate of 0.5 ml/min, and 1.0-ml fractions are collected into tubes containing 1 ml of 1% BSA. The fractions containing [125I]insulin are pooled, the pool is diluted with phosphate buffer 2 (40 mM phosphate buffer, pH 7.4, 0.5% BSA, and 0.01% thimerosal) to yield a working solution containing approximately 90,000 dpm per 100/zl.

Titration Curve To Determine Appropriate Dilutions of Antisera for Radioimmunoassays Carcinoembryonic Antigen (CEA). Twelve disposable glass test tubes (1.5 × 15 cm, Packard Instrument Co.) are labeled appropriately, and 5 ml of EDTA buffer (3.7 mM EDTA, pH 6.5, 0.017% sodium azide, and 0.002% BSA) are added to each tube. Two-hundred microliters of 50 mM borate buffer, pH 8.4, containing 10% group A human plasma and 0.01% thimerosal are added to the two blank tubes, and 200/zl of five increasing dilutions (appropriate dilutions must be determined experimentally for each antiserum) in the same buffer of anti-CEA serum are added in dupli-

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cate to the other 10 tubes. Then 25 ~1 of 125I-labeled CEA are added to all tubes. The contents of the tubes are mixed, and then the tubes are incubated at 45 ° for 30 min in a H20 bath. At the end of the incubation period, 2.5 ml of the Z-gel slurry are added rapidly to each tube, and the tubes are mixed on a Vortex mixer. The Z-gel is collected by centrifugation (1000 g for 6 min) at room temperature, and the supernatant is decanted. The pellet of Z-gel is washed once by resuspension with a Vortex mixer in 5 ml of 0.1 M ammonium acetate (NH4Ac), pH 6.25, followed by recentrifugation. The supernatant is decanted again, and the amount of 1251 associated with the pellet of Z-gel is determined in a gamma scintillation spectrometer. Under these conditions, approximately 70% of the ~25I in the preparation of 125I-labeled CEA binds to Z-gel in the presence of excess antibody, and less than 10% binds nonspecifically to Z-gel in the absence of antibody. A dilution of antiserum that will bind approximately 60% of the antibody-precipitable azsI-labeled CEA is used in the radioimmunoassay. a-Fetoprotein (AFP). Titration curves with anti-AFP serum and [I~5I]AFP are prepared using the procedure described for CEA with the following changes. The amount of EDTA buffer per tube is reduced to 2.5 ml, and in addition the EDTA buffer is made 1% (v/v) with respect to normal goat serum. Fifty microliters of p25I]AFP are added to each tube, and the tubes are incubated at 37° for 90 min. Approximately 70% of the 1251 in the [IzSI]AFP preparation binds to the Z-gel under these conditions in the presence of excess antibody; however, approximately 30% of the 1251binds nonspecifically to Z-gel in the absence of antibody. A dilution of antiserum that will bind approximately 60% of the [125I]AFP in the incubation mixture is used in the radioimmunoassay. Insulin. Twelve glass test tubes are labeled, and 0.2 ml of phosphate buffer 1 is added to each tube. The two blank tubes receive 0.1 ml of phosphate buffer 2, and the remaining I0 tubes receive in duplicate 0.1 ml of five increasing dilutions of anti-insulin serum in phosphate buffer 2. A constant amount (0.1 ml) of [125I]insulin in phosphate buffer 1 is added to each tube, and the tubes are incubated at 37° for 45 min. The reaction is stopped by the addition to each tube of 5 ml of the Z-gel slurry followed by 10 ml of 0.1 M NI-I4Ac (pH 6.25). The contents of the tubes are mixed by inversion, and the Z-gel is collected by centrifugation (1000g for 5 min) at room temperature. The supernatant is decanted, and the pellet is washed by resuspension in 10 ml of 0.1 M NH4Ac followed by recentrifugation. The supernatant is decanted, and the amount of ~2zI associated with the pellet of Z-gel is measured. Approximately 80% of the 1251in the preparation of [~5I]insulin binds to Z-gel in the presence of excess antibody, but only 10% binds in the absence of antibody. A dilution of the

[20]

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antiserum that will bind to the Z-gel albproximately 50% of the [x2sI]insulin is used for the preparation of standard inhibition curves.

Preparation of Standard Inhibition Curves Carcinoembryonic Antigen (Fig. IA). Five mililiters of EDTA buffer is added to each of 10 glass test tubes and then 10, 25, 50, and 100/~1 (1.25, 3.125, 6.25, and 12.5 ng) of the CEA standard are added in duplicates to 8 of the tubes. Two tubes receive no nonradioactive CEA. Twenty-five microliters of the appropriate dilution (see titration curve) of anti-CEA serum are added to each tube; the tube contents are mixed and then incubated in a H20 bath for 30 min at 45 °. After this initial incubation period, the tubes are removed from the bath, and 25 tzl of ~25I-labeled CEA are added to each tube. The tubes are returned to the 45 ° bath and are incubated for an additional 30 min. The tubes are removed from the bath, Zgel is added to stop the reaction, and the tubes are processed as described for the titration curve. ~-Fetoprotein (Fig. 1B). Standard inhibition curves for AFP are prepared essentially as described for CEA with some modifications. The

A

40

501"

B

20,

C

1

i g

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o

(2_

I

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I

I

4

Corclnoembryonlc

I 8

I

I 12

Antigen rig/tube

4 -

0

I

I

4

I

I

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I

I

12

I

1

16

Q- Fetoproteln ng/tube

I

I

0

i 4

I

I 8

ImLulln /~Iu/tube

FIG. 1. Standard inhibition curves for the measurement of carcinoembryonic antigen (A), ct-fetoprotein (B), and insulin (C) by radioimmunoassays utilizing Z-gel. The total amounts of ~25I in the tubes used for the preparation of the inhibition curve were 90,000 cpm, 79,000 cpm, and 45,000 cpm for carcinoembryonic antigen (CEA), ,x-fetoprotein (AFP), and insulin, respectively, with a gamma scintillation counter efficiency of 45%.

I

304

RADIO1MMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS'

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amount of EDTA buffer is reduced to 2.5 ml per tube, and 1% normal goat serum is added; 10, 25, 50, and 100/zl (1.8, 4.5, 9.0, and 18.0 ng) of the AFP standard are added in duplicate to 8 tubes. Two tubes with no nonradioactive AFP are included in the assay. Fifty microliters of the appropriate dilution of anti-AFP are added to each tube, the contents of the tubes are mixed, and then the tubes are incubated at 37° for 90 min. The tubes are removed from the bath, and 50/xl of [125I]AFP are added to each. After a second incubation period of 90 min at 37°, 1 ml of Z-gel is added to each tube; then the tubes are processed as described (see titration curve). Insulin (Fig. 1C). Standard inhibition curves are prepared using 10 tubes, each of which contains 0.1 ml of buffer 2 and 0.1 ml of the appropriate dilution of anti-insulin serum. Four pairs of tubes receive 0.1 ml of the insulin standard diluted so as to contain 1.0, 2.5, 5.0, or 10.0/zlU of insulin. The tubes are incubated at 37° for 2 hr, and then 0.1 ml of [~25I]insulin is added to each. After an additional incubation period of 45 min at 37°, 5 ml of Z-gel are added and the tubes are processed as described (see titration curve). Comments This radioimmunoassay for CEA has been widely used clinically to quantitate the level of CEA in perchloric acid extracts of plasma. 9 The sensitivity of this assay is approximately 0.5 ng of CEA per milliliter of plasma. '° Multiple analyses on the same sample have established standard deviations of 0.76 to 1.03 for plasma samples with CEA titers from 0 to 5.0 ng/ml, of 1.12 to 2.31 for plasma samples with titers from 5.1 to 10 ng/ml, and of 2.29 to 5.47 for samples with higher titers. 1° a-Fetoprotein can be quantitated in serum samples of 25/A using the Z-gel,procedure; however, the accuracy and reproducibility of the procedure have not been studied extensively. The sensitivity and reproducibility of the radioimmunoassy with Z-gel for insulin are comparable to those for other published procedures. 3 Z-gel can be used theoretically to separate a variety of antigens from their antibody complexes provided certain conditions can be satisfied; i.e., if a pH can be found at which the antigen and its antibody complex carry opposite net charge. Z-gel is a negatively charged gel at pH 6.25; it will bind positively charged molecules, e.g., proteins at a pH below their isoelectric pH. I n the three radioimmunoassays described in this report, Z-gel at a pH of 6.25 is used to adsorb the positively charged antibody g H. J. Hansen,J. J. Snyder,E. Miller,J. P. Vandevoorde,O. N. Miller, L. R. Hines, and J. J. Burns, Human Pathol. 5, 139 (1974). 10T. M. Chu and G. Reynoso,Clin. Chem. 18, 918 (1972).

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molecules (p! > 7) with their associated antigens, while leaving the negatively charged free antigens (CEA, AFP, and insulin) in solution. Experience has shown that NH4 ÷ is the preferred cation as Na ÷ and K ÷ decrease the ability of Z-gel to adsorb antigen-antibody complexes. H It should be possible to use Z-gel as a simple, convenient, and inexpensive reagent for the separation of antigen from antigen-antibody complexes in any radioimmunoassay for which the investigator can establish experimental conditions that meet the charge requirements of Z-gel, provided that nonspecific adsorption to Z-gel is not a problem and that Z-gel does not dissociate the antigen-antibody complex, as was noted in the case of angiotensin. H 11 j. p. Vandevoorde and H. J. Hansen, unpublished data.

[21] M i c r o f i l t r a t i o n a s a M e a n s o f S e p a r a t i n g Free Antigen from Antigen-Antibody Complexes in I m m u n o a s s a y B y SYLVIA R . CHALKLEY a n d A . R E N S H A W

In immunoassay techniques, efficient separation of free antigen from the antigen-antibody complexes is essential for reproducible results and maximum sensitivity. Microfiltration under suitable conditions gives efficient separation and ease of handling large or small numbers of samples. The technique of microfiltration is more adaptable to mechanization than centrifugation. Various methods of microfiltration are described here that use either disposable microfilters, 1'2 discrete filter disks, 3-5 or continuous filtration through glass fiber paper tape. ~ i S. R. Chalkley and A. Renshaw, Clin. Chim. Acta 62, 377 (1975). 2 C. J. Sanderson, G. H. Taylor, and R. Geary, J. Immunol. Methods 4, 17 (1974). a G. A. Taylor, C. J. Sanderson, R. Geary, and J. C. Meakin, Lab. Pract., August, 521 (1975). 4 j. O'Brien, S. Knight, N. A. Quick, E. H. Moore, and A. S. Platt, J. lmmunol. Methods 27, 219 (1979). s K. D. Bagshawe, Lab. Pract., September, 573 (1975). A. Pollard and C. B. Waldron, Technicon Symp. 1, 49, (1966).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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305

molecules (p! > 7) with their associated antigens, while leaving the negatively charged free antigens (CEA, AFP, and insulin) in solution. Experience has shown that NH4 ÷ is the preferred cation as Na ÷ and K ÷ decrease the ability of Z-gel to adsorb antigen-antibody complexes. H It should be possible to use Z-gel as a simple, convenient, and inexpensive reagent for the separation of antigen from antigen-antibody complexes in any radioimmunoassay for which the investigator can establish experimental conditions that meet the charge requirements of Z-gel, provided that nonspecific adsorption to Z-gel is not a problem and that Z-gel does not dissociate the antigen-antibody complex, as was noted in the case of angiotensin. H 11 j. p. Vandevoorde and H. J. Hansen, unpublished data.

[21] M i c r o f i l t r a t i o n a s a M e a n s o f S e p a r a t i n g Free Antigen from Antigen-Antibody Complexes in I m m u n o a s s a y B y SYLVIA R . CHALKLEY a n d A . R E N S H A W

In immunoassay techniques, efficient separation of free antigen from the antigen-antibody complexes is essential for reproducible results and maximum sensitivity. Microfiltration under suitable conditions gives efficient separation and ease of handling large or small numbers of samples. The technique of microfiltration is more adaptable to mechanization than centrifugation. Various methods of microfiltration are described here that use either disposable microfilters, 1'2 discrete filter disks, 3-5 or continuous filtration through glass fiber paper tape. ~ i S. R. Chalkley and A. Renshaw, Clin. Chim. Acta 62, 377 (1975). 2 C. J. Sanderson, G. H. Taylor, and R. Geary, J. Immunol. Methods 4, 17 (1974). a G. A. Taylor, C. J. Sanderson, R. Geary, and J. C. Meakin, Lab. Pract., August, 521 (1975). 4 j. O'Brien, S. Knight, N. A. Quick, E. H. Moore, and A. S. Platt, J. lmmunol. Methods 27, 219 (1979). s K. D. Bagshawe, Lab. Pract., September, 573 (1975). A. Pollard and C. B. Waldron, Technicon Symp. 1, 49, (1966).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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Materials and Methods

Filter Paper Glass fiber paper, Whatman grade GF/B (W. & R. Balston Ltd., Maidstone, Kent, U. K.) is the most suitable for use in immunoassay. This has an optimum flow rate of 10 ml/min through a disk 1 cm in diameter, and the pore size is such that all particles over 0.5/xm 3 are retained. The filter should be prewashed immediately before use with a protein solution, such as aqueous 0.25% albumin or gelatin in order to minimize protein adsorption. 10--

9

8

7

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FIG. 1. Effect of washing the CRC thimble, i--U, (2ZNa). From Chalkley and Renshaw. 82

i

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Precipitate (1251); o - - o , supernatant

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This filter paper is fragile when wet and must be supported. Glass filter paper has a high diffusion ratio giving an even flow throughout the filter matrix under negative pressure. An adequate flow rate is achieved with one hole 0.8 mm in diameter for an area 6 mm ~ and a negative pressure of 15 psi (83 kN/m2). Under these conditions the precipitate is spread evenly over the surface of the filter. When glass fiber paper tape is used, this can be strengthened by the application of a rubber solution along each edge, 6 or with an under tape of strong filter paper having coarse filtration properties. After drying, the tape must be sealed on the upper surface with transparent cellulose tape. The volume of the wash used on the filter does not appear to be critical above a minimum volume, which is 0.4 ml for a disk 1 cm in diameter (Fig. 1). If this volume is used to wash the container in which the reaction has taken place, the amount of precipitate on the filter increases, presumably owing to recovery of residual precipitate. The amount of supernatant retained on the filter decreases sharply and becomes constant at about 0.1% of the original volume, after 0.4 ml of wash has passed through the filter. Increasing the volume above 0.4 ml does not remove more supernatant, nor does it remove precipitate from the filter.

CRC Thimbles The disposable microfilters, "CRC thimbles" (W. & R. Balston Ltd., Maidstone, Kent, U. K.) shown in Fig. 2 consist of a glass fiber filter disk 1 cm in diameter with an effective filter area of 33 mm 2, sealed between two polypropylene cylinders. The outer cyclinder has a base perforated

O

2

I

I 1 cm

FIG.2. Explodedviewof CRC thimble. 1, Innercylinder;2, glassfiberfilterdisc; 3, outer cylinder with perforated base. From Chalkleyand Renshaw.~

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RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

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by seven holes, which supports the glass fiber, and a groove just below the upper rim into which a corresponding projection on the inner cylinder locks. The CRC thimbles are adaptable for use in basic manual apparatus or may be incorporated into automatic equipment. Apparatus A manual microfiltration apparatus (Fig. 3) has been made by the present authors for use with a CRC thimble. The apparatus is constructed of stainless steel with neoprene rubber seals. A hollow base holds a test tube for collection of the filtrate, if required, and has a connection for a vacuum pump. The top, which fits onto the base with an O ring, has on its upper surface a well to hold the CRC thimble and a support for the removable filter cover. The cover has a probe that is inserted into the solution to be filtered. When negative pressure is applied to the vacuum connection and the CRC thimble is held in place by the filter cover, the airtight seals enable solution to pass through the probe. A prototype apparatus (Fig. 4) developed by Chalkley and Renshaw, 1 utilizing the CRC thimble will automatically filter the contents of 100 tubes in pairs. The basis of the apparatus is a box that contains tubing and a sequence programmer and holds in place electrical and mechanical com-

2

|

I

6

FIG. 3. Microfiltration apparatus. 1, Stainless steel base; 2, Stainless steel top; 3, neoprene rubber seal; 4, vacuum connection; 5, CRC thimble; 6, test tube for collection of filtrate; 7, airtight filter cover with probe; 8, airtight seating.

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FIG. 4. Automated filtration apparatus. I, Processing turntable; 2, upper sample tube turntable; 3, lower turntable holding collecting tubes for CRC thimbles; 4, vertical cylinders for loading CRC thimbles.

310

RADIOIMMUNOASSAYSAND IMMUNORADIOMETRIC ASSAYS [21]

ponents; three turntables and five delta pumps transfer liquids from the sample tubes onto the microfilters, and the others add liquid directly to the tubes or to the microfilters. The apparatus will (a) load paired microfilters onto the processing turntable; (b) prewash them with protein solution; (c) transfer the contents of the paired sample tubes on the first circular tray onto the microfilters; (d) wash both the tubes and the microfilters; (e) drop the microfilters into the tubes in the second circular tray. When the fiftieth pair of microfilters have been dropped into their tubes, the apparatus switches itself off. In cell culture techniques, the need to harvest cells from the culture medium has resulted in the development of harvesters that will filter a large number of small-volume samples. Some of these are suitable for immunoassay work. Various harvesters that can filter twelve samples at a time have been developed by Sanderson and his colleagues. 2"3 The original model transferred the contents of a microplate onto CRC thimbles, and the later versions transfer the contents of either tubes or microplates onto glass fiber filter disks. Ganged peristaltic pumps were used in both models. The microplate, or row of 12 tubes, is carried on a sliding platform, which locates positively by a ball and spring, allowing each row of wells or tubes to fit under an intake manifold. The vacuum manifold in the original version holds CRC thimbles in recesses in the top. Each recess connects with the cavity of the vacuum manifold. The manifold slides into position under the outlets from the peristaltic pump, and in this position the cavity connects to the vacuum line. In later models, the vacuum manifold consists of two separate Perspex blocks2 The lower block has a row of 12 sharpedged platforms on its upper surface, each of which is perforated by 8 holes joining the cavity connected to the vacuum line. The upper block has 12 holes, which are shaped to give an exact fit over the platforms on the lower block. For use, a strip of glass fiber filter paper is laid over the lower block and the top block is pressed down onto it, so that 12 disks are cut and clamped into place between the two blocks. A simplified version of this is manufactured by Ilacon Limited, Tonbridge, Kent, U. K. Two other filtration modules, which both formed part of automated radioimmunoassay systems, have been developed. 5,6 One of these, 6 was based on conventional continuous flow techniques. At the filtration stage, a continuously moving strip of glass fiber filter paper was strengthened and wetted, the reaction mixture was filtered, and the precipitate was washed by two streams of buffer. The strip was dried and overlaid with cellulose adhesive tape before being counted as it passed between two end-window radioactivity detectors and wound onto a take-up spool. In the second system, 5 glass fiber filter disks are mounted at intervals over perforated segments of a flexible plastic carrier tape. The contents of five

[21]

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MICROFILTRATION IN RADIOIMMUNOASSAY

~ ~ | I II

311

MINIMUM RETENTION FOR GFB FILTER

i I I I I I I I I

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PARTICLE SIZE (~rn 3) FXG. 5. Particle size distribution after 6 hr final incubation, o - - o , Standard; n - - l l , human serum. GFB, Whatman grade GF/B glass fiber paper. From Chalkley and Renshaw. 62

reaction tubes at a time are transferred onto these disks, which have previously been wetted with protein solution. Washing solution is pumped into the reaction tubes and transferred onto the filters, which are also washed directly. A pressure plate bears on the lower surface of the filters and provides negative pressure for the transfer. When the filtration is complete, the pressure plate is lowered, and the filter tape and reaction tubes then move on five places. The filter tape is dried, overlaid with adhesive cellulose tape, and wound onto a take-up spool. When the full cycle is completed, the tape is rewound onto a supply spool, ready to be counted in a separate radioactivity detector module.

Preparation of Samples Microfiltration is ideal for use with immunoassay methods that use pre-precipitated antibody, 7 solid phase techniques where the antibody is r K. D. Bagshawe, C. E. Wilde, and A. H. Orr, Lancet 1, 1118 (1966).

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bound to Sephadex or cellulose, 8 or chelation of the first antigen-antibody complex by staphylococcal protein A instead of a second antibody. 9 In these methods the solid phase particles are all larger than the minimum retention size of the glass fiber filter. Although the coated iron particles produced by Technicon Corporation (Ardsley, New York) are intended to be separated from the mixture by magnetic methods, 1° these could be separated as easily by microfiltration. In double-antibody methods where the precipitating antibody is the last reagent to be added, the final incubation must be at least 17 hr, 1 unless accelerators have been added. H Tests have suggested, x that precipitates form at different rates in the presence or the absence of serum. This is shown by the particle size distribution found in reaction mixtures containing standard solution or human serum after an incubation period of 6 hr (Fig. 5) or 17 hr (Fig. 6). In a comparison of 94 3OrMINIMUM RETENTION FOR GFB FILTER

2o

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FIG. 6. Particle size distribution after 17 hr final incubation, o - - o , human serum. From Chalkley and Renshaw. 82

Standard; i - - m ,

8 L. E. M. Miles and C. N. Hales, in "'Protein and Polypeptide Hormones" (M. Margoulies, ed.), Proceedings of the International Symposium, Liege, p. 61. Excerpta Med. Found., Amsterdam, 1968. 9 M. E. Soergal, F. L. Schaffer, J. C. Sawyer, and C. M. Prato, Arch. Virol. 57, 271 (1978). 1o L. S. Hersh and S. Yaverbaum, Clin. Chim. Acta 63, 69 (1972). 11 B. Morris, personal communication, 1979.

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RADIOIMMUNOASSAY

eP' • °qa

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LOG TSH (H.U/rnt) CENTRIFUGED F i G . 7. C o r r e l a t i o n o f T S H v a l u e s after 17 hr final i n c u b a t i o n . F r o m C h a i k l e y and R e n shaw.8 2

samples, 1 which were assayed in duplicate and then separated by either microfiltration or centrifugation after a final incubation of 17 hr (Fig. 7), the correlation was 0.91 (p < 0.001) and the equation of the regression line, y = 1.01 × - 0 . 3 4 does not differ significantly from the line of identity. Immunoassay methods involving adsorption of antigen by charcoal particles were found to be unsuitable for use with microfiltration techniques. The fine particles quickly block the glass fiber, with a sharp drop in the flow rate, so that variable amounts of the supernatant are retained with the precipitate.

Measurement of Radioactivity The CRC thimble and glass fiber filter disks are suitable for use in radiolabeled techniques. With y-ray counting, the microfilters are placed in containers (for example, disposable cellulose acetate tubes) and sealed

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before being measured in a well-type counter. Where fl-emitting isotopes have been used, liquid scintillation counting is most appropriate, although the glass fiber disks could be counted in a planchet counter. For liquid scintillation, the microfilters should be placed in screw-top counting vials and dried before the addition of the chosen scintillant. The polypropylene CRC thimble becomes translucent in toluene-based scintillation fluid, and tests have shown that the counts acquired are similar whether counted with the thimble intact or with the filter removed and counted separately .2 The orientation of the thimble in the vial does not significantly affect the counts. The filter tape is most conveniently counted with two end-window detectors, one on either side of the tape. Although it is possible to cut the tape into suitable lengths to be counted in a well-type counter, this is tedious and the position of each sample must be marked exactly. Conclusion Microfiltration using glass fiber filter disks is an efficient alternative to centrifugation for the separation of free antigen from antigen-antibody complexes. When small numbers of samples are involved in manual methods or for mechanized aparatus, microfiltration has advantages over centrifugation. For immunoassay techniques, cellulose acetate filters offer no advantage over glass fiber filter paper, and the slow flow rate and the handling problems of cellulose acetate when compared with glass fiber make it less suitable for use when large numbers of samples are involved. The retention characteristics of GF/B filter paper are adequate for most immunoassay techniques. However, in receptor assays, TM the particle sizes are close to the minimum retention size of the GF/B filter paper, and cellulose acetate filters are being used for filtration of these samples.

~2 p. C u a t r e c a s a s , Proc. Natl. Acad. Sci. U. S. A. 69, 318 (1972).

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[22] G e l C e n t r i f u g a t i o n : S e p a r a t i o n o f F r e e L a b e l e d Antigen from Antibody-Bound Labeled Antigen B y JENS LARSEN a n d WIGGO FISCHER-RASMUSSEN

Separation of protein-bound steroid from free steroid by gel filtration on stationary columns with Sephadex G-25 was elaborated by Murphy and Pattee 1 for estimation of cortisol in plasma. Giese et al. 2 developed the quicker method of gel centrifugation for separation of prelabeled antigen from antibody-bound labeled antigen in a radioimmunoassay (RIA) for angiotensin I. Before using this method of separation for the successful estimation of estrogens in serum, 3,4 we found it necessary to evaluate the completeness and the precision of the gel centrifugation. Material and Methods Solvents and R e a g e n t s

Spectrograde diethyl ether, benzene, and methanol, and analytical grade absolute ethanol were obtained from Merck AG, (Darmstadt), as were crystalline estradiol-17/3, estrone, and estriol. Other materials were human albumin fraction V (Sigma Chemical Company), Merthiolate (British Drug House), Sephadex LH-20 and Sephadex G-25 fine (AB Pharmacia, Sweden), Insta-Gel (Packard Instrument Company, U.S.A.), counting vials of polyethylene (Scintitec Laboratorieudstyr A/S, Denmark). [2,4,6,7-3H]Estrone (115 Ci/mmol), [2,4,6,7-~H]estradiol-17fl (100 Ci/mmol), and [2,4,6,7-3H]estriol (112 Ci/mmol) were purchased from New England Nuclear Corporation (Boston, Massachusetts). Human [125I]7-globulin (64/zCi/ml) was obtained from the Department of Nuclear Medicine, Rigshospitalet, Copenhagen. Antibodies. The antibody A-1 against estrone and estradiol-17/3 was raised in a sheep against estradiol-17fl coupled at C-17 to bovine serum albumin. At 50% displacement of tritiated estradiol-17/3, the cross-reac1 B. E. P. Murphy and C. J. Pattee, J. Clin. Endocrinol. 24, 919 (1964). J. Giese, M. J~rgensen, M. D. Nielsen, J. O. Lund, and O. Munck, Scand. J. Clin. Lab Invest. 26, 355 (1970). 3 j. Larsen and W. Fischer-Rasmussen, Ugeskr. Laeg. 138, 1082 (1976). 4 j. Larsen, W. Fischer-Rasmussen, P. Henger, and B. Ovlisen, Ugeskr. Laeg. 138, 1086 (1976).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1960by Academic Press, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-18197(L1

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tivity of the antibody with estrone was 52% and with estriol 4%. By means of a Scatchard 5 plot the affinity constant for estradiol-17/3 was calculated to be 1.76 x 10TM 1/M and for estrone to be 1.00 x 10TM 1/M. The antibody A-2 specific for estradiol-17/3 was raised in a rabbit against estradiol-17/3 coupled at C-6 to human serum albumin. The cross reaction with estrone was 6%, and with estriol 0.1%. By means of a Scatchard plot the affinity constant was calculated to 5.70 × l09 1/M with respect to estradiol-17B. The antibody A-3, highly specific for estriol, was obtained from a sheep by immunization with estriol coupled with human serum albumin at C-6. The cross-reaction against estrone was less than 0.01%, and against estradiol-17/3, 0.1%. Preparation o f Materials

Gelatin buffer was prepared from glass-distilled water containing phosphate buffer (10 mM, pH 7.0), sodium chloride (0.14 M), Merthiolate (0.01%), and gelatin (0.1%). Albumin buffer was prepared by addition of human albumin (0.1%) to gelatin buffer. Preparation of Sephadex G-25 fine minicolumns was performed in the following way: The barrels of 2-ml polyethylene syringes (50 x 9 mm) were furnished with a circular filter of Whatman No. 41 paper. After swelling for at least 3 hr in surplus of gelatin buffer Sephadex G-25 fine was packed into the syringes leaving the upper 4 mm of the barrels empty. Rubber caps were placed on the nozzles of the syringes, which were stored at 2° and used within 8 days. Method in Detail

Estrone, estradiol-17/3, and estriol have been estimated in serum by means of RIA including a step of separation with Sephadex G-25 fine minicolumns. After diethyl ether extraction from 1-ml serum samples and chromatography on Sephadex LH-20 columns, estrone and estradiol-17/3 were estimated after incubation by the method of Hotchkiss et al. ~ with antibody A-1. In order to obtain quicker methods without chromatography, the more specific antibodies A-2 and A-3 have been used to estimate estradiol-17/3 and estriol, respectively. These two antibodies were incubated directly with the residues after extraction of serum with dichioromethane. G. Scatchard, Ann. N. Y. Acad. Sci. 51, 660 (1949). 6 j. Hotchkiss, L. E. Atkinson, and E. Knobil, Endocrinology 119, 177 (1971).

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After incubation for 1 hr at 2°, the separation of antibody-bound antigen from free antigen was performed by Sephadex G-25 fine minicolumns pretreated in the following way. The rubber caps of the cold Sephadex G-25 fine columns were removed, and the columns were coated by application of 500/zl of ice-cold albumin buffer on the top surface of the gel particles and subsequent spinning of 320 g for 5 min at 2 °. Aliquots of 600/zl of the cold incubates were transferred to the albumin-coated columns and subsequently 200 ~l of ice-cold albumin buffer were applied in order to wash down residue of the samples on the inside wall of the syringes into the gel. The columns were kept cool for 5 min before centrifugation at 2° at 320 g for another 5 min. During centrifugation the free steroid was retained in the gel particles and the fraction bound to antiserum was spun out directly into the counting vials in which the minicolumns were suspended by their wings. To each counting vial 10 ml of Insta-Gel were added, and the counting was performed in a Nuclear Chicago model Isocap/300 liquid scintillation system to a level of at least 104 counts. Results

Completeness of Separation 1. As much as possible of the antibody with the bound fraction of the labeled estrogen should pass through the Sephadex G-25 fine minicolumns. As an indicator of this passage, human [125I~-globulin in gelatin buffer was transferred to the Sephadex G-25 fine columns and treated as incubates. Two series, each with eight samples, were used with 3 ng and 150 ng of y-globulin, respectively. These amounts of y-globulin were chosen as the amount of antibody used in the assay in one vial was estimated to be 15 ng. The total counting activity was determined by transfer of identical amounts of [l~5I]y-globulin to four control vials for each of the two series. After gel centrifugation the counting activity was measured of both the column eluates and the used Sephadex G-25 fine minicolumns, which were placed directly in y-counting vials and counted in a Packard TriCarb scintillation spectrometer Model 3003. From the results in Table I it is seen that more than 97% of the radioiodinated y-globulin passed through the Sephadex G-25 minicolumns. 2. As little as possible of the free fraction of estrogen should pass through the Sephadex G-25 fine minicolumns. This was estimated by incubation of tritiated estrogen without antibody followed by centrifugation through the Sephadex G-25 fine minicolumns. As seen in Table II, 1% or less of the tritiated estrogen applied will pass through the minicolumns.

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TABLE I PASSAGE OF HUMAN [~25I]y-GLOBULIN THROUGH THE SEPHADEX G-25 FINE MINICOLUMNS [12~I]y-Globulin in control vials (mean cpm a - SD, n=4)

[~25I]y-Globulin in column effluent (mean cpm - SD, n=8)

Recovery in column effluent (mean % ± SD, n=8)

Rest activity in columns after use (mean % ± SD, n =8)

431,972 ± 1204 (approx. 150 ng) 11,495 - 106 (approx. 3 ng)

421,254 -- 2386

97.5 ± 0.6

1.3 - 0.3

11,197 --- 109

97.4 - 0.9

1.6 ± 0.2

cpm = counting activity as counts per minute.

T A B L E II PASSAGE OF TRITIATED ESTROGEN THROUGH THE SEPHADEX G-25 FINE MINICOLUMNS AFTER INCUBATION WITHOUT ANTIBODY A m o u n t of tritiated estrogen applied to minicolumns (mean cpm a _+ SD, n = 4)

Tritiated estrogen in column effluent (mean cpm ± SD, n = 8)

RIA including Sephadex LH-20 chromatography Estrone, 2447 ___ 42 Estradiol-17fl, 1878 - 32

16.2 - 1.8 (0.7 - 0.1%) 18.8 ± 6.5 (1.0 --- 0.4%)

RIA without Sephadex LH-20 chromatography Estradiol-17/3, 8292 - 175 Estriol, 10,338 ± 193

84.8 --_ 8.5 (1.0 --- 0.1%) 23.3 +-- 2.9 (0.2 -+ 0.03%)

a cpm = counting activity as counts per minute.

3. During the gel centrifugation of incubates with antibody it was of major importance that the fraction of tritiated estrogen bound to antibody should not be contaminated by the free fraction, which will later pass through the columns by continued elution (Fig. 1). This was investigated by repeated centrifugation of estrogen samples from standard curves at 400 and 1000 pg levels after application of 250/zl of albumin buffer. Table III shows that approximately 0.5% of the amount of tritiated estrogen in the sample was eluted. However, after repeated application of 500/zl of albumin buffer and gel centrifugation somewhat more tritiated estrogen appeared in the effluent. 4. After application of the incubates onto the minicolumns, the equilibrium obtained during incubation is changed by the stripping effect to

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1000. 750 500 250

ml ALBUMIN BUFFER FIG. I. Counting activity (cpm, ordinate) of minicolumn effluent and volume (milliliters, abscissa) of albumin buffer applied onto the minicolumn. Centrifugation was repeated after every application. The first peak represents antibody-bound tritiated estrogen: the second peak represents e]ution of tritiated free estrogen retained in the minicolumn.

TABLE III RESULTS OF TWO REPEATED SEPHADEX G-25 FINE CENTRIFUGATIONS WITH ALBUMIN BUFFER AFTER CENTRIFUGATION OF THE SAMPLES Albumin buffer Sample

250/zl

500 p.1

12.8 - 3.6 0.36

23.5 ± 3.9 0.67

14.0 --_ 2.2 0.46

23.3 -+ 4.9 0.76

52.2 - 4.1 0.63

286.0 -+ 20.9 3.45

51.1 ± 6.0 0.45

130.5 ± 9.0 1.16

RIA including Sephadex LH-20 chromatography Estrone samples (n = 4), 3511 cpm a, 400 pg Mean cpm ± SD Percentage Estradiol-17/] samples (n = 4), 3056 cpm, 400 pg Mean cpm - SD Percentage

RIA without Sephadex LH-20 chromatography Estradiol-17/3 samples (n = 4), 8292 cpm, 1000 pg Mean cpm - SD Percentage Estriol samples (n = 4), 11,255 cpm, 1000 pg Mean cpm - SD Percentage a cpm, counting activity as counts per minute. t h e a d v a n t a g e o f t h e f r e e f r a c t i o n o f t r i t i a t e d e s t r o g e n . T h i s is r e t a i n e d in t h e m i n i c o l u m n a n d t h e a n t i b o d y - b o u n d f r a c t i o n in t h e c o l u m n e f f l u e n t is reduced. Tables IV and V show that after contact of the incubates with the minicolumns for 5 min the stripping effect of the minicolumns during the next 5-min period will be less pronounced than after further contact.

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TABLE IV INFLUENCE OF THE DURATION OF CONTACT OF THE INCUBATES WITH THE M1NICOLUMNS ON THE AMOUNT OF ANTIBODY-BOUND TRITIATED ESTRONE AND ESTRADIOL-17fl PASSING THROUGH THE SEPHADEX G - 2 5 FINE MIN1COLUMNS a Estrone Time of incubate on microcolumn at 2 ° (rain) 0 5 10 15 20 30 60 120

E s t r a d i o l - 1713

0 pg, n = 4 (cpm b - SD)

100 p g , n = 4 ( c p m -+ S D )

1319.4 1169.5 1134.1 1044.5 1012.2 933.9 867.8 804.4

499.1 448.1 430.2 398.6 371.2 343.3 299.4 304.0

-+ -+ ---+ -+ -+

22.7 22.9 24.2 34.8 20.1 24.3 22.8 7.9

-+ --+ --+-

23.7 15.0 12.6 11.4 20.6 10.7 10.5 11.4

0 pg, n = 4 (cpm - SD) 1081.3 1005.3 982.1 948.9 872.0

-+ _+ _+ ---

100 p g , n = 4 ( c p m -+ S D )

28.5 23.1 18.2 16.0 25.8

8 0 2 . 2 --- 2 3 . 6 7 8 4 . 5 --- 2 4 . 8

571.6 492.5 487.1 482.9 478.1 418.7 404.1 399.8

-+ -+ -+ -+ -+ ---+

15.8 17.3 10.5 9.3 20.4 22.4 10.5 20.8

a A n t i b o d y A - 1 w a s i n c u b a t e d w i t h u n t r i t i a t e d e s t r o n e a n d e s t r a d i o l - 1 7 / 3 in a m o u n t s o f 0 p g a n d 100 p g a n d w i t h t h e s a m e t r i t i a t e d e s t r o g e n , r e s p e c t i v e l y . b cpm = counting activity as counts per minute.

TABLE V INFLUENCE OF THE DURATION OF CONTACT OF THE INCUBATES WITH THE MINICOLUMNS ON THE AMOUNT OF ANTIBODY-BOUND TRITIATED ESTRADIOL-17/3 AND ESTRIOL PASSING THROUGH THE SEPHADEX G - 2 5 FINE MINICOLUMNS a E s t r a d i o l - 17/3 Time of incubate on microcolumn a t 2 ° (rain) 0 5 10 15 20 30 60 120

0 pg, n = 4 ( c p m -+ SD) 2471.8 2400.2 2367.7 2257.6 2165.6 2107.3 2011.0 1960.1

-+ -+ + + -+ + -+ -+

33.1 45.1 48.1 14.3 43.3 53.7 32.8 33.9

Estriol

500 p g , n = 4 ( c p m --+ S D ) 418.2 397.3 391.3 385.7 362.9 347.5 328.3 327.6

+ + + + -+ -+ + -+

13.0 45.1 11.2 12.7 11.4 8.4 14.7 17.7

0 pg, n = 4 (cpm + SD) 1838.0 1764.4 1711.6 1595.5 1446.2 1237.4 1175.4 1123.2

+ + + -+ -+ -+ -+ -+

19.1 8.4 11.2 31.3 47.6 49.3 21.4 46.9

500 pg, n = 4 ( c p m --- S D ) 1131.6 1010.3 989.5 927.1 852.3 812.3 754.5 700.9

+ + + + -+ -+ -+ -+

31.4 34.7 14.9 33.3 33.9 16.0 29.1 32.5

a A n t i b o d y A - 2 w a s i n c u b a t e d w i t h u n t r i t i a t e d e s t r a d i o l - 1 7 f l a n d a n t i b o d y A - 3 w a s inc u b a t e d w i t h u n t r i t i a t e d e s t r i o l b o t h in a m o u n t s o f 0 a n d 5 0 0 p g a n d w i t h t h e s a m e tritiated estrogen, respectively. b Cpm = counting activity as counts per minute.

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TABLE VI PRECISION OF SEPHADEX G-25 FINE CENTRIFUGATION ESTIMATED BY APPLICATION TO 20 MINICOLUMNS OF SAMPLES FROM ONE MAJOR INCUBATION VOLUME OF EACH ESTROGEN, RESPECTIVELY

Estrogen (pg/vial)

Measured value Coefficientof (mean +- SD) variation(%)

RIA including Sephadex LH-20 chromatography Estrone, 250 Estradiol-17/3, 250

246.9 - 6.3 239.6 - 6.4

2.5 2.7

240.4 ± 7.2 945.6 - 51.5

3.0 5.4

RIA without Sephadex LH-20 chromatography Estradiol-17/3, 250 Estriol, 1000

Precision Major incubation volumes were prepared for each of the estrogen assays, and from each incubate 600-/~1 samples were applied onto 20 minicolumns that were centrifuged together. Table VI reports the values that were measured with coefficients of variation between 2.5% and 5.4%.

Practicability One hundred Sephadex G-25 fine minicolumns are prepared during half an hour. By means of the Sephadex G-25 fine gel centrifugation procedure, about 100 separations are performed in 1 hr. Comments The reliability of the separation step in RIA of estrogens by means of gel centrifugation using Sephadex G-25 fine minicolumns is demonstrated by the following observations. 1. More than 97% of [l~sI~/-globulin will pass the minicolumns. This is supposed to apply also to the antibody-bound fraction of tritiated estrogen, 2. Only 1% or less of the free fraction of tritiated estrogen will escape retention in the minicolumns, 3. The counting activity is low in the column efluent, appearing by repeated application of albumin buffer and centrifugation immediately after the passage of the antibody-bound fraction of tritiated

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estrogen and before the elution of the unbound fraction retained in the minicolumns, 4. The stripping effect of the minicolumns is present only to a low degree and kept constant by a fixed period of 5 min from application of incubate before centrifugation. Abraham 7 reported that using an antibody with an affinity constant of more than 109 1/M the stripping effect should be negligible in a RIA including charcoal as a separating agent. Lindberg et al. 8 were not able to use dextran-coated charcoal in their estriol RIA because of an initial binding of only 3%, but changing of the separation step to include precipitation with ammonium sulfate gave an initial binding of 81%. It is regarded as an advantage to use only one type of separation technique for different assays in the laboratory, and the versatility of the Sephadex G-25 fine minicolumn method is demonstrated as the affinity constant of the estriol-antibody A-3 was only 5.10 x 108 1/M. The precision of separation by the minicolumns was reduced with a low affinity constant but still reasonable with a coefficient of variation of 5.4% in the estriol assay. 7 G. E. Abraham, Acta Endocrinol. (Copenhagen) Suppl. 183 (1974). 8 B. S. Lindberg, P. Lindberg, K. Martinsson, and E. D. B. Johansson, Acta Obstet. Gynecol. Scand. Suppl. 32 (1974).

[23] T h e T a l c - R e s i n - T r i c h l o r o a c e t i c Acid Test for Screening Radioiodinated Polypeptide Hormones By BARBARAB. TOWER, MORTON B. SIGEL, RUSSELL E. POLAND,

W. P. VANDERLAAN, and ROBERT T. RUBIN A rapid, reliable physicochemical method for predicting the immunologic performance of ~q-labeled hormones in radioimmunoassays (RIA) has been developed: the talc-resin-TCA test. 1 The search for an accurate physicochemical indicator of the quality of a labeled hormone has been undertaken for three decades,2-8 but most techniques have had limB. B. Tower, M. B. Sigel, R. T. Rubin, R. E. Poland, and W. P. VanderLaan, Life Sci. 23, 2183 (1978). S. A. Berson, R. S. Yalow, S. S. Schreiber, and J. Post, J. Clin. Invest. 32, 746 (1953). S. A. Berson, R. S. Yalow, A. Bauman, M. A. Rothschild, and K. Newerly, J. Clin. Invest. 35, 170 (1956). 4 p. Sonksen and S. Refetoff, in "Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 89. Churchill-Livingstone, Edinburgh, 1971. METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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estrogen and before the elution of the unbound fraction retained in the minicolumns, 4. The stripping effect of the minicolumns is present only to a low degree and kept constant by a fixed period of 5 min from application of incubate before centrifugation. Abraham 7 reported that using an antibody with an affinity constant of more than 109 1/M the stripping effect should be negligible in a RIA including charcoal as a separating agent. Lindberg et al. 8 were not able to use dextran-coated charcoal in their estriol RIA because of an initial binding of only 3%, but changing of the separation step to include precipitation with ammonium sulfate gave an initial binding of 81%. It is regarded as an advantage to use only one type of separation technique for different assays in the laboratory, and the versatility of the Sephadex G-25 fine minicolumn method is demonstrated as the affinity constant of the estriol-antibody A-3 was only 5.10 x 108 1/M. The precision of separation by the minicolumns was reduced with a low affinity constant but still reasonable with a coefficient of variation of 5.4% in the estriol assay. 7 G. E. Abraham, Acta Endocrinol. (Copenhagen) Suppl. 183 (1974). 8 B. S. Lindberg, P. Lindberg, K. Martinsson, and E. D. B. Johansson, Acta Obstet. Gynecol. Scand. Suppl. 32 (1974).

[23] T h e T a l c - R e s i n - T r i c h l o r o a c e t i c Acid Test for Screening Radioiodinated Polypeptide Hormones By BARBARAB. TOWER, MORTON B. SIGEL, RUSSELL E. POLAND,

W. P. VANDERLAAN, and ROBERT T. RUBIN A rapid, reliable physicochemical method for predicting the immunologic performance of ~q-labeled hormones in radioimmunoassays (RIA) has been developed: the talc-resin-TCA test. 1 The search for an accurate physicochemical indicator of the quality of a labeled hormone has been undertaken for three decades,2-8 but most techniques have had limB. B. Tower, M. B. Sigel, R. T. Rubin, R. E. Poland, and W. P. VanderLaan, Life Sci. 23, 2183 (1978). S. A. Berson, R. S. Yalow, S. S. Schreiber, and J. Post, J. Clin. Invest. 32, 746 (1953). S. A. Berson, R. S. Yalow, A. Bauman, M. A. Rothschild, and K. Newerly, J. Clin. Invest. 35, 170 (1956). 4 p. Sonksen and S. Refetoff, in "Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 89. Churchill-Livingstone, Edinburgh, 1971. METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

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ited success in yielding consistently reliable results. 5'e'a'x° With the present t a l c - r e s i n - T C A test, however, we have successfully anticipated the RIA behavior of x25I-labeled human (h) and rat (r) prolactin (PRL), growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) prepared from more than 100 iodinations. Direct immunoreactive assessment of a25I-labeled hormones by the setting up of standard curves or serum standards may be costly and time consuming. Similarly, the excess antibody test may be wasteful of limited reagents and may provide only incomplete information. 5 The t a l c - r e s i n TCA test, on the other hand, provides an immediate, accurate answer as to whether one should commit valuable samples and technical time to an RIA, using a given labeled hormone preparation. The t a l c - r e s i n - T C A test is based on three different physicochemical properties of iodohormones: adsorption to talc, exclusion from resin, and precipitation by trichloroacetic acid (TCA). The test patterns are distinct for monomeric iodohormone, aggregated iodohormone, and 125I-labeled salts (or free 1251). Thus, a usable iodohormone may be identified quickly and accurately by its meeting the criteria of greater than 90% talc adsorption, less than 25% bound to resin, and greater than 90% TCA precipitation. In addition, the talc and TCA percentages should agree within 3%. Radioiodinated human and rat PRL, TSH, GH, and L H have been prepared by different iodination procedures. All purified monomeric iodohormones producing sensitive and reliable RIA results showed the characteristic talcresin-TCA test results of >90% talc adsorption, <25% resin binding, and >90% TCA precipitation. T h e T a l c - R e s i n - T C A Test

Reagents: Talc tablets, 100 mg, Ormont Drug & Chemical Co., Englewood, New Jersey

s W. M. Hunter, in "Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 1. Churchill-Livingstone, Edinburgh, 1971. 8 R. S. Yalow and S. A. Berson, Trans. N. Y. Acad. Sci. 28, 1033 (1966). 7 H. Pinto, B. L. Wajchenberg, O. Z. Higa, I. Torres de Toledo e Souza, R. S. Werner, and R. R. Pieroni, Clin. Chim. Acta 60, 125 (1975). 8 H. Pinto, A. C. Lerario, I. Torres de Toledo e Souza, B. L. Wajchenberg, E. Mattar, and R. R. Pieroni, Clin. Chim. Acta 76, 25 (1977). 9 S. A. Berson and R. S. Yalow, in "Radioisotope Techniques in the Study of Protein Metabolism," p. 29. International Atomic Energy Agency Technical Reports Series No. 45, Vienna, 1965. 1o F. C. Greenwood, in "'Principles of Competitive Protein Binding" (W. D. Odell and W. H. Daughaday, eds.), p. 288. Lippincott, Philadelphia, Pennsylvania 1971.

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Bio-Rad anion exchange resin, Dowex AG 1-xl0, 200-400 mesh, chloride form, was used as received. However, this resin has been discontinued by the manufacturer, but we have obtained comparable results by using the Bio-Rad AG l-x8 resin, 200-400 mesh, chloride form, as a replacement (Bio-Rad catalog No. 1401451). A plastic 1.0 ml syringe may be used to measure the resin. Trim off tip of syringe at the zero graduation. Retract the plunger to measure 0.25 ml, and tamp syringe into resin. Dispense resin into test tube by pushing clown syringe plunger (0.25 ml should equal 150 mg resin). Trichloroacetic acid (TCA), 10%, reagent grade RIA assay buffey with 1% bovine serum albumin (BSA) added (1% BSA) lZSI-labeled hormone diluted in 1% BSA to l04 cpm or the counts normally added to each assay tube Procedure

1. Prepare three 12 × 75 mm test tubes, each containing 1 ml of diluted 125I-labeled hormone. 2. To the first tube, add 1 talc tablet. 3. To the second, add 150 mg (0.25 ml) of resin. 4. To the third, add 1 ml of 10% TCA. 5. Vortex each tube; walls of the talc and resin tubes may be washed down with an additional 1 ml of 1% BSA. Centrifuge 10-20 min at 2000-2500 rpm. 7. Carefully decant supernatants into clean 12 × 75 mm tubes. 8. Count the supernatant (super.) and precipitate (ppt) for each of the three tests. . Calculate the percentage of precipitate for each test as .

% ppt =

ppt × 100 ppt + super.

General Considerations

As mentioned, optimal RIA results are obtained from monomeric iodohormones showing greater than 90% adsorbed to talc, less than 25% bound to resin, and greater than 90% precipitated by TCA, with the talc and TCA results within 3% agreement. Any change in these physicochemical parameters indicates a change in the structure of the iodohormone. Since a relatively minor change in the structure of a polypeptide hormone may lead to drastically altered immunoreactivity, it follows that rigidly adhering to the optimum t a l c - r e s i n - T C A test guidelines to screen

[23]

TALC--RESIN--TCA TEST FOR HORMONES

325

the iodohormones before use will eliminate a major cause of unreliable assays. Talc will adsorb the iodohormone, and it has been used in RIA to separate bound from free hormone 1' and to purify iodohormones. TM Hormonal aggregates will easily dissociate from talc, ~3 and thus more than 90% of the labeled hormone should remain adsorbed to talc. Less than 90% talc adsorption indicates the formation of either free iodide or basic aggregates. Since acidic aggregates also may adsorb to talc, the talc and TCA percentages should agree within 3%. The Dowex AG 1-xl0 ion exchange resin used in our test has the capacity to remove both acidic aggregates and free [~25I]iodide salts. This resin has been used to monitor the iodination reaction 14-17 and to remove free iodide formed during storage of the purified labeled material/s TCA precipitation of protein has been the standard test used to monitor the quality of radiolabeled material for in vivo studies 2,3 and for use in RIA. TM The nonprecipitable material can be either free iodide or acid-soluble peptide fragments. In either case, material showing less than 90% TCA precipitability will have a markedly decreased immunoreactivity. We again stress that for optimal RIA results the TCA and talc percentages should agree within 3%.

Results

The glucose oxidase-lactoperoxidase (GO-LPO) iodination method of Tower et al. 2° was used to produce [~25I]hPRL. After purification on a column of Sephadex G-100, the major peaks of iodinated material were screened for use in RIA by the t a l c - r e s i n - T C A test (Fig. 1). Peak III material obtained from the Sephadex G-100 gel filtration purification was the 11 G. Rosselin, R. Assan, R. S. Yalow, and S. A. Berson, Nature (London) 212, 355 (1966). 1~ R. S. Yalow and S. A. Berson, Nature (London) 212, 357 (1966). 13 p. Cuatrecasas and M. D. Hollenberg, Biochem. Biophys. Res. Commun. 62, 31 (1975). 14 A. S. McFarlane, in "Radioisotope Techniques in the Study of Protein Metabolism," p. 1. International Atomic Energy Agency Technical Reports Series No. 45, Vienna, 1965. 1~ T. Chard, M. J. Kitau, and J. Landon, J. Endocrinol. 46, 269 (1970). le T. Goodfriend, in "Protein and Polypeptide Hormones" (M. Margoulies, ed.), p. 624. Excerpta Med. Found., Amsterdam, 1969. 17 R. J. Lefkowitz, J. Roth, W. Pricer, and I. Pastan, Proc. Natl. Acad. Sci. U. S. A. 65, 745 (1970). is M. A. Lesniak, J. Roth, P. Gordon, and J. R. Gavin, Nature (London), New Biol. 241, 20 (1973). 19 A. Von Zur Muhlen, in "Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 83. Churchill-Livingstone, Edinburgh, 1971. s0 B. B. Tower, B. R. Clark, and R. T. Rubin, Life Sci. 21, 959 (1977).

PERCENT OF ADDED COUNTS PRECIPITATED Talc Resin TCA "X'optimol >90---% <25% >90% Peak [ 74 Peak ]] 44 47 95 *Peak l"rT 98 22 34 Peak ~. 35 94

"~

5 --

]~ A

4 -

/ I I

/ /

x Q. 2

20

50

40

50 60 70 FRACTION ( 2 m l )

80

90

FIG. 1. Gel filtration profile of ~=SI-labeled human prolactin, p=5I]hPRL, (VLS No. 4) iodinated by the glucose oxidase-lactoperoxidase ( G O - L P O ) method and purified on a 2 x 50 cm Sephadex G-100 column. Elution solvent was 50 mM phosphate buffer, pH 7. l, containing 0.15 M NaCl, 0.1% sodium azide, and 0.1% crystalline bovine serum albumin. After chromatography, fractions of the major peaks were checked with the talc-resin-trichIoroacetic acid (TCA) test. Peak III, the material suitable for use in radioimmunoassay, showed >90% talc adsorption, <25% resin binding, and >90% TCA precipitation. Peaks I and II are aggregated hormone, and peak IV is dissociated l=SI-labeled salts and peptid¢ fragments (Tower et al.1).

PERCENTOF ADDED COUNTS PRECIPITATED Talc Resin TCA ~oplimol >9o~ <25=/= >90% 94

Tn'

5-

95

4x ~3a. 2-

20

I

30

I

40

I

I

I

50 60 70 FRACTION ( I ml)

I

80

I

90

FIG. 2. Gel filtration profile o f ~25I-labeled human prolactin, [I=S[]hPRL ( V L S N o . 3) iodinated by the chloramine-T method and purified on a 1.5 x 30 cm Sephadex G-100 column, precoated with b o v i n e serum albumin. E l u t i o n solvent was 10 m M phosphate buffer, p H 7.5, containing 0.15 M N a C I and 1 : 10,000 M e r t h i o l a t e . The labeled h o r m o n e was purified just p r i o r to use. The t a i c - r e s i n - t r i c h l o r o a c e t i c acid ( T C A ) test results showed that peak ] I [ material was suitable f o r use in radioimmunoassay ( T o w e r et al.t).

[23]

TALC--RESIN--TCA TEST FOR HORMONES

327

TALC-RESlN-TRICHLOROACETIC ACID (TCA) TEST RESULTS FOR 125-I-LABELED HORMONES PREPARED BY THE GLUCOSE OXIDASE--LACTOPEROXIDASE(GO-LPO) IODINAT1ON METHOD Peak III 125I-hormone preparation (NIH batch no.) a hGH (HS1394) hTSH (AFP 1) hPRL (VLS 4) hPRL (AFP 1) hLH (LER960) rGH (I-2) rGH (1-3) rTSH (1-4) rPRL (1-2) rPRL (1-3) rLH (1-4)

Optimal:

Talc

Resin

>90%

<25%

TCA >90%

97 95 98 96 93 96 97 98 90 93 96

3 20 22 19 11 13 15 9 22 18 13

96 94 95 96 91 97 98 98 93 94 93

a Peak III material from 2 x 50 cm G-100 gel filtration column, as in Fig. 1. Hormone preparations were obtained from the National Pituitary Agency, through NIAMDD or Dr. A. F. Parlow.

usable monomeric iodohormone which produced reliable radioimmunoassays. The chloramine-T iodination method of Hunter and Greenwood 21 also was used to produce [125I]hPRL, as shown in Fig. 2. Again, peak III of the G-100 column purification yielded talc-resin-TCA results within the defined limits of usable material, which was verified by reliable RIA results. 22 Iodo-hPRL prepared by either iodination method thus will yield reliable RIA results if the talc-resin-TCA test shows >90% adsorbed to talc, <25% bound to resin, and >90% precipitated by TCA (agreeing with talc -+3%). The G O - L P O iodination and G-100 gel filtration purification of human and rat PRL, TSH, LH and GH all yielded results similar to those shown in Fig. 1. The peak III iodohormone that gave reliable assay results always fell within the aforementioned limits when screened by the talc-resin-TCA test (see the table). Discussion

Storage Labeled Hormone. Two-milliliter fractions of the peak III material prepared by the G O - L P O iodination method (Fig. 1) should be stored at 21 W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). ~ Y. N. Sinha, F. W. Selby, U. J. Lewis, and W. P. VanderLaan, J. Clin. Endocrinol. Metab. 36, 509 (1973).

328

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS %

n7

PERCENT OF ADDED COUNTS PRECIPITATED Talc Resin TCA ~optimal >90~/. < 2 5 % >90%

54

[23]

Peak I Peak ~

58 43

56 47

92 75

~PeakTFr Peok'rV

90 33

22 95

93 (2

o_ t.) 2 1-

20

30

40

50

60

70

80

90

FRACTION (2ml)

FIG. 3. Elution pattern of nSl-labeled rat prolactin, [12~I]rPRL (AFP 1-2), prepared by the glucose oxidase-lactoperoxidase iodination procedure and purified as described in Fig. I for human PRL. The unlabeled hormone was stored as a dry powder, desiccated, and weighed out just prior to iodination; this resulted in a maximum yield of peak III material. The peak III monomeric hormone is suitable for use in radioimmunoassay (cf. Fig. 4) (Tower et al.~).

- 20 ° with 50 mg of resin added to each tube. Extra BSA may be added for protection; 0.1 ml of 0,5% BSA in phosphate-buffered saline, for example, may be added to each tube. The radiolysis of aqueous solutions produces radicals that attack the iodohormones, and BSA is the traditional scavenger added to counteract this process. 6 A note of caution about the BSA used with iodohormones: some lots of BSA contain substances, believed to be enzymes, that will degrade the labeled preparation33 This degradation will be apparent by an increased deiodination rate, an increase in aggregate formation, or a combination of both. Careful appraisal of each lot of crystalline BSA will eliminate the "bad" lots. Unlabeled Hormone. Rat prolactin (AFP 1-2) was labeled by the G O LPO method and purified by gel filtration on Sephadex G-100 (Fig. 3). This material resulted in a high yield of peak III material. The rPRL was stored as received from Dr. A. F. Parlow, through the NIAMDD rat hormone distribution program. The rPRL was weighed out and diluted just before iodination. Contrast this storage procedure with the results shown in Fig. 4. Here the same rPRL 1-2 preparation was diluted in PO4 buffer, aliquoted, and frozen before iodination. After 1 month of storage, an ali=a A. E. Freedlender, in "Protein and Polypeptide Hormones '' (M. Margoulies, ed.), p. 608. Excerpta Med. Found., Amsterdam, 1969.

[23]

TALC--RESIN--TCA TEST FOR HORMONES

5-

x

PERCENT OF ADDED COUNTS PRECIPITATED Talc Resin TCA %ptimal >90~, <25% >90% Peak I 60 53 92 Peak ~T 63 55 91

I 1"1" ~ A /

%

/

I

~

4-

329

Peok 177"

82

60

88

t

3-

13_ 2I-

20

i

i

50

40

50

60

i

i

~

70

80

90

FRACTION (2ml}

FIG. 4. Elution pattern of l=5I-labeled rat prolactin, [125I]rPRL (AFP I-2), prepared by the glucose oxidase-lactoperoxidase iodination method and purified as described in Fig. 1 for human PRL. Before this material was labeled, it had been diluted in phosphate buffer to 100/~g/ml, aliquoted into separate reaction vials, and stored at - 2 & for I month. During storage, the unlabeled hormone apparently converted to aggregate, as the talc-resin-(TCA) test indicated that no usable monomeric hormone remained available for iodination (cf. Fig. 3) (Tower et al.1).

quot was defrosted and iodinated by the G O - L P O method. Most of peak III had converted to the unusable, aggregated peak I or peak II material, and the t a l c - r e s i n - T C A test results showed that no material obtained from this iodination could be used for RIA. This was confirmed with an antibody binding check on the peak III material; less than 10% of the expected immunoreactivity remained. Therefore, in order to obtain a maximum yield from the valuable pituitary hormone preparations, they must be stored as a dry, lyophilized powder, in a desiccator, at 4°. The preparation should be weighed out just before use and diluted immediately before iodination with cold phosphate buffer. The phosphate buffer must not contain azide or any other enzyme inhibitors, as they will completely inhibit the G O - L P O iodination reaction. The shelf life of any unlabeled hormone preparation is not known. Interbatch differences in the quality of unlabeled human PRL preparations have been noted. ~a Some materials will produce reliable RIAs for years, whereas others will show a rapid decline in immunoreactivity and an increase in aggregate formation. The careful monitoring of the behavior of the labeled hormone by the t a l c - r e s i n - T C A test will detect subtle changes in the hormone preparations. ~4 V. Fang, J. Armstrong, and I. G. Worsley, Clin. Chem. 24, 941 (1978).

330

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[23]

125Ifor Iodination Interbatch differences in the quality of the radioiodine purchased from commercial suppliers have been apparent, zS-2r The use o f " b a d " iodine can result in several different problems, including a low incorporation rate of iodine and an increased formation of aggregated iodohormone either during the iodination reaction or upon storage. We have requested Union Carbide Corp. (Tuxedo Park, New York) to supply us with Na~25I at a concentration of <400 mCi/ml in dilute NaOH. Since this request for Na~ZSI at a lower concentration, we have not noted any difficulties with 36 lots of radioiodine. The NalZSI in dilute NaOH gives reliable iodination results for as long as 2 months when stored just as received from the supplier, at room temperature.

Iodination Method Reliable, sensitive RIAs can be obtained regularly with the G O - L P O iodination method. 2° A brief outline of the procedure follows. Reagents 12sI for protein iodination, carrier-free, diluted by supplier to <400 mCi/ml (Union Carbide Corp., Tuxedo, New York). Highly purified lactoperoxidase (LPO), at least 90 U/mg with an A412/A~8o >0.8 (P-L Biochemicals, 1037 West McKinley Ave., Milwaukee Wisconsin). Weigh out and dilute just before use. Glucose oxidase (GO): any purified preparation containing the lowest possible catalase contamination. Type VII from Sigma Chemical Co. (St. Louis Missouri) is satisfactory. Weigh out and dilute just before use. Hormones for iodination: the currently distributed N I H materials. The hormones should not be diluted and aliquoted before storage. They should be stored dry, as received, in a desiccator at 4 ° and weighed out (4-7/~,g) and diluted in cold PO4 buffer just prior to iodination. Phosphate buffer, azide free, 0.1-0.5 M, is suitable for the iodination reaction, but the enzymes and hormone should be diluted in 10-50 mM buffer, as the higher molarity may cause aggregation. There must be no azide or other enzyme inhibitors in the buffers used for the iodination reagents. 25 y . Miyachi, A. C h r a m b a c h , R. Mecklenburg, and M. B. Lipsett, Endocrinology 92, 1725 (1973). ~e B. Schneider, R. Straus, and R. S. Yalow, Diabetes 25, 260 (1976). ~r W. M. H u n t e r , Br. Med. Bull. 30, 18 (1974).

[23]

T A L C - - R E S I N - - T C A TEST FOR H O R M O N E S

331

fl-D-(+)-Glucose: any reagent quality is suitable (e.g., Sigma Chemical Co., St. Louis Missouri). This is the specific substrate of the glucose oxidase. The glucose should be diluted just before being added to the reaction mixture. If the diluted material is allowed to stand, it will slowly levorotate to the inactive a-isomer. Crystalline BSA; check for purity before use (see above). Equipment Hamilton syringe, 5 ~1, with handle No. 85N. The syringe is used to pierce the rubber septum of the iodine shipping vessel. The top never should be taken off the iodine vessel. Micropipettes, Coming No. 7099 series Tuberculin syringe, 1 ml, connected by a short piece of rubber tubing to a micropipette mouthpiece. This makes a disposable unit for dispensing each reagent with the micropipettes and allows a gentle bubbling technique to be used. 02 is a necessary ingredient for the iodination reaction. Culture tubes, 6 × 50 mm; these are the reaction vials (12 × 75 mm culture tubes also may be used). Disposable 10 ml pipette-column of Sephadex G-50M Well-type gamma counter Hand monitor for assessing contamination Powder-free gloves Fume hood with > 100 cubic feet per minute draw, in which the iodination reaction is performed. Pasteur pipettes for transfer of iodination reaction mixture to Sephadex G-50M column. Plastic-backed paper to line the floor of the hood. Several layers should be used and removed, one layer at a time, to be discarded in the radioactive waste if spillage occurs during the procedure. Protocol sheets, prepared before the procedure and available for reference during the iodination. Procedure. Add, in order, to the reaction tube: 0.3-0.5 mCi of 1251; 40/zl of phosphate buffer; 10/~1 of LPO, 100/zg/ml in phosphate buffer; 40/.d (4-7/zg) of hormone; 10-20/zl of GO, 10/~g/ml or 100/Lg/ml in phosphate buffer; and 20 ~1 of 1% fl-o-(+)glucose in H~O. Wait 5-30 min, then stop the reaction by adding phosphate buffer containing 0.1% azide. Transfer the iodination reaction to a Sephadex G50 column and wash the reaction vial with 5% BSA-phosphate buffer, transfering the washes to the column. Start the column flow after all the washes and transfers have been completed. Collect 0.4-ml fractions in each of 20 tubes. Count each fraction for 0.1 min. Save the first (protein) peak, and dis-

332

RADIO|MMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[23]

card the second (1251) peak into the radioactive waste. The first peak must be purified on a 2 x 50 cm Sephadex G-100 column, or by other purification methods of your choice. Clean up the hood and monitor the area for contamination promptly upon completion of the iodination. We have been impressed with the favorable radiation safety aspects of the GO-LPO iodination technique. Since we have been using this technique, personnel monitoring for radiation contamination both by bioassay (thyroid counts) and by dosimeter readings have not been above background (NAB). Discussion. Iodohormones prepared by the G O - L P O iodination method are the most stable labeled preparations we have used. Standard curves obtained from a purified hPRL preparation (VLS No. 4) on the day of iodination and after 3 months of storage at - 20° are illustrated in Fig. 5. When stored properly, the stable G O - L P O iodohormones should give reliable, reproducible RIA results without further purification. However, chloramine-T-labeled hormones must be rechromatographed after storage, just prior to use. Regular monitoring of the quality of stored iodohormones before use in an RIA is easily accomplished with the talcresin-TCA screening test.

Exceptions The classical parameters of >90% talc adsorption and TCA precipitation, with ---3% agreement between the talc and TCA values, and <25% binding to resin are appropriate for polypeptide hormones with a certain chemical composition, including most anterior pituitary hormones. We have found, so far, two exceptions to the above parameters: the S variant form of human growth hormone (hGH-S) 2s and human FSH (all preparations). These are acidic proteins and show a decreased adsorption to talc and an increased binding to resin. If standardized, reproducible profiles can be obtained from hormones with parameters differing from the normal t a l c - r e s i n - T C A test profiles, then a valid predictive test will still be possible. The verifying support must come from the RIA data. The standard t a l c - r e s i n - T C A parameters of greater than 90% talc and TCA precipitability (___3%), with less than 25% bound to resin, was not established until RIA results confirmed the prediction from more than 100 iodinations. Therefore, caution should be exercised in establishing outlying parameters of the t a l c - r e s i n - T C A test profile for aberrant hormones. A different anion exchange resin may be substituted for the AG 1-xl0 ~s M. B. Sigel, W. P. VanderLaan, E. F. VanderLaan, and U. J. Lewis, 106, 92 (1980).

Endocrinology

[23]

TALC-RESIN-TCA

333

TEST FOR HORMONES

I00 80 o m

60 I-Z b.I W

20

0

i

Ol

i

OI

I

I0

ng hPRL

FIG. 5. Identical standard curves obtained from human prolactin (hPRL) (VLS No. 4) iodinated by the glucose oxidase-lactoperoxidase method and purified as described in Fig. 1. One standard curve (©--©) was obtained with freshly prepared peak III iodoprolactin on the day of iodination. The other standard curve ( O - - O ) was obtained from peak III material from the same iodination, which then had been stored at - 2 0 ° for 3 months before use in radioimmunoassay. Before storage, 50 mg of resin and 100/~l of 5% BSA had been added to each 2-ml fraction of the peak III material. Both standard curves were obtained with AFP No. 1 hPRL antiserum and AFP No. l hPRL standard. In both instances the nonspecific binding was 3% and the initial binding (B/T) was 32% (Tower et al.~).

resin (200-400 mesh, chloride form) for testing a protein with different chemical properties (lower or higher molecular weight, charge, isoelectric points, etc.). The AG 1-xl0 resin, and its manufacturer's substitute, AG 1-xS, have been suitable for most anterior pituitary ~25I-labeled hormones because of their affinity for free ~25I and their ability to bind acidic aggregates.

Screening of Commercially Prepared lodohormones On occasion, iodinations are not performed by the user. The talcresin-TCA test is a simple technique for validating the suitability of a25Ilabeled hormones obtained commercially, which often are expensive. As only the equivalent of three assay tubes of iodohormone are required for this simple screening test, it is not unreasonable to check the quality of every batch of commercially labeled material before its use, including iodohormones supplied with RIA kits. The t a l c - r e s i n - T C A test has been of great assistance in setting up a reliable glucagon assay. [x25I]Glucagon obtained from New England Nuclear (Boston, Massachusetts) was checked; it yielded t a l c - r e s i n - T C A

334

RADIOIMMUNOASSAYS AND

IMMUNORADIOMETRIC

ASSAYS

[24]

test results of > 9 0 % , < 2 5 % , and > 9 0 % , respectively. When the c o m m e r cial iodoglucagon deteriorated to the point where the test p a r a m e t e r s were < 9 0 % , > 2 5 % , and < 9 0 % , the material b e c a m e unusable and a new lot was purchased.

Application to Bioassays and Radioreceptor Assays T h e optimal R I A p a r a m e t e r s o f the t a l c - r e s i n - T C A test also apply to i o d o h o r m o n e s used in bioassays and radioreceptor assays (RRA). The talc and T C A testing has been used to m o n i t o r the degradation o f 125I-labeled h o r m o n e used with prolactin, 29 insulin, z° and growth h o r m o n e TMreceptors. Strict a d h e r e n c e to the limits o f > 9 0 % talc adsorption, < 2 5 % resin binding, and > 9 0 % T C A precipitation provides a rigorous quality control for the p r e s e n c e of bioactive and receptor-affinitive m o n o m e r i c iodohormone. Acknowledgments We are grateful to the National Pituitary Agency, NIAMDD, and to Drs. A. F. Parlow and U. J. Lewis for generous supplies of hormones and antisera. It is a pleasure to acknowledge the technical assistance of Ms. Mary Thomas and Mr. John Hammond and the secretarial asistance of Mrs. Debbie Hanaya. This research was supported by the Office of Naval Research Contract N00014-77-C-0245 and NIMH grants MH 28380 and MH 29491. R. T. R. is the recipient of NIMH Research Scientist Development Award MH 47363. 39 R. P. C. Shiu and H. G. Friesen, Biochem. J. 140 301 (1974). 30 p. F r e y c h e t , R. Kahn, J. Roth, and D. M. Neville, Jr., J. Biol. Chem. 247, 3953 (1972).

[24] Labeled Antibodies and Immunoradiometric

Their Use Assay

in the

By C. N. HALES and J. S. WOODHEAD Antibodies labeled by a variety of techniques and with different substances have been used for studies of antigens for very m a n y years. S o m e of the earliest e x a m p l e s o f labels include radioactive isotopes, fluorescent dyes, electron dense atoms, ferritin, and e n z y m e s . Radioactive iodine was used as a label m a n y years ago, the iodine being incorporated into unpurified plasma proteins. The main p u r p o s e o f these earlier investigations was the qualitative identification and localization o f various antigens. The use of antibodies and high specific activity radioisotopes in the production of antigen assays with a very high degree o f sensitivity was METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 19~0by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

334

RADIOIMMUNOASSAYS AND

IMMUNORADIOMETRIC

ASSAYS

[24]

test results of > 9 0 % , < 2 5 % , and > 9 0 % , respectively. When the c o m m e r cial iodoglucagon deteriorated to the point where the test p a r a m e t e r s were < 9 0 % , > 2 5 % , and < 9 0 % , the material b e c a m e unusable and a new lot was purchased.

Application to Bioassays and Radioreceptor Assays T h e optimal R I A p a r a m e t e r s o f the t a l c - r e s i n - T C A test also apply to i o d o h o r m o n e s used in bioassays and radioreceptor assays (RRA). The talc and T C A testing has been used to m o n i t o r the degradation o f 125I-labeled h o r m o n e used with prolactin, 29 insulin, z° and growth h o r m o n e TMreceptors. Strict a d h e r e n c e to the limits o f > 9 0 % talc adsorption, < 2 5 % resin binding, and > 9 0 % T C A precipitation provides a rigorous quality control for the p r e s e n c e of bioactive and receptor-affinitive m o n o m e r i c iodohormone. Acknowledgments We are grateful to the National Pituitary Agency, NIAMDD, and to Drs. A. F. Parlow and U. J. Lewis for generous supplies of hormones and antisera. It is a pleasure to acknowledge the technical assistance of Ms. Mary Thomas and Mr. John Hammond and the secretarial asistance of Mrs. Debbie Hanaya. This research was supported by the Office of Naval Research Contract N00014-77-C-0245 and NIMH grants MH 28380 and MH 29491. R. T. R. is the recipient of NIMH Research Scientist Development Award MH 47363. 39 R. P. C. Shiu and H. G. Friesen, Biochem. J. 140 301 (1974). 30 p. F r e y c h e t , R. Kahn, J. Roth, and D. M. Neville, Jr., J. Biol. Chem. 247, 3953 (1972).

[24] Labeled Antibodies and Immunoradiometric

Their Use Assay

in the

By C. N. HALES and J. S. WOODHEAD Antibodies labeled by a variety of techniques and with different substances have been used for studies of antigens for very m a n y years. S o m e of the earliest e x a m p l e s o f labels include radioactive isotopes, fluorescent dyes, electron dense atoms, ferritin, and e n z y m e s . Radioactive iodine was used as a label m a n y years ago, the iodine being incorporated into unpurified plasma proteins. The main p u r p o s e o f these earlier investigations was the qualitative identification and localization o f various antigens. The use of antibodies and high specific activity radioisotopes in the production of antigen assays with a very high degree o f sensitivity was METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 19~0by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

[24]

LABELED-ANTIBODY ASSAY METHODS

335

Radioimmunoassay

[125!Ag + Ag + Ab'~ lmmunoradiometric

~AgAb+II251]AgAb

Assay

Flo. 1. Outline of the reactions involved in radioimmunoassay and immunoradiometric assay techniques. Ag, antigen; Ab, antibody.

pioneered by Yalow and Berson I in their establishment of the technique of radioimmunoassay. As a result of this work and certain theoretical considerations, Miles and Hales 2,a decided to investigate whether, for soluble antigens, assays of equivalent or even greater sensitivity and with certain practical advantages might not be produced by the production of high specific activity purified radiolabeled antibodies. The technique so derived was termed an immunoradiometric assay to emphasize that the procedure was different from, and indeed the converse of, radioimmunoassay (Fig. 1). The distinction is important because the theory, practice, and optimization of the procedures are very different. Unfortunately, subsequently some commercial and other immunoradiometric assays have been labeled "radioimmunoassays," causing unnecessary confusion. Subsequent to the establishment of the immunoradiometric assay the procedure was developed further. The next step was the development of the two-site immunoradiometric assay. 4"5 The objective of this procedure was to use a specific antibody on a solid phase to extract antigen. This offers the possibility of purification and concentration. If, while the antigen remains bound, a second (labeled) antibody is bound at another site on the antigen, then the second antigen can be measured as a function of the amount of bound labeled antibody (Fig. 2). The fact that for measurement the antigen must be bound simultaneously to two different antibodies at two sites on the antigen offers considerable potential for increasing the specificity of the method. To emphasize this attribute and to distinguish the method from other procedures, such as the so-called i R. S. Yalow and S. A. Berson, Nature (London) 184, 1648 (1959). 2 L. E, M. Miles and C. N. Hales, Nature (London) 219, 186 (1968). 3 L. E. M. Miles and C. N. Hales, Biochem. J. 108, 6111 (1968). 4 G. M. Addison and C. N. Hales, Horm. Metab. 3, 59 (1971). 5 G. M. Addison and C. N. Hales, in "Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 447. Churchill-Livingstone, Edinburgh, 1970.

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'Ag 1251 Ab"

phaSe[Ab,

I

mAb'

FIG. 2. Diagram of the reaction steps in the two-site immunoradiometric assay. Ab' and Ab" represent antibodies directed against different antigenic determinants in the antigen Ag.

"sandwich" immunological techniques, this type of immunoradiometric assay was termed a "two-site" assay. Unfortunately, a number of workers have failed to appreciate this important distinction and have used the terms "sandwich" and "two-site" assay as though they are interchangeable, again giving rise to confusion. The term "sandwich" in reference to immunological detection and assay systems has become rather indiscriminately applied to a number of techniques that differ in principle and in purpose. Because of this and the resultant confusion that has arisen, it is probably best that the term be avoided altogether. Coons 6 in a review in 1956 referred to two types of layering technique for the measurement of antibodies using fluorescent labeled antibodies. In one, cells bearing antibodies were allowed to react with antigen and the bound antigens were used to bind fluorescent labeled antibodies, thus labeling antibody-producing cells. This test has been referred to subsequently as a sandwich assay of antibody. Coons also described a method for the detection of soluble antibody using tissue antigens for their extraction. The bound antibodies were then detected by the use of a fluorescent labeled anti-immunoglobulin. This technique may also be described as a sandwich assay in the sense of possessing multiple layers. However, it has become more usual to refer to methods whereby antigens or antibodies are detected by the use of an anti-immunoglobulin as "indirect" techniques, and we have employed this terminology here. Iodinated anti-immunoglobulins were used for the measurement of human antibodies to red cells 7 and subsequently for the measurement of IgE antibodies to allergens/Yet another situation in which the term sandwich has been used is in a radioimmunoassay of antibodies.9 In this technique tubes are coated with antigen and used to extract antibodies. The antibodies thus bound are measured by reaction with labeled antigen. This method, unlike the indirect methods, does not indicate the type of antibody bound. A further development of the immunoradiometric assay was the in6 A. 7 N. s L. S.

H. Coons, Int. Rev. Cytol. 5, 1 (1956). Costea, R. Schwartz, M. Constantoulakis, and W. Dameshek, Blood 20, 214 (1962). Wide, H. Bennich, and S. G. O. Johnsson, Lancet 2, 1105 (1967). E. Salmon, G. Mackey, and H. H. Fudenberg, J. lmmunol. 103, 129 (1969).

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337

Indirect lmmunoradiometric Assay

Ag + Abl[125I]antilgGI

AgAb([lZSIJantiIgO)

Inclirect Two-site Immunoracliometric Assay

~

Ab' ~i'AgAb"+f 125I]antilg0

N'~'~Ab, ~----IphaseIii' AgAb"[125 I] ant i lg 0

FIG. 3. Diagramsof the reactions in the indirect immunoradiometric and indirect two-site immunoradiometric assays. Ab' and Ab" represent antibodies directed against different antigenic determinants in the antigen Ag. direct immunoradiometric assay (Fig. 3). In this method the antibody used for measuring antigen ("first" antibody) is not directly labeled, but indirectly through the use of a labeled antibody to the immunoglobultn of the same animal species as that in which the first antibody is raised. The labeled anti-immunoglobulin antibody may be allowed to react with the first antibody before or after its reaction with antigen. It may also be used in conventional 1° or two-site 1~ immunoradiometric assays. In this chapter are described all these variations of the immunoradiometric assay. Since the range of actual and potential applications of the techniques is very large indeed, it is not possible in most instances to provide "recipes" that can be applied indiscriminately to any of these applications. We have therefore structured each section so that it begins by describing the principles underlying the procedure to be described, illustrates those aspects of the procedure that require attention in its application to different systems, and finally includes a full practical account of one example of the procedure. We hope that this will allow the construction of new assays with the minimum of difficulty. Preparation of Immunoadsorbents The need for extreme purity of the labeled antibody used as a reagent in an immunoradiometric assay, plus the physical and chemical similar~op. Beck and C. N. Hales, Biochem. J. 145, 607 (1975). 11S. J. Rainbow,J. S. Woodhead, D. K. Yue, S. D. Luzio, and C. N. Hales, Diabetologia 17, 229 (1979).

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ities that exist between antibody molecules and other IgG molecules present in serum, necessitates the adoption of special purification procedures. Fortunately, affinity chromatography using immobilized antigen (immunoadsorbent) is capable of providing a very high degree of purification in a single step. The starting material for such a purification of a specific antibody may be the IgG fraction derived from serum by a precipitation TM and/or by a chromatographic TM procedure, but this is not really necessary since an adequate purification can be achieved even when starting with such a crude protein mixture as whole serum. It is possible to achieve this rapid purification only by careful attention to the preparation and properties of the immunoadsorbent used to extract the specific antibody molecules. Immunoadsorbents can be prepared in a very wide variety of ways. The criteria to be satisfied in the choice of an immunoadsorbent are as follows: a chemically inert solid phase; high capacity for binding antigen; stability of the antigen-solid phase bond; low nonspecific uptake by the immunoadsorbent of protein other than antibody; and a high antibody-binding capacity. These criteria are satisfied by powdered cellulose-based immunoadsorbents. They can be prepared in such a finely divided state that there is a very large surface available for reaction. Furthermore diazonium derivatives of cellulose react with a variety of sites on proteins (e.g., primary amino groups, tyrosine, histidine) thus allowing for the presentation of the bound antigenic protein in a variety of configurations. Since it is often not known which part of the protein antigen is providing the antigenic determinant for a particular antibody, this versatility of orientation increases the possibility that many different antibodies to a single antigen will be capable of reacting with the immunoadsorbent. Aminocellulose

The overall method on which most of the preparation of immunoadsorbent is based is that of Gurvich e t a l . la (Fig. 4). The initial preparation of cellulose powder can be carded out by refluxing cotton wool with ethanol and acetyl chloride. However, a more convenient alternative is to use commercially available microgranular cellulose (Whatman Ltd., Springfield Mill, Maidstone, Kent). Five grams of cellulose are suspended in 20 ml of 90% ethanol containing 0.5 g of sodium acetate and 1.4 g of nitrobenzyloxymethyl pyridinium chloride (British Drug House, Poole, Dorset, U.K.). Sodium acetate is dissolved first in 2 ml of water, and the 12 C. A. Williams and M. W. Chase, in "'Methods Immunology and Immunochemistry (C. A. Williams and M. W. Chase, eds., Vol. 1, p. 307 (1967). 13 A. E. Gurvich, O. B. Kuzorela, and A. E. Tumanova, Biokhimiya 26, 934 (1961).

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4•--•]

CH2OCH20-cell ulose

Cellulose powder 4'

J

NO2 CI" m-nitrobenzyloxymethyl pyridinium chloride

I dr'etdhuic°tn 'otne

aminocellulose I suspension Initrous J acid

ammoniacal cupric hydroxide

Ilulose CH2OCH20-cellulose *N~

+polypeptide

~

H2OCH20-cellulose

~ ~CH2OCH20-cellulose N-~Polypeptide

FZG. 4. Outline of the series of chemical reactions involved in the preparation of a cellulose-based immunoadsorbent.

pyridinium reagent in 18 ml of ethanol. These are mixed in a petri dish with the cellulose. The slurry is dried at 70° in an oven, and then reaction is carried out by heating for a further 40 min at 125°. The resulting nitro derivative of cellulose is pale brown. It is washed three times with 200 ml of benzene in a sintered glass funnel fitted to a Biichner flask and finally sucked dry. The powder is then reduced by suspending it in 150 ml of water containing 30 g of sodium dithionite. The reaction is carded out for 30 min at 55-60 ° in a water bath with occasional shaking. The resulting aminocellulose is washed in a sintered glass funnel with alterntting water and 30% acetic acid until no smell of H2S remains. The solid is air dried and then pulverized in a mortar. The resulting powder is stable for several months stored over silica gel at 4°.

Diazocellulose The aminocellulose powder is initially dissolved in ammoniacal cupric hydroxide and reprecipitated to produce a flocculent suspension. In this form it is converted to the diazonium derivative with nitrous acid and immediately allowed to react with protein. The following quantities are adequate for the binding of up to 500 mg of protein assuming a molecular weight of the protein of approximately 100,000. For small polypeptides (molecular weight in the region of 10,000 or less) these quantities would only be sufficient to react with 50 mg of peptide.

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Cupric chloride, 1.5 g, is dissolved in 5 ml of water. To the green solution an excess (75 ml) of freshly prepared 1 M NaOH is added with constant stirring. The resulting blue precipitate is washed with water on two layers of Whatman No. 41 filter paper on a Biichner funnel until the pH of the wash falls below 9 ( - 100 ml). The precipitate is dissolved in 40 ml of ammonia solution (specific gravity, 0.880) to form a saturated solution. After stirring for at least 15 min the excess cupric hydroxide is removed by centrifugation. To the 40 ml of saturated solution of ammoniacal cupric hydroxide, 0.5 g of amino cellulose are added with constant stirring. After 15 min any undissolved aminocellulose is removed by centrifugation and the supernatant is decanted into 1.5 liter of water. Then 10% H~SO4 (v/v) is added to the dark blue solution until it is almost colorless and the pH is below 4. At this point the aminocellulose forms a white precipitate. After it has stood for 1 hr to allow the precipitate to settle, the supernatant is decanted and the precipitate is washed four times in water by centrifugation to remove all traces of copper. If in the later washes the suspension proves difficult to sediment, a few drops of 1 M hydrochloric acid are added. The aminocellulose is finally suspended in 50 ml of 2 M hydrochloric acid and cooled in an ice bucket; 2 ml of 1% sodium nitrite are added to generate nitrous acid. The oxidant may then be detected by its blackening starch iodide paper. It is allowed to react with the amino cellulose for 20 min at 4°, and then solid urea is added until the starch iodide test is almost negative (approximately 5 g of urea). The diazocellulose is washed by centrifugation at 4° three times in water and twice in 0.2 M borate buffer, pH 8.2. It is important that all the washing solutions be kept at 4 °. The diazocellulose is finally suspended in 0.2 M borate buffer, pH 8.2, to give a concentration of approximately l0 mg/ml. At this pH the diazocellulose is pale yellow-green. A test of the success of the preparatioi~ is given by adding a little of the suspension to a small spatula end of/3-naphthol suspended in 1 ml of water, whereupon a bright orange color should be generated. It is advisable to carry out this test before committing valuable protein to the next stage of the preparation. Reaction with Protein

The reaction of diazocellulose with protein at a variety of sites occurs spontaneously at high pH. In practice a pH of 8.2 has been found to be generally satisfactory and avoids problems of precipitation and denaturation of some proteins at higher pH. The reaction is carried out by adding a solution of protein at 10-20 mg/ml (or even higher concentration if possible) in ice-cold borate buffer pH 8.2 to diazocellulose at 20 mg/ml in the same buffer and at 4° (the starting quantity of aminocellulose is as-

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sumed to be recovered completely at this stage). Less soluble or more scarce protein may be made to react at lower concentrations, but with the aim of keeping the concentration as high as possible. The reaction is allowed to continue at 4 ° overnight in the dark with occasional shaking. Conjugation is shown by a change in color of the suspension from pale yellow to a deeper yellow. Very little reaction with/3-naphthol can be demonstrated at this stage. If much reaction is demonstrated, an excess of glycine may be added to react with remaining diazo groups. Finally the supernatant may be removed after contrifugation for recovery of any unreacted protein. It is essential that the final preparation be free of anything but covalently bound protein. Furthermore, it is necessary to avoid protein preciptation at any stage of the reaction because the (unbound) precipitate will be recovered in the cellulose pellet, and if it goes into solution later will greatly interfere with the use of the immunoadsorbent. The washing procedure used is in principle as vigorous as the stability of the protein allows. For example, stable polypeptides, such as insulin and parathyroid hormone, can be subjected to alternating washes in 0.1 M borate, pH 8.2, and a 1 : 1 : 1 (v/v/v) mixture of phenol, acetic acid, and water, using three washes of each. A final water wash removes all the phenol and a suitable final suspension medium is 50 mM Veronal buffer, pH 8.0, containing sodium azide (200 p,g/ml) bovine serum albumin (1 mg/ml), and nonimmune rabbit or guinea pig IgG (20/zg/ml) buffers referred to below as NIRG or NIGP, respectively. When less stable proteins of higher molecular weight are used, it is necessary to restrict washing to a buffered saline solution to avoid denaturation. However, since such proteins show a much lower nonspecific binding to cellulose, this procedure is satisfactory.

Characterization and Storage It is important that some attempt be made to establish the suitability of the immunoadsorbent before use or storage. Uptake of protein onto the cellulose may be measured directly by the Folin reaction 14 and provides a good indication of the success of the preparation. The amount of protein bound appears to be related to the molecular weight of the protein, being higher on a molar basis for small polypeptides. This feature may be due to the greater ability of small molecules to gain access to all the reactive sites (there is approximately one active site per 40 glucose residues). At the same time the immunoreactivity of the bound polypeptide seems to decrease with decreasing molecular weight. This phenomenom may be ex14 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

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plained in the same way, since the large IgG molecules may not be able to penetrate to all the available antigenic sites when a molecule like insulin is bound. Consistent with this explanation is the observation that an IgG immunoadsorbent showed full immunological reactivity. It may be expected that some loss of immunoreactivity will occur consequent upon the binding blocking some antigenic sites. This in turn will vary with the antigen and antibody. To date, using a wide range of polypeptides, production of an immunoadsorbent does not appear to lead to an important loss of immunoreactivity. Once an immunoradiometric assay has been established, the simplest and most meaningful characterization of a new immunoadsorbent is to compare its ability to bind labeled antibody with that of a previous batch of immunoadsorbent. Immunoadsorbents may be stored for months at 4 ° without great loss of activity although the suitability of this means of storage will vary with the protein. Probably the major cause of loss of activity, especially for the otherwise very stable polypeptides such as insulin, is the growth of microorganisms that occurs eventually despite precautions taken. There is always a small release of free polypeptide (in terms of a few parts per million) from immunoadsorbent. Since for many procedures the immunoadsorbent is used in considerable excess and since free polypeptide probably reacts with antibody more readily than that bound to cellulose, it is important to keep contamination with unbound material as low as possible. It is essential therefore to wash the immunoadsorbent three or four times before use. Preparation of Labeled Antibodies

Purification of Antibody In immunoradiometric assays the use of labeled antibody as the reagent for antigen measurement imposes the need for a very high degree of purity of the antibody. This situation is the converse of that posed by specificity in a radioimmunoassay. In the latter the antiserum used may be markedly heterogeneous provided only that the purity of the labeled antigen selects from the mixture of antibodies present an immune reaction that has the specificity required. Therefore, in an immunoradiometric assay in order to achieve sufficient purity of the antibody it is essential to prepare immunoadsorbent from a highly purified polypeptide. Isolation of antibody is achieved by the incubation of undiluted antiserum with immunoadsorbent at 4 ° for 24-96 hr with continuous gentle mixing. The ratio of immunoadsorbent to antiserum varies with the titer

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of the serum and its availability. It is therefore possible to give here only the general principles guiding selection, Ideally one wishes to know the molar content of reactive antigen in the immunoadsorbent and the moles of antibody in the antiserum. The reaction of antibody with immunoads o r b e n t m a y then be set up in such a way as to provide a minimum of 10-20/zg of antibody protein for iodination. If the supply of antiserum is abundant, it is preferable to have a two- to fourfold excess of antibody. This will ensure maximum reaction with the immunodsorbent and may also allow for a certain selection toward higher affinity antibody binding to take place. If the availability of antiserum and immunoadsorbent allows, it is preferable to prepare enough antibody-immunoadsorbent complex so that it can be aliquoted for successive iodinations, thus increasing the standardization of the procedure. After reaction with antibody the immunoadsorbent is washed with buffer six times to remove non-antibody protein. The success of the reaction is then determined by measuring the total protein present 14 and comparing it with the protein on the unreacted immunoadsorbent. Antibody uptake is assumed to be the difference between these two figures. Iodination

The incorporation of iodine into antibody while bound to the immunoadsorbent presents certain advantages. First, it provides for stabilization of the antibody and protection of the antigen-binding site from iodination. Second, as the material being iodinated is insoluble, the removal of reagents and termination of the reaction is rapidly and gently achieved by washing through a filter. Third, subsequent washing at neutral pH will remove damaged antibody molecules if their affinity for antigen has been reduced. In practice IgG molecules have proved to be reasonably stable under a variety of iodination conditions. For this reason the rapid and convenient chloramine-T iodination procedure described by Greenwood e t a l . ~5 is most frequently used. Although stable to iodination, IgG molecules do not withstand the incorporation of much in excess of one atom of ~5I per molecule, possibly owing to the effects of "decay catastrophe. ''1e'~7 The following procedure details the preparation of labeled anti-IgG. Modification of the procedure for the labeling of other antibodies can ~5 F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. 89, 114 (1963). ~e R. S. Yalow, in "Protein and Polypeptide Hormones" (M. Margoulies, ed.), p. 605. Excerpta Med. Found., Amsterdam, 1969. 17 G. M. Addison, Ph.D. Thesis "Preparation and Properties of Labeled Antibodies," University of Cambridge, 1971.

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readily be achieved using the principles listed above. In practice it has proved very difficult to iodinate satisfactorily amounts of antibody protein less than l0/~g. However, new procedures for labeling have been produced since this figure was originally deduced empirically using chloramine-T, 18-2° and we have not systematically tested whether any of these procedures would allow for satisfactory labeling of smaller amounts of antibody protein. When it is necessary to label less than 1 p,g, it is in any case preferable to switch to an indirect labeling procedure (see below). Iodination of Antibody to Rabbit IgG. The well washed antibodycoated immunoadsorbent is suspended in 30/~l of 0.2 M phosphate buffer pH 7.4 in an amount sufficient to provide not less than 10/~g of antibody protein in an LP3 tube (Luckham Ltd., Victoria Gardens, Burgess Hill, Sussex, U.K.); 1-2 mCi of 125I (IMS 30 Radiochemical Centre, Amersham, U.K.) is added using a 10-/~l automatic pipette set aside for this purpose, followed by l0/~l (2.5 mg/ml) of chloramine-T, and the preparation is mixed by shaking. This entire procedure is carried out in a fume cupboard behind a screen of lead bricks. After exactly 15 sec the reaction is terminated by the addition of 10 p,l (6 mg/ml) of sodium metabisulfite. Both the chloramine-T and sodium metabisulfite solution should be fresh solutions.

Other Labels There is increasing interest in the use of nonisotopic labels in immunoassay. The main advantages of such labels in labeled antibody techniques are the expected longer shelf life of the reagent and the ability to use a detection apparatus that may for one reason or another prove to be more convenient--e.g., for automation or cheapness. Some alternatives that are being explored are bacteriophages, 21 "spin labels, ''22 enzymes, 2a fluorescent compounds, 24 and luminescent compounds. 25 It is important to recognize among all the claims for the advantages of one or another of these alternative labels that radioisotopes themselves is j. I. Thorell and B. G. Johnsson, Biochim. Biophys. Acta 251, 363 (1971). 19 A. E. Bolton and W. M. Hunter, Biochem. J. 133, 529 (1973). 20 M. A. K. Markwell and C. F. Fox, Biochemistry 17, 4807 (1978). 21 j. Haimovich, E. Hurwitz, N. Novik, and M. Sela, Biochim. Biophys. Acta 207, 125 (1970). 22 R. K. Leute, E. F. Ullman, A. Goldstein, and L. A. Herzenberg, Nature (London)236, 93 (1972). 2a E. Engvall and P. Perlmann, Immunochemistry 8, 871 (1971). 24 R. C. Aalberse, Clin. Chim. Acta 48, 109 (1973). 25 j. S. A. Simpson, A. K. Campbell, M. E. T. Ryall, and J. S. Woodhead, Nature (London) 279, 646 (1979).

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have very useful properties. 125Iis not a dangerous isotope, especially in the quantities used in routine assays. It has a reasonable half-life (60 days), and the short shelf life of labeled antigens is more often a feature of the effects of iodination and oxidation on the molecule than a result of simple radioactive decay of the isotope. These difficulties are greatly reduced with radioactively labeled antibodies, and it should be possible to use such a reagent for at least two isotope half-lives (see below). The need to prepare a fresh reagent two or three times a year is not excessive. Another very great advantage of radioactive isotopes is the sensitivity, speed, accuracy, and automation of their detection. Few if any of the alternative labels listed above can match these characteristics. Nevertheless, in one system employing enzyme labeling the need for separation of the reagents is avoided. This is the so-called homogeneous enzyme immunoassay method, 2e and it has obvious attractions in its simplicity of manipulation.

Elution o f Labeled Antibodies There are a number of different methods for the disruption of the antibody-antigen reaction. Most of these depend upon the ability to disrupt ionic bonds and include urea, guanidine-HC1, chaotropic ions, salt solutions, and extremes of pH. Since all these conditions are capable of denaturing proteins, it is important that they should only be used to the most limited degree necessary to produce the desired effect and having done so should be rapidly reversible. In practice acid pH has proved to be extremely convenient and satisfactory for this purpose. Unbuffered diluted acid may be used to reduce gradually the pH of elution of antibody, and elution into strong buffer immediately reverses the affect. In this way it is possible to limit the exposure of antibody to denaturing conditions to a few seconds. A further advantage of the stepwise reduction of pH is that antibodies of lower affinity are more readily dissociated and are removed at a higher pH. Thus it is possible during elution to enrich selectively the labeled antibodies with those of a higher affinity. A difficulty in the elution of antibody can arise if the antigen itself is oligomeric, since the conditions needed for the elution of antibody may also cause the elution of monomers of the antigen. If these are not irreversibly denatured by elution, they will merely recombine with antibody once the pH is restored to neutrality. One way of avoiding this problem (observed in the production of labeled antibodies to ferritin) is extreme care in the selection of pH for the elution of antibodies, but avoiding the 2e K. E. Rubenstein, R. S. Schneider and E. F. Ullman, Biochern. Biophys. Res. Commun. 47, 846 (1972).

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dissociation of antigen (in the case of ferritin, pH 2.6). Another theoretical possibility not yet explored would be to produce a monomeric immunoadsorbent by selection of the conditions for conjugation or by subsequent elution of all but the bound monomers. Most of the labeled antibodies used to date in immunoradiometric assays have been eluted at pH 2.0. In a few cases the recovery of antibodies has been measured and may be as high as 80%. Certain losses are inevitable and even desirable. Damage will occur due to iodine, radiation, and oxidation, and it is the aim of the procedure selectively to discard these along with antibodies of lower affinity. Although further radioactive material can be eluted by the reduction of pH below 2, such material has never been satisfactory for assays in the authors' hands. It is not certain whether this represents antibodies of still higher affinity that have been irreversibly damaged by the extreme pH or small amounts of tightly adsorbed nonspecific protein or even breakdown of the immunoadsorbent itself. Naturally, because the immunoadsorbent is itself protein it is impossible to eliminate iodination of this protein. Elution Procedure. After iodination and its termination by the addition of sodium metabisulfite, approximately 1 ml of NIGP buffer is added by a Pasteur pipette to the iodination reaction vessels. The entire contents are then transferred to a filter funnel containing a washed Whatman No. 41 filter paper. The iodinated immunoadsorbent is then washed with 200 ml of NIGP buffer to remove the iodination reagents and any damaged antibody. The next wash is with 100 ml of unbuffered HCI pH 3.0 (prepared by adding HCI to water on a pH meter). Both these washes are recovered separately so that aliquots can be counted later to determine the efficiency of incorporation and the distribution of counts between different fractions. The actual elution of usable antibodies is achieved by a further wash of 4 ml of unbuffered HC1 pH 2.0 directly into 4 ml of double strength NIGP buffer. A second pH 2.0 wash may then be carried out into a second batch of double strength buffer, but in practice the recovery of counts in this fraction may be very low, in which case it is discarded (at < 20% of the counts in the first pH 2.0 wash). If greater amounts of radioactivity are eluted in the second pH 2.0 wash, it is worth keeping and testing the activity of this separately. The precise elution pattern and characteristics of the iodinated antibodies vary a little from one antigen-antibody system to another and also between iodinations. The distribution pattern of counts between the four washes gives an early indication of the likely success of the procedure. If 20% or more of the total counts recovered are recovered in the first pH 2.0 wash, then usually the iodination yields a satisfactory product. A certain amount of radioactivity remains uneluted in the filter funnel. Much of this must rep-

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resent iodine uncorporated into the antigen on the immunoadsorbent. The relative proportion of radioactivity thus retained has not been systematically investigated. The most important practical point in the elution procedure is to ensure thorough and careful washing of the immunoadsorbent pellet. This is achieved by adding each wash slowly so that the pellet is not disturbed and so that the continuous slow flow of washing fluid maximizes its effect. At no time should a large volume of wash be allowed to build up in the filter funnel.

Characterization and Storage of Labeled Antibodies The most important feature of the labeled antibody obviously is its ability to react with antigen with a high affinity. A quick and immediate test of its capacity to bind to antigen is to determine what percentage binds back to a fresh batch of immunoadsorbent. This is best tested by doubling dilutions of freshly washed immunoadsorbent, since the amount of radioactivity bound to immunoadsorbent goes on increasing slightly at even very high concentrations of immunoadsorbent. By using several dilutions of immunoadsorbent the shape of the binding curve can be determined as well as an amount of immunoadsorbent that will allow the production of a satisfactory assay (in practice the amount that binds at least 50%, and preferably 80% or more, of the total radioactivity). The immunoadsorbent suspension at 1 mg of cellulose per milliliter is washed three times and then doubly diluted to 1/64. The labeled antibody is diluted to the region of 5000 cpm in 100 ttl and added in this volume in quadruplicate to 50-/zl aliquots of immunoadsorbent suspension in polyethylene tubes (Beckman EET or Sarstedt 46/6). The reaction is carried out for 20 min at room temperature. After centrifugation for 1 min in a Beckman Microfuge (model B), 100-p,l aliquots of supernatant are removed to counting tubes (e.g., Luckham LP3) using a diluter (e.g., LKB model 2075). In order to measure the total radioactivity used, four tubes are set up containing NIGP buffer instead of immunoadsorbent. If the labeled antibody preparation proves to be satisfactory by the above rapid test it is usual to store it prior to its evaluation in an assay. It has been found that a very satisfactory and convenient method of storage is to bind the labeled antibody back onto immunoadsorbent (the appropriate amount required may be judged from the immunoadsorbent dilution curve). The rebound antibody is then frozen in aliquots that contain enough radioactivity to produce a single assay. Since one iodination nearly always provides more label than will be used in subsequent assays, before the preparation of more labeled antibody it is advisable to err on

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the side of excess antibody per aliquot. In order to keep counting times reasonably short (1-5 min) it is convenient to have 5000-10,000 cpm in each assay tube. The recovery of reeluted antibody is usually in the region of 50-80% in the first pH 2.0 wash, so that to have sufficient antibody for a 100-tube assay would require a total of approximately 2 x 106 cpm per aliquot. For preparation of labeled antibody for storage the calculated amount of immunoadsorbent (see below) is washed and added to one or both (pooled) pH 2.0 washes and allowed to react with gentle mixing overnight at 4 °. The antibody-bound immunoadsorbent is then washed and resuspended in a total volume of 8 ml of NIGP buffer. The aliquot size for storage is determined by the size and number of assays to be performed, the radioactivity incorporated, and the total counts required per assay tube. The final, and of course most important, characterization of the preparation is provided by the results of the first assay carried out after reelution of antibody (see below). T h e Immunoradiometric Assay Direct

The basic principle of this procedure is the reaction of antigen with excess labeled antibody and the measurement of the amount of labeled antibody-antigen complex. To do this it is necessary to remove unreacted labeled antibody by the subsequent addition of a large excess of immunoadsorbent. In theory the amount of labeled antibody used should not matter, since it is in excess and the unreacted material is removed. In practice any preparation of labeled antibody contains a certain amount of radioactive material that will not react with antigen either in the free state or in the immunoadsorbent. Therefore the unbindable background count rate against which changes are measured increases with the total amount of labeled antibody preparation added. For maximum sensitivity it is advisable to use as little labeled antibody as allows for a convenient and accurate count. The time allowed for the reaction of antibody and antigeh also depends upon the sensitivity required, being usually between 24 and 96 hr. The factors determining the time allowed for the final reaction with immunoadsorbent are (a) the speed of reaction with immunoadsorbent and (b) the rate of dissociation of the labeled antibody-antigen complex. The reaction with immunoadsorbent is normally very fast owing to the large excess of immunoadsorbent. Unless automated separation is used, it is advisable to wait until the reaction is virtually complete (normally 0.5-

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1 hr) so that any small variations in the timing of the separation do not produce a significant effect. It is theoretically possible that during the reaction with immunoadsorbent labeled antibody may dissociate from the soluble immune complex and, because of the large excess of immunoadsorbent, preferentially bind to the latter, thus reducing the reaction with free antigen. In practice, for most high affinity antibody-antigen systems the amount of dissociation in a 4-hr period is negligible. This may not be true for hapten-antibody complexes, which often dissociate quite rapidly. In this situation it may still be possible to produce a satisfactory assay, but the time of exposure to immunoadsorbent will have to be reduced to a few minutes. Typical Assay Protocol for the Measurement of Rabbit IgG. A 50-/zl solution of freshly eluted 125I-labeled anti-(rabbit)-IgG (approximately 5000 cpm) is dispensed into a series of 0.4-ml plastic microfuge tubes (EET23, Beckman-Spinco); 50 p,l of standard rabbit IgG (0-5 mM) in NIGP buffer are added, and the incubation is carded out for 24 hr at 4 °. Fifty microliters of immunoadsorbent in NIGP buffer at a suitable concentration determined as above are added at 4 ° and incubated for 2 hr at 4 °. After centrifugation at 15,000 rpm in a Beckman microfuge for 1 min, 100-/zl samples of the supernatant are removed into plastic tubes and counted.

Two Site The principle underlying this procedure is that, prior to a direct immunoradiometric assay, antigen is extracted onto antibody that has been previously bound or adsorbed onto a solid phase. The immunoradiometric assay is carried out by combining a labeled antibody with the antigen while the latter is bound to solid phase on antibody. Preparation of Solid Phase Antibody. There are many chemical and physical methods by which antibodies may be linked to different solid phase materials. Unfortunately relatively little work has been carded out to investigate systematically the properties of the different methods and materials. The main desirable characteristics are (a) convenience of handling and standardization both in preparation and in utilization; (b) adequate amount of antibody bound and stability of the preparation both in terms of the binding to solid phase and on storage; (c) a low degree of nonspecific binding on the subsequent addition of antigen and labeled antibody. The method originally described by Catt and Tregear ~7for the physical adsorption of antibodies onto plastic tubes is simple to carry out, and the 27 K. Catt and G. W. Tregear, Science 158, 1570 (1967).

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tubes are moderately convenient to handle. The antibody-coated tubes are also reasonably stable on storage. The disadvantages of the method are the variable degree of coating with different antisera, the low density of antibody uptake, and the tediousness of preparing and washing large numbers of tubes both in preparation and in the assay itself. Commercial preparations of antibodies bound to a glass solid phase are available in immunoassay " k i t s , " but the details of the methods of preparation and precise properties are not available. The known ability of antibodies to bind nonspecifically to plastics would suggest that alternative materials such as glass would provide a much better solid phase support than plastic. The capacity of a plastic surface to bind antibody can be considerably increased by the prior purification of antibodies. Preparation of Antibody-Coated Plastic Tubes. Antisera are diluted in sodium hydrogen carbonate buffer (50 mM, pH 9.6), and 200/~1 are added to polyethylene tubes [Cat. No. 700 from W. Sarstedt (U.K.) Ltd., 165 Scudamore Road, Leicester, LE3 IUQ]. After standing 24 hr at room temperature, the solution is removed and the tubes are washed three times with 50 mM sodium barbitone buffer, pH 8.0, containing 150 mM sodium chloride, 5 g of bovine serum albumin per liter, and sodium azide. Batches of tubes are stored at - 20°. It is essential that an assay be carried out with a single batch of tubes to avoid variation due to any difference in coating between batches. Under these conditions of storage the tubes are stable for several months. The optimum dilution at which to use an antiserum for tube coating varies with the antiserum and its titer. In our experience it is difficult to obtain good tube coating at concentrations of antiserum very much in excess of 1/1000. It may be possible to obtain a perfectly satisfactory tube coating by recovering the dilute antiserum used on one occasion and reusing it on a second. It has not proved possible to reuse antisera three times, but again the success of such maneuvers will depend on the dilution used in the initial tube coating and the titer of the antiserum. For certain assays it may be essential to produce tubes with a very high antigen binding capacity. This situation arises when the antigen to be measured exists in the presence of a large amount of a different compound that is capable of reacting with the extracting antibody. An example of such a situation is provided by the measurement of proinsulin. Measurements of proinsulin in plasma or pancreatic extracts are nearly always carried out in a 10-20-fold or more excess of insulin. The initial extraction of proinsulin onto an insulin antibody must be carried out by a sufficient excess of insulin antibody to ensure complete extraction of both proinsulin and insulin (see below). In order to achieve this large capacity, the insulin antibodies are initially purified on an insulin immunoadsorbent. Ten milliliters of guinea pig anti-insuiia serum (binding capacity 1-

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2 IU of insulin per milliliter) are allowed to react with an insulin immunoadsorbent (assuming that the insulin on the latter is 20% immunoreactive and allowing an equivalence of antibody binding capacity) for 24 hr at 4° on a rotating turntable. The cellulose is collected by centrifugation and washed on a filter paper (Whatman No. 41) successively with 100 ml of NIGP buffer, 50 ml of dilute HC1 pH 3.0, and 5 ml of dilute HC1 pH 2.0. The final eluate is collected in a glass container containing 0.3 ml of sodium carbonate (0.1 M) and then diluted to 50 ml with sodium hydrogen carbonate buffer (50 mM, pH 9.6). The coating of tubes by this solution of purified insulin antibodies is carried out as above. Two-Site Immunoradiometric Assay Procedure. The solution or plasma to be assayed is allowed to react with the antibody-coated tube in a volume equal to that used in the initial tube coating procedure and at 4°. The time of the reaction depends on the sensitivity to be achieved and varies between overnight and 4 days. After this reaction the tubes are washed and allowed to react with labeled antibody at 4° and over the same range of times. An important feature determining the sensitivity and precision of the procedure is the amount of nonspecific binding of labeled antibody to the tube. For this reason the choice and concentration of protein to be used as carrier of the labeled antibody preparation is very important and may have to be determined empirically. It should be possible by the use of suitable concentrations of carrier albumin, y-globulin, or serum to keep the background count rate (the counts per minute on the tube in the absence of antigen) down to or below 1% of the total counts added. This allows for the detection of quite small changes in the amount of counts bound to the tube and hence enhances the sensitivity of the procedure. Two-Site Assay Protocol for the Measurement of Human ot-Fetoprotein. Purified antibodies to human a-fetoprotein (AFP) are prepared by allowing 1 ml of high titer antiserum (binding capacity approximately 125/~g of AFP per milliliter) to react with 1 mg of AFP immunoadsorbent (protein uptake 120/zg per milligram of cellulose) for 48 hr at 4°. Elution of high affinity antibody is carded out as described above for the preparation of purified insulin antibody. The antibody solution is diluted to 100 ml in 50 mM sodium hydrogen carbonate buffer (pH 9.6) and used to coat 500 plastic tubes as described above. A standard curve is prepared from dilutions of a stock (400 ng/ml) solution of AFP in 50 mM phosphate buffer containing 0.9% sodium chloride, 0.1% bovine serum albumin, and 0.01% sodium azide. The standard solutions (range 12.5-400 ng/ml) are diluted with an equal volume of normal male serum, and 200/zl of each are incubated overnight in antibody-coated tubes. The tube contents are then aspirated using a suction line fitted with a Pasteur pipette. Labeled antibody is prepared as described above by iodination, using

352

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1 mCi of 125I, of a complex prepared by extracting 100/zl of antiserum with 100/xg of AFP immunoadsorbent. Label containing approximately 20,000 cpm in 200/zl is incubated in each of the assay tubes for a period of 4 - 6 hr; after removal of unreacted material, the tubes are washed twice with the albumin-containing phosphate buffer and counted. Depending on the requirements of the assay, it may be necessary to adjust the concentration of coating antibody or the reaction periods, since saturation of the solid phase results in the appearance of the "high dose hook" phenomenon, where a paradoxical fall in binding occurs at high antigen doses. 2s

Indirect Immunoradiometric Assay The difficulty of labeling small amounts of often scarce antibodies directly with 1251led to the development of the indirect two-site immunoradiometric assay procedure. 1°'11 In this procedure an antibody to the IgG of the species in which the reagent or primary antibody is raised is labeled with 1251. Thus if it is desired to carry out an indirect immunoradiometric assay of growth hormone using as the reagent antiserum a rabbit anti-growth hormone serum, it is necessary to label an antibody to rabbit IgG to carry out an indirect immunoradiometric assay. Using the same basic principle, it is also possible to carry out a two-site indirect immunoradiometric assay. In addition to the ability to work with very small amounts of reagent antibody, the indirect assay principle has the advantage that one labeled antibody may be utilized for a wide variety of assays provided only that the reagent antibody for all the different assays is raised in the same species. The materials used for the production of the labeled antibody are easy to prepare in large quantities (e.g., rabbit IgG and, say, a sheep antiserum to this) and c a n be carefully characterized at each stage before committing scarce materials to the assay itself. If the two-site assay procedure is used, this also avoids the use of an immunoadsorbent separation step with consequent economy in the use of the primary antigen. Indirect Immunoradiometric Assay of Human Growth Hormone (hGH). The method used is that described by Beck and Hales. 1° 125I-labeled anti-rabbit IgG is prepared as described previously. Rabbit antihGH antibodies are combined with a human growth hormone immunoadsorbent by incubating the latter in amounts containing 50-500/zg of human growth hormone with 100-500/zl of the rabbit antiserum for 1-3 ~a L. E. M. Miles, C. P., Bieber, L. F. Eng, and D. A. Lipschitz, in "Radioimmunoassay and Related Procedures in Medicine," p. 149. International Atomic Energy Agency, Vienna, 1974.

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days at 4 °. lzsI-labeled anti-rabbit IgG was eluted as above in sufficient quantity to provide approximately 50/zCi of radioactivity. This is allowed to react with the human growth hormone immunoadsorbent-antibody complex (containing 450 ng or more of rabbit antiserum IgG as measured by protein uptake) for 48 hr at 4 °. After washing, the antibodies complexed to the human growth hormone immunoadsorbent were eluted as described above. Recombination of the dissociated rabbit anti-hGH antibodies and the 125I-labeled anti-rabbit IgG is allowed to proceed at 4 ° for 24 hr. The recombined labeled complex is then used in an immunoradiometric assay as described above. Indirect Two-Site Immunoradiometric Assay o f Human Proinsulin. The method used is that described by Rainbow et al. n Plastic tubes coated with purified guinea pig anti-insulin antibodies are prepared as described above; 200-/zl samples containing human proinsulin are added to these coated tubes and incubated at 4° for 24 hr. After removal of the sample, tubes are washed twice with 400/~1 of NIGP buffer. Rabbit antibody to human C-peptide is diluted to 1/1000 in 50 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride, 10 g of bovine serum albumin per liter, and 100 mg of guinea pig IgG per liter; 200/zl are added to each tube. After a further 24 hr of incubation at 4° the tubes are washed twice as previously and 200/zl of ~25I-labeled sheep anti-rabbit IgG (10,000 cpm) are added in the same buffer as that used for diluting the Cpeptide antiserum. After a final 24 hr of incubation and two further washes as above, the tubes are counted. Automation The need for assays that are readily available on a large scale and also provide a rapid turn round has prompted a move toward partial or complete automation of the procedures involved. One fully automated system at present available in the United Kingdom is the Kemtek 3000 (Kemble Instrument Co., Burgess Hill, Sussex, U.K.). The system comprises computer-controlled input/dilution reagent addition and multichannel counting modules; it also provides analysis of data using standard curve-fitting methods. Assay separation is provided by an off-line filtration unit where samples are transferred to glass figure pads contained in a continuous nylon strip and are washed in situ. This system has yielded major advantages in the routine application of immunoradiometric assays. First, filtration using glass fiber pads provides an efficient means of separating the excess antibody (i.e., that bound to immunoadsorbent) from antigen-antibody complex. Second, the use of a time delay in addition of immunoadsorbent, coupled with an appropriate wash sequence when filtration takes

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place, means that it is possible to select an incubation period for the reaction with immunoadsorbent that is identical for all assay tubes. As a result the technique can be used to assay samples in extremely large batches. Because of the high technical demands of a manually operated system, assay size has always been limited by factors such as operator fatigue, leading to problems of drift and pipetting error. It is now common practice in our laboratory to assay parathyroid hormone by a fully automated system in batches of 60 samples with appropriate controls at intervals of 20 samples. Patient samples are input at the appropriate dilution, this process taking approximately 1 hr. On the reagent addition unit, labeled antibody is added, as well as plasma to standards and buffer to plasma samples. Reagent addition takes 10-15 min. After incubation at 4° for 72 hr, the assay tubes are allowed to adjust to room temperature (the Kemtek 3000 does not have means of refrigeration), and immunoadsorbent is added, with a l0 sec delay between each addition. When addition is complete (30-40 min), filtration is commenced with a cycle that takes 50 sec for its five simultaneous transfers plus washing. The filters are dried and counted. It should be noted that this procedure differs from the standard immunoradiometric technique in that it is the radioactivity not bound to antigen that is counted. The data therefore resemble those derived from a classical radioimmunoassay and can be processed by the Kemtek computer using a polynomial cubic analysis. 20 The Kemtek 3000 also enables two-site assays to be automated although washing is confined to the final separation stage. It is thus not possible to accommodate as yet those assays (e.g., proinsulin) that require intermediate washing stages to remove potentially reacting materials. Nor is it possible to analyze directly the data obtained from a two-site assay, it being necessary to transfer the recorded counts to an alternative datahandling device by means of paper tape. Even though the development of automated labeled antibody assays is still at an early stage of development, it is clear that automation provides the key to rapid and precise analyses that can be carried out on large numbers of samples. Discussion Immunoradiometric assays and related assays using alternative labels provide a variety of methods with different characteristics. The particular method to be chosen will very much depend upon the type and availability 29 j. S. Woodhead, J. S. A. Simpson, S. J. Davies, H. Foster, and C. J. Davies, in "Quality Control in Clinical Endocrinology" VIII Tenovus Workshop, (D. W. Wilson, S. J. Gaskell and K. W. Kemp, eds.). Alpha Omega, Cardiff, Wales, 1980. In press.

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of antigen. The other two important criteria determining selection are the sensitivity and precision required. The main disadvantages of the direct immunoradiometric assay are the need for relatively large quantities of antibody for labeling and of antigen for the preparation of immunoadsorbent to use in the separation procedure. More recent iodination procedures should be explored as a means of overcoming the first problem although the indirect immunoradiometric assay bypasses it at the expense of increasing the complexity and length of the assay. The problem of immunoadsorbent preparation can be reduced by the use of less pure antigen for the immunoadsorbent used in the assay for separation. It is often relatively easy to obtain reasonable quantities of polypeptides containing approximately 20-30% of the desired antigen. Such preparations are perfectly adequate for the production of immunoadsorbent for the separation step of the assay itself. Only small amounts of highly purified antigen are needed for the extraction of antibodies for iodination. The direct two-site immunoradiometric assay has considerable advantages by comparison. It avoids the need for immunoadsorbent in the separation of the assay. The antibody used for extraction may be one that is not sufficiently specific for a one-site radioimmunoassay or immunoradiometric assay because of the great gain in specificity provided by the simultaneous recognition of two sites on the antigen molecules. A l~urther advantage in addition to specificity is sensitivity due to the very low background (1% of total count or less) against which changes in count rate are measured. Also arising from this feature is the very wide range of concentrations over which the assay can be conducted without any loss of precision.~8 The indirect two-site assay provides a great economy in the use of reagent antibody at the expense of an additional reaction step. It also allows for the production of one or two iodinated antibodies that will cope with virtually any assay. It seems likely that, if the procedure can be speeded by the judicious use of high temperatures and better geometry of the reaction container, it will prove to be a very satisfactory assay procedure.

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[25] 1 2 ~ I - L a b e l e d P r o t e i n A: R e a c t i v i t y w i t h I g G a n d U s e a s a T r a c e r in R a d i o i m m u n o a s s a y B y JOHN J. LANGONE

Protein A (PA) is a cell wall component produced by over 98% of the strains of Staphylococcus aureus that have been tested. 1 It was reported first by Verwey, 2 then rediscovered by Jensen nearly two decades later. 3,4 Its name emphasizes the chemical class, since PA originally was believed to be a polysaccharide, but shown later to be a protein with little or no carbohydrate. 5 The outstanding functional property of PA is its ability to bind specifically to the Fc region of immunoglobulin molecules, especially IgG.4,6, 7 Early results indicated that PA interacting with IgG could produce immunological reactions in vitro and in vivo that are typically induced by antibody and antigen. 8,9 Consequently, the nature of the reaction between PA and IgG has been the subject of intensive investigation. In recent work complexes formed in the fluid phase between PA and hemolytically active rabbit IgG have been separated and characterized in terms of functional activity and molecular composition. 1°'11 Protein A has also been used as a probe in nuclear magnetic resonance 12 and fluorescence quenching ~3 experiments to study the effects of antigen binding at the Fab sites on the conformation of the Fc part of IgG antibody. Significantly, PA bound to the Fc region of IgG does not inhibit the reaction between antigen and antibody. This property had led to the use of labeled PA as an analytical tool for the quantitative determination of IgG. A. Forsgren, Infect. Immun. 2, 672 (1970). 2 E. F. Verwey, J. Exp. Med. 71, 635 (1940). 3 K. Jensen, Ph.D. Thesis, Munksgaard, Copenhagen, 1959. 4 j. W. Goding, J. Imrnunol. Methods 20, 241 (1978). 5 j. Sj6quist, B. Meloun, and H. Helm, Eur. J. Biochem. 29, 572 (1972). e A. Forsgren and J. Sj6quist, J. lmmunol. 97, 822 (1966). Ii J. J. Langone, M. D. P. Boyle, and T. Borsos, J. Immunol. Methods 18, 281 (1977). s j. Sj6quist and G. St~hlenheim, J. Immunol. 103, 467 (1969). g G. T. Gustofson, G. St~lhlenheim, A. Forsgren, and J. Sj6quist, J. Immunol. 100, 530 (1968). lo j. j. Langone, M. D. P. Boyle, and T. Borsos, J. Immunol. 121, 327 (1978). n j. j. Langone, M. D. P. Boyle, and T. Borsos, J. Imrnunol. 121, 333 (1978). ~2 C. Wright, K. J. Willan, J. Sjodahl, D. R. Burton, and R. A. Dwek, Biochem. J. 167, 661 (1977). ~a j. K. Wright, J. Engel, D. Ch. Brandt, and J.-C. Jaton, FEBS Lett. 90, 79 (1978).

METHODS IN ENZYMOLOGY, VOL. 70

Copyright© 1980by AcademicPress, Inc. All fightsof reproductionin any form reserved. ISBN 0-12-181970-1

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125I-LABELED PROTEIN A

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This report will describe the preparation of radiolabeled PA and emphasize recent advances in the use of the ~*SI-labeled compound in immunoassays of fluid-phase and cell-bound antibody and antigen. For a general discussion of the properties of PA and a summary of earlier applications of labeled PA, the reader is referred to the review by Goding. 4 Preparation of Labeled P A Pure PA is available commercially from Pharmacia Fine Chemicals, (Piscataway, New Jersey), who also produce PA bound covalently to Sepharose 4B, and from Sigma Chemical Co. (St. Louis, Missouri). PA has been used mainly as an indicator of antibody bound to cell-surface antigen. ~4,~s For this purpose, it has been labeled with a fluorescent tag (fluorescein~e), with ferritin, ~r or with radionuclides, r,~4,~5,z°,~ ~5I-, ~3q_, and all-labeled PA have been prepared, and their suitability as analytical reagents has been demonstrated. Iodination Procedures General Considerations

For PA, the conditions of the iodination reaction can be crucial. ~25I(or 131I)-labeled PA generally has been prepared by labeling tyrosine residues by use of the chloramine-T method. ~5,19.22Lactoperoxidase also has been used. 14,~8However, since all four tyrosine groups of PA are necessary for maximum functional activity, loss of Fc binding ability results from incorporation of more than 1 iodine atom per molecule. ~5,19 Loss of activity also results from chemical damage unless limited amounts of chloramineT are used and the reaction time is kept short. When this method is used, 15,~9experimental details must be followed exactly. Alternatively, the Bolton-Hunter reagent 23 can be used to prepare [125I]PA/This is the method of choice. The procedure is simple, and re14 p. Biberfeld, V. Ghetie, and J. Sj6quist, J. Immunol. Methods 6, 249 (1975). ]5 G. Dorvai, K. I. Welsh, and H. WigzeU, J. lmmunol. Methods 7, 237 (1975). ]6 V. Ghetie, H. A. Fabricius, K. Nilsson, and J. Sjoquist, Immunology 26, 1081 (1974). ]r C. L. Templeton, R. J. Douglas, and W. J. Vail, FEBS Lett. 85, 95 (1978). ]8 j. j. Marchalonis, Biochem. J. 113, 229 (1969). 19 p. M. Zeltzer and R. C. Seeger, J. lmmunol. Methods 17, 163 (1977). 20 p. R. Lambden and P. J. Watt, J. Immunol. Methods 20, 277 (1978). 2] R. L. Wilder, C. C. Yuen, B. Subbarro, V. L. Woods, C. B. Alexander, and R. Mage (1980). J. Immunol. Methods 28, 255 (1979). ~ W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 22 A. E. Bolton and W. M. Hunter, Biochem. J. 133, 529 (1973).

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suits are reproducible in terms of stability, functional activity (i> 85%), and specific activity (100 _+ 10 Ci/mmol) of the product. This reagent labels e-amino groups of lysine residues. Protein A has 52 lysines, and introduction of > 1 atom of 1251per PA molecule does not appear to affect adversely the functional activity. 7 Unless otherwise indicated, [lZSI]PA prepared by this method was used in the experiments described in this report. Chloramine- T Method ~s'z2 Reagents

Buffer: sodium phosphate 0.15 M, pH 7.5 NalZSI (New England Nuclear or Amersham/Searle), 1 mCi PA, 50/zg (50 p,l of 1 mg/ml buffer) Chloramine-T, 4/.~g (5/zl of 0.8 mg/ml buffer) Sodium metabisulfite, 5/zg (5/zl of 1 mg/ml buffer) Ovalbumin, 5%, 0.25 ml in 0.15 M phosphate, pH 7.1 Procedure. To the Na125I add the PA, followed by chloramine-T. Shake the reaction mixture for 1 min at 23 °, then add sodium metabisulfite followed by ovalbumin. [a25I]PA is isolated by chromatography on a column (1.5 × 30 cm) of Sephadex G-25. Although the authors 1~ suggest that the product be stored at - 2 0 °, precipitation and inactivation can occur under these conditions. Storage at 4° in 0.04% sodium azide is recommended, r Bolton-Hunter Reagent n,zz Reagents

Buffer: sodium phosphate, 50 mM, pH 8.0 125I-labeled Bolton-Hunter reagent, 0.2 mCi in benzene-0.2% dimethylformamide (Amersham/Searle, > 1400 Ci/.mmol) PA, 50/~g (100/~1 of 0.5 mg/ml buffer) Glycine, 100/zg (100/zl of 1 mg/ml buffer) Procedure. Evaporate the solution of Bolton-Hunter reagent to dryness under a gentle stream of dry nitrogen or air. Add the PA and shake the mixture occasionally during 15 min at 23 °. Add the glycine and incubate the mixture for an additional 30 min. [125I]PA is isolated by chromatography on a column (1.5 x 30 cm) of Sephadex G-25 wet-packed and eluted with VBS-gel (Veronal-buffered isotonic saline containing 0.15 mM Ca 2÷, 1 mM Mg ~+, and 0.1% gelatin, pH 7.2). Fractions of 12 ml are collected, and the pooled product is stored at 4 ° in the presence of 0.04% sodium azide. The specific activity is reproducibly 100 ___ 10 Ci/mmol, and the product is functionally stable for at least 8 weeks.

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When 2/~g of PA were iodinated under similar conditions, the specific activity ranged between 5000 and 10,000 C i / m m o l ) T M Tritiation Procedures

General 3H-Labeled PA has been prepared by two different methods. 2°,21 These reagents have not been widely tested, but reportedly are functionally stable. They have been used to detect antibody bound to antigen on the cell surface 2° or immobilized to a solid support. 2' Since [3H]PA may be a useful alternative to [12sI]PA, the syntheses are included here.

[3H]Acetyl-PA Z° Reagents Buffer: sodium phosphate, 0.3 M pH 7.2 25 mCi [3H]acetic anhydride (Amersham; 10 txmol, 2.5 Ci/mmol) PA, 5 mg in 0.45 ml of buffer Procedure. Add the PA solution to [3H]acetic anhydride and allow the mixture to incubate at room temperature for 2 hr. Collect 3H-labeled acetyl-PA by chromatography on a column (1.5 × 25 cm) of Sephadex G-25 fine by elution with 10 mM sodium phosphate, pH 7.2. Collect 0.5 ml fractions at a flow rate of 10 ml/hr. The specific activity is approximately 25 Ci/mmol. This procedure should primarily label ~-amino groups of lysine residues. [3H]PA by Reductive Methylation 21 Reagents Buffer: borate, 0.20 M pH 9.0 Formaldehyde: 1.8 mg (62 tzmol); 0.5 ml of 3.7% aqueous solution PA, 10 mg in 1.0 ml of buffer NaB3H4 (specific activity 60 Ci/mmol): 15/zmol as 0.3 ml of 3 Ci/ml solution in 0.1 M sodium hydroxide. Use immediately after preparation. Procedure. Add formaldehyde solution to PA followed by NaB3H4. After the mixture has stood at 4° for 30 min, [zH]PA is isolated by chromatography on Sephadex G-25M that was prewashed with 15% BSA and PBS, pH 7.4. The authors 21 mix the product with 10% fetal calf serum or 2.5% BSA and store aliquots in liquid nitrogen. Thawed samples report24L. Levine, I. Alam, and J. J. Langone,Prostagl. Med. 2, 177 (1979).

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edly are stable for over a month at 4 °. E-amino groups of lysine residues are methylated by this procedure. Quantitation of Fluid-Phase IgG Using ['25I]PA 7 General Considerations

Two properties of [x25I]PA make it a useful analytical reagent: specificity and the ability to react with antibody without inhibiting antigen-antibody binding. Although PA reacts mainly with IgG, the specificity is not absolute either in terms of Ig class, subclass, or species. 4 However, for practical purposes, the reaction with IgG is the important one, since IgG generally is the principal class of antibody produced against antigens and haptens in hyperimmunized animals. Principle

The principle of the method is illustrated in Eq. (1). RalgGb - *PA RalgGb + IgGf+ *PA ~ IgGf- *PA

(1)

Fluid-phase IgG (IgGf) and rabbit IgG bound covalently to polyacrylamide beads (RaIgGb) compete for a limited amount of [125I]PA (*PA). Binding of [x2sI]PA is determined in the absence of standard IgGf. The degree of competition, calculated as percentage of inhibition of maximum binding, is plotted as a function of IgGf added. This standard curve is used to determine the concentration of IgG in a test sample based on the observed percentage of inhibition. All rabbit IgG reportedly binds to PA, ze so rabbit IgG is a useful substrate for comparing the relative reactivity of IgG from different species. Procedure Optimal A m o u n t s o f Reagents. The functional purity of [125I]PA and the optimal amounts of beads and tracer to use for routine assay of fluidphase (PA reactive) IgG are determined from the binding curves shown in Fig. 1. Increasing amounts of beads (0.1 ml; 5-200/~g of beads corresponding to 15-600 ng of rabbit IgG) are incubated for 60 min at 30° with [125I]PA (0.1 ml). The beads are washed with two 3-ml portions of buffer by centrifugation at 1500 g (4°) for 5 min or by filtration on polycarbonate filters, and the radioactivity in the bead pellets is determined. In this ex-

~5I. Alam, J. J. Langone,and L. Levine,Prostagl. Med. 2, 167 (1979). 26I. Lind, I. Live, and B. Mansa,Acta Pathol. Microbiol. Scand. Sect. B 80, 702 (1970).

[25]

lzsI-LABELED PROTEIN A i

24 22 20

361

,

r

cpm Added: 28,300_.....t][

1 /ag Beads= 3 ng IgG ~

~

j f

x 12

14,200

E 6

~

7,300___________--

4 2 "

' ; 40 2's

5'0

160

26o

Rabbit IgG BeadsAdded (pg) FIG. 1. Binding of l=5I-labeled protein A (PA) to immobilized rabbit IgG. Increasing amounts of beads (0.1 ml; corresponding to 15-600 ng of IgG) were incubated for I hr at 30° with either 28,1)01) (0-----0), 14,2111)(O---O), or 7301) cpm (&--&) of [~sI]PA. The beads were washed with two 3-ml portions of buffer, and radioactivity in the bead pellets was determined.

ample the functional purity is approximately 85%, based on maximum uptake of radiolabel. For routine work it is convenient to use an amount of beads that will bind approximately 10,000-15,000 cpm. Assay oflgG. The ability of IgG from different species to inhibit the binding of [~25I]PA to immobilized rabbit IgG was tested with concentrations of beads and tracer established from Fig. 1. A representative protocol is shown in Table I. In addition to determination of maximum binding (mixture 7) and inhibition by different amounts of IgG (mixtures 1-6), control samples include binding of [~25I]PA in the presence of the highest concentration of test sample and in buffer alone, both with no beads present (mixtures 8 and 9). These controls normally are 250-300 cpm out of 40,000 cpm added. Typical inhibition curves are shown in Fig. 2 for rabbit, swine, mouse, and rat IgG. The amount of IgG required to inhibit binding by 50% can be used to compare the relative specificity of [~25I]PA. These results are summarized in Table II for the 12 species tested. Reactivity of IgG ranges over a factor greater than 103 and agrees with the available data on PA specificity .4 This procedure, in which dilutions of test sample are analyzed along with standard IgG, has been used to determine the concentration of IgG in normal human, rabbit, and guinea pig serum. Levels shown in Table III agree well with available reported values.

ir~--

~l

I~ o

~

I~

~rj

~

~J

=

~

z

z L~

E

~ !

~

-=

[25]

lzsI-LABELED PROTEIN A I

]

363 I

100 L9 z Q z 80

RABB,T

_/

MOOSE

en

60 ii

o z

_o 4O t~

z -

20

10

100

1,000 NANOGRAMS ]gG ADDED

10,000

100,000

FXG. 2. Inhibition of nSl-labcled protein A (PA) binding to 20/~g of rabbit IgG Immunobeads by differing amounts of rabbit ( H ) , swine (O--©), mouse (A--&), and rat (A--A) IgG. Out of 37,000 cpm of [~25I]PA added, approximately 10,800 cpm were bound. From Langone. 27

TABLE II INHIBITION OF BINDING OF lsSI-LABELED PROTEIN A TO IMMOBILIZED RABBIT IGG BY IGG FROM DIFFERENT SPECIESa

Species

IgG required to inhibit by 50% (ng)

Rabbit Human Guinea pig Pig Dog Cow Mouse Horse Sheep Goat Rat Chicken

60 60 60 135 290 3,000 4,500 5,000 40,0O0 > 100,00& > 100,000c > 100,00(F

a Reproduced from Langone27 with permission. b Inhibition at this level: 45%. c Inhibition at this level: < 1 5 % .

364

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS [25] TABLE III LEVELS OF I G G IN NORMAL SERA

Species Sample

Human

1

10.5 --- 0.5 9.0 ± 0.5 7.3 ± 0.3 8.9 ± 0.8 10.3 ±- 0.5

2 3 4 5

Rabbit 3.8 5.2 4.3 4.7 5.3

-+ 0.1 ± 0.1 ± 0.1 ± 0.3 ± 0.2

Guinea pig (strain 2)a 3.3 2.0 5.9 5.9 5.9

--- 0.3 ± 0.1 ± 0.1 ± 0.4 ± 0.1

a Langone et al. r Immunoassay

o f F l u i d - P h a s e A n t i g e n s a n d H a p t e n s 2r

Principle

125I-labeled protein A as a t r a c e r for I g G has b e e n e x t e n d e d to a general i m m u n o a s s a y m e t h o d f o r fluid-phase antigen and hapten. T h e steps i n v o l v e d are s u m m a r i z e d in Eqs. (2) and (3). AB + (Lb + Lf) ~ (Ab - Lb) + (Ab - Lf)

(2)

Ab - Lb + *PA ~ Ab - Lb -- *PA

(3)

I m m o b i l i z e d (Lb) and free (Lf) ligand c o m p e t e for I g G a n t i b o d y binding sites [Eq. (2)]. T h e a m o u n t o f Lf p r e s e n t will determine the a m o u n t o f a n t i b o d y b o u n d to Lb, After the b e a d s are w a s h e d , (excess) [125I]PA is a d d e d and a s e c o n d i n c u b a t i o n carried out. T h e a m o u n t o f [12sI]PA b o u n d is a quantitative m e a s u r e o f a n t i b o d y , and indirectly o f Lf. A s t a n d a r d inhibition c u r v e is o b t a i n e d u n d e r optimal a s s a y conditions using k n o w n a m o u n t s o f h o m o l o g o u s Lf and used to relate o b s e r v e d inhibition to conc e n t r a t i o n o f Lf in test samples. Immobilized

L i g a n d s 27

L i g a n d s are c o u p l e d by amide b o n d s to p o l y a c r y l a m i d e b e a d s that h a v e free c a r b o x y l ( I m m u n o b e a d s ) o r a m i n o g r o u p s (Affi-Gel 701; BioRad). Reagents

Buffer: s o d i u m p h o s p h a t e , 3 m M , p H 6.35 Ligand: 0 . 1 - 1 . 0 mg o f protein o r 0 . 1 - 0 . 3 m g o f h a p t e n dissolved in 0.5 ml o f buffer 27 j. j. Langone, J. Immunol. Methods 24, 269 (1978).

[25]

1251-LABELED PROTEIN A

365

Beads: 100 mg of Immunobeads or 500 mg of Affi-Gel 701 washed and suspended in 9.0 ml of buffer 1-Ethyl-3-(3-dimethylaminopropyi)carbodiimide (EDAC), 6 mg (0.3 ml of 20 mg/ml buffer) Procedure. Mix the ligand and bead suspension at 4 °, then add EDAC. Allow the mixture to rock at 4° for 4-25 hr. In the cold, wash the beads with three 20-ml portions of coupling buffer, three 20-ml portions of 5 M guanidine hydrochloride, pH 7.2, then several times with phosphate-buffered saline, pH 7.2. After the beads have stood in this last buffer at 4 ° for 2 hr, they are washed twice with 20 ml of VBS-gel and resuspended in 25 ml of this buffer containing 0.04% sodium azide and stored at 4 °. Generally, beads are stable for several months and can be prepared with reproducible activity.

General Assay Procedure Optimal Bead and Antibody Concentrations. Working concentrations are determined from curves similar to those shown in Fig. 1. Reagents Beads, serially diluted (0.1 ml) Antibody--either diluted whole serum or IgG fraction (0.1 ml) [125I]PA, 40,000-50,000 cpm (0.1 ml) Procedure. Add antibody to increasing amounts of beads and incubate the mixture at 30° for 60 min. Wash with two 3-ml portions of buffer. Add [125I]PA, carry out a similar incubation and washing procedure, and determine the number of counts per minute bound. Controls include a set of tubes containing increments of beads with no antibody (0.1 ml buffer), antibody alone plus buffer (0.1 ml), and buffer alone (0.2 ml). Typical binding curves shown in Fig. 3 were obtained by incubating increasing amounts of immobilized human chorionic gonadotropin (HCG) (2.5-100 ~g of beads) and rabbit antiserum diluted either z~, 5r~o, 2~o, or 6r-~. Note that binding of [125I]PA to the beads in the absence of antibody is insignificant. Excess [125I]PA. At the concentrations of antibody and immobilized ligand used, sufficient [125I]PA must be added to saturate the receptor sites on the bound IgG. A set of tubes containing replicate samples of antibodycoated beads are prepared by the procedure described above. Increasing amounts of [~2H]pA (0.1 ml) are added to the bead pellets, and the mixtures are incubated at 30 ° for 60 min. The beads are washed, and bound radioactivity is determined. Typical binding curves are shown in Fig. 4 for four sets of anti-HCG (25-~) coated-HCG beads that were treated with [~25I]PA ranging between 2900 and 100,000 cpm. When 40/~g of beads were used, excess [~25I]PA was not present, even when 100,000 cpm was

366

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

28

I

I

[25]

I

ANTI-HCG 1/250 24

,.;'- 20 _

~,

O

1/750

_

II1

1/2,250 8

4 J-]p" ~ 0 IIr~ 1 2

1/6.750 e~ 5

t5 ~,, NO ANTIBODY 10 20 RELATIVE CONCENTRATION OF IMMOBILIZED HCG

~d] 40

FIG. 3. Binding of l=5I-labeled protein A (PA) (0.1 ml, 38,000 cpm added) to differing amounts of human choriomic gonadotropin (HCG) beads that were treated with rabbit antiHCG diluted either rk~ (O---Q), r ~ (©--©), r ~ (A--A), or ~ (&--&). Binding of [12sI]PA to beads in the absence of antibody (I-q--l-q) was also determined. The beads were incubated with antibody for 60 min at 30°, washed, and then incubated with [nsI]PA for 60 min at 30° before the number of counts per minute bound was determined. A relative concentration of 1 = 2.5/~g of HCG beads. From Langone. 27

added. However, with 13/zg of beads, approximately 35,000 cpm was sufficient to saturate the Fc antibody binding sites. As less beads were used, less [125I]PA was required to reach a saturating dose as the maximum binding decreased. Standard Inhibition Curves: Sensitivity and Specificity. The ability of homologous ligand to inhibit antibody binding is measured as inhibition of [125I]PA binding. Table IV shows a sample protocol including the necessary controls. In the initial reaction [Eq. (2)] beads (0.1 ml) and antibody (0.1 ml) are incubated at 30° for 60 min in the presence of different amounts of inhibitor (0.1 ml) or in buffer to determine maximum binding (tubes 1-9). The beads are washed twice, then incubated again with [x25I]PA (40,000 cpm, 0.1 ml) [Eq. (3)]. Radioactivity bound to the beads is determined, and inhibition curves are plotted. Typical results shown in Fig. 5A were obtained using a constant amount of HCG beads (13 p.g) and

[25]

]25I-LABELED PROTEIN A 50

i

40; z2;

i

367 i

!

i

~

---, 20 X

18

a Z 0

16

<

12

14

10

8

4.3 ~g

4

-

1.4~ug 2

'];-

0

I

10

20

30

I

40 50 50 70 CPM 12SI-pA ADDED (X 10-3)

I

I

I

80

90

100

FIG. 4. Binding of differing amounts of [125I]PA to different amounts of antibody-coated HCG beads. Aliquots (50/.d) of either 1.4 v-g ( V - - I ' ) , 4 . 3 / , g ( A - - A ) 13 v,g ( O - - O ) , or 40/zg(O---O) of human chorionic gonadotropin (HCG) beads and 0.1 ml of rabbit anti-HCG diluted z ~ were incubated at 30° for 60 min. The beads were washed with buffer, incubated with differing concentrations of [t25I]PA (0.1 ml) for 60 min at 30°, washed again, then counted. From LangoneY

three different concentrations of rabbit anti-HCG. The sensitivity of the assay increased as less antibody was used. Maximum binding decreased from 14,200 to 4,250 cpm. Inhibition curves also can be obtained by varying the amount of beads and holding the antibody concentration constant (Fig. 5B). By this procedure optimal binding and sensitivity can be achieved and amounts of reagents adjusted to conserve either antibody or immobilized ligand. Once optimal conditions are established, relative effectiveness of compounds related to the homologous ligand to act as inhibitors is tested in exactly the same way to determine antibody specificity. Analysis of Test Samples: Potential Problems. Normally, analysis of physiological fluids (e.g., serum and urine) is straightforward. Potential problems encountered so far are the following. 1. Nonspecific sticking of IgG to the reaction tube resulting in high background binding of [I~sI]PA. This can occur when concentrated ( < ~

I Z

c~

0 a~ 0 < Z 0

I ~

Z 0 "1"

~

~

.=0

Z

< o < 0 Z

r~ Z >

r~ < r~ Z

.~=~ °~

[25]

lgSI-LABELED PROTEIN A 100

I

A 80

I

ANTI-HCG • 11250 o 1/750

369

I

JA~/"

50

40 Z

z

~u

o z

20

100

B

k-

~:

T

0

80

I

I

l

I

I

i

..]

HCG BEADS (/~g) •

13

50

20

1.0

10

I

I

100

1,000

-3 NANOGRAMS HCG ADDED (= 1U X 10 )

FIG. 5. Inhibition of the binding or rabbit anti-HCG to human chorionic gonadotropin (HCG) beads by differing amounts of HCG as measured by inhibition of [12M]PA binding. (A) Effect of varying antibody concentration on assay sensitivity. Inhibition curves were obtained using 13/~g of HCG beads and anti-HCG diluted ~ ( H ; 14,200 cpm bound), r ~ (O---O; 8000 cpm bound), or r,~n (L~--/x; 4250 cpm bound). (B) Effect of varying bead concentration on assay sensitivity. Inhibition curves obtained using anti-HCG diluted ~-~ and either 40/~g ((3---0; 23,500 cpm bound), 13/~g ( H ; 14,200 cpm bound), or 4.3 p.g (/x--/x; 6000 cpm bound) of HCG beads. In each case, 38,000 cpm of [1=sI]PA were added. From Langone.27

dilution) serum from a species with PA-reactive IgG (Table II) is analyzed. To avoid this problem, samples are transferred to new tubes before counting. Alternatively, IgG can be removed by absorption of the sample with PA-Sepharose. zs ~s j. j. Langone, M. D. P. Boyle, and T. Borsos, Anal. Biochem. 93, 207 (1979).

370

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

[25]

2. Nonspecific sticking of serum lipid to beads when concentrated serum (< ~o dilution) or culture fluid is analyzed. High binding of radiolabel will result. This problem also can be solved by preabsorbing the sample as indicated above. When a preabsorption is carried out, control samples should be included to account for possible absorption of the target molecule. Absorption Procedure ~s Reagents PA-Sepharose stock suspension: Suspend 1.5 g PA-Sepharose (Pharmacia) in 10 ml of VBS-gel. Allow the mixture to rock at room temperature for 30 min, then wash the beads with two 10-ml portions of buffer. Collect the beads by centrifugation or filtration, and resuspend them in 5.3 ml of 0.15 M saline. One milliliter of suspension is equivalent to 2 mg of PA. Serum, 1 ml, centrifuged at 3000 g for 5 min Procedure. Centrifuge 1 ml of PA-Sepharose suspension and discard the supernatant liquid. Add serum, vortex, and allow the mixture to incubate at room temperature for 60 min. Collect the serum by centrifugation (1500 g for 5 min), being sure to remove all the beads. This procedure reproducibly absorbs > 99% of the PA-reactive IgG from human or rabbit serum containing up to 12 mg of IgG/ml. 28 Examples HCG in Urine. ~7 Optimal assay conditions for HCG were established by the procedure described above. Levels of HCG in the urine of women who were in the second or third trimester of pregnancy are shown in Table V. The concentrations are given as HCG equivalents because luteinizing hormone, which also is produced during pregnancy, crossreacted in the anti-HCG immune s y s t e m Y No immunoreactivity was detected in urine from females who were not pregnant, nor in the urine from male subjects. Immunoglobulin Levels in Serum. 27.~s Similar immunoassays were developed for human IgM and IgE. Levels of these Igs were determined in the sera of normal individuals and are shown in Table VI. The IgE analyses were performed on samples that were absorbed with PA-Sepharose as described above to remove components responsible for nonspecific (lipids) and specific (IgG) interference in the assay. The IgE levels were comparable to values obtained for the same samples by double-antibody RIA. 28 The concentration of IgG in these sera (Table VI) was determined by the procedure described above [Eq. (1)].

[25]

125I-LABELED PROTEIN A

371

TABLE V LEVELS OF HUMAN CHORIONIC GONADOTROPINS (HCG) EQUIVALENTS IN URINE a

Concentration of HCG equivalents (/~g/ml = IU/ml) c

Subject b D.S. C.B. E.M. C.L. D.L. Da. L. M.B. J.L.

6.8 ± 0.8 15.8 ± 0.5 13.5 +-- 0.0 <0.05 a <0.05 d <0.05 d <0.05 a <0.05 a

a Langone.27 b D. S. and C. B. were in the second trimester of pregnancy, E. M. in the third trimester. C. L., D. L., and Da. L. were females who were not pregnant; M. B. and J. L. were male subjects. c Urine samples from pregnant women were centrifuged at 3000 g for 5 min and diluted and ~ ; 0.l-ml aliquots were analyzed in duplicate. Undiluted samples (0.1 ml) from other subjects were analyzed. a Not detected in undiluted urine (i.e., <10% inhibition in the immunoassay).

TABLE VI IMMUNOGLOBULIN LEVELS IN NORMAL HUMAN SERA

Subject A B C D E

IgGa (mg/ml) 12.0 11.5 8.3 9.5 11.3

--- 0.3 ± 0.2 + 0.7 ± 0.0 ± 0.4

IgMb (mg/ml) 0.57 1.15 1.00 1.74 1.11

--- 0.4 ± 0.20 ± 0.06 ± 0.6 ± 0.06

IgE ° (ng/ml) 123 ± 8 158 ± 3 150 ± 5 130 ± 5 112 ± 10

a Langone.27 b Langone et al. 2a Remarks

This assay method appears to be general and has been applied to the a n a l y s i s o f h a p t e n s a s w e l l a s a n t i g e n i c m a c r o m o l e c u l e s . 24,25,2r,29 T h e ~ J. J. Langone and L. Levine, Anal. Biochem. 9S, 472 (1979).

372

R A D I O I M M U N O A S S A Y S AND I M M U N O R A D I O M E T R I C

ASSAYS

['95]

TABLE VII COMPOUNDS FOR WHICH ASSAYS HAVE BEEN DEVELOPED Antigens

Haptens

Human I g G a,b Human IgMb Human IgEe Goat IgGb

Methotrexate b Leucovorina Indomethacin s Thromboxane B2c

HCGb

PGD2c

13,14-Dihydro PGE2c 5,6-Dihydro PGI2c 6-Keto PGF1,c 15-Hydroxy-9a, 1la(epoxymethano)prosta-5,13-dienoic acidc 15-Hydroxy-1la,9a(epoxymethano)prosta-5,13-dienoic acidc

a Langone e t al. 7 b Langone.2r c Levine et al. 24 d Langone and Levine.29 e Langone e l al. zs i Alam et al. 25 m e t h o d o l o g y is exactly the same. Table V I I lists the c o m p o u n d s for which assays have been reported. U s e of [125I]PA as a tracer for IgG antibody is independent o f antibody specificity. Thus i m m u n o a s s a y s can be d e v e l o p e d for substances for which stable, i m m u n o r e a c t i v e radiolabeled derivatives o f high specific activity are not readily available. Use of a single tracer obviates the need to prepare, purify, and store large n u m b e r s o f radioactive ligands in laboratories where r a d i o i m m u n o a s s a y is a routine analytical method. A n a l y s i s of C e l l - B o u n d A n t i b o d y General

Considerations

125I-labeled PA (by the chloramine-T method) originally was used to detect antibody bound to cell-surface antigens. 14,~5,3° This has b e c o m e a routine procedure that has received special attention in t u m o r immunology to detect tumor-associated antigens on the surface o f c a n c e r cells. 4 In the general procedure, cells are incubated with antiserum, then wi~h [~25I]PA. U n d e r appropriate conditions, fluid-phase antigen can be determined by steps analogous to Eqs. (2) and (3), except that the t u m o r cells are the source of bound ligand [Lb, Eq. (2)]. 7 Binding o f [~zsI]PA o f k n o w n specific activity allows a reliable quantitative m e a s u r e of cell-bound antigen p r o v i d e d the antibody is essentially IgG and produced in an appropriate species (e.g., rabbit) with PA-reactive Ig. The investigator should keep in mind that the bivalency o f PA for IgG m a y influence the results. 30 K. I. Welsh, G. Dorvall, and H. Wigzell, Nature (London) 254, 67 (1975).

[25]

125I-LABELED PROTEIN A 14

I

//

12

Q

x

I

Anti L -10

I

373

// //"

100 A

90

80

g~ = ¢,-

70

8

E

60

O li1

~ 1-

,-

50

-o

40

~

3O

~

E

E Q.

4

f I

I

12 4

1

8

Anti L-1 Anti L-1 I

20

O

• I

,,// ,It/

16 32 Relative Antibody Concentration

•

10

I

64

FIG. 6. Binding of nSI-labeled protein A (PA) (0.1 ml; 26,000 cpm added) to 105 line-10 (L-10) tumor cells sensitized with increasing amounts of rabbit antiserum to line-10 cells (H) or to the antigenically distinct line-I (L-l) ((3---0). These determinations of total bound IgG are compared to the cytotoxic (complement-fixing)activity of the antisera. Cytotoxicity was measured as uptake of the vital dye trypan blue followingincubation of the antiline-10 (A--A) or anti-line-1 (b--&) sensitized line-10cells with human complement. A relative concentration of 1 corresponds to a rh~ dilution of antibody. From data given in Langone et al. al

Binding o f A n t i b o d y to Tumor Cell A n t i g e n s

By following the procedure outlined, but using cells instead of beads as the source o f antigen, the binding o f specific IgG antibody to cellular antigens can be determined directly. In a typical experiment, 31 105 line-10 guinea pig hepatoma cells (0.1 ml) are incubated at 30° with increasing dilutions o f rabbit antiserum (0.1 ml) specific for line-10 antigens. The cells are washed with two 3-ml portions o f buffer, then incubated with [nsI]PA (0.1 ml; 26,000 cpm) for 30 min at 30 °. The cells and washed, and bound radioactivity is determined. The binding curve is shown in Fig, 6 along 3~j. j. Langone, M. D. P. Boyle, and T. Borsos, J. Natl. Cancer Inst. 60, 411 (1978).

O O

8 0 o

Z

t

e_o ~..=~

I

I

I

c'q ¢'q c-,I

O u

0

'7

m Z 0

~" ,A e4 ,A I

I

I

I

I

I

I

I

e~

~ e.i --" e,i Z

O

E

.r,

8 ,.A -z

ZZZ

0 Z

z~"~

E

2

o

O

e~ z

e ~ ~..._. "v

7 ,'77

,m

O

r..

O

ZZZZ

.<

m

"7 0

F,, z

~

c'q ~

c-4

~ ~ ..~ ,..~

z

[25]

Ig5I-LABELED PROTEIN A

375

with the curve obtained when 105 antigenically distinct guinea pig line-1 hepatoma cells were used. These results, which measure total IgG antibody, are compared to the complement-dependent cytotoxic activity of the antiserum, which measures only complement-fixing antibody.

Measurement of Cell-Bound IgM Antibody Although IgG is usually the predominant class of antibody produced in hyperimmunized animals, in some cases relatively high levels of IgM are produced. This is the case with rabbit antiserum against line-1 cells. 31 Cell-bound IgM can be determined by an extension of the usual procedure. 3~ There are four steps: 1. Anti-target cell antiserum (e.g., rabbit anti-line-l) is absorbed with PA-Sepharose as described above to remove IgG antibody. 2. The target cells are incubated with absorbed antiserum as described above to allow fixation of IgM antibodies. 3. These antibody-coated cells are treated in the usual way with a second (primarily IgG) antiserum directed against the IgM of the first species (e.g., goat anti-rabbit IgM). 4. The washed cells from step 3 are incubated with [125I]PA. The degree of binding of radiolabel is an indirect measure of IgM antibodies. Results of IgM determinations in rabbit antisera to line-1 and line-10 are shown in Table VIII along with the IgG levels obtained by measuring direct binding of [12sI]PA to cells treated with unabsorbed antisera. Goat anti-rabbit IgM was used as the second antibody (step 4), since goat IgG complexed to cell-bound antigen will bind PA efficiently even though fluid-phase goat IgG will not. Even after booster injections, antiline-1 is predominantly IgM while anti-line-10 is IgG. These results agree with the class of complement-dependent cytotoxic antibody determined semiquantitatively.31

8~ M. D. P. Boyle and J. J. Langone, J. Natl. Cancer Inst. 62, 1537 (1979).

376

RADIO1MMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[26]

[26] The RAST Principle and the Use of Mixed-Allergen RAST as a Screening Test for IgE-Mediated Allergies

By T. G. MERRETT and J. MERRETT An atopic person has reaginic antibodies directed against common environmental allergens, ~ and since immunoglobulin E (IgE) is the most important reaginic antibody in man its measurement is relevant to both clinical allergists and immunologists. When normal serum immunoglobulin levels are compared (see the table) it can be seen that more sensitive methods are required for the quantification of total IgE than for other immunoglobulins. Clearly, sensitivity will be an even more important characteristic of methods designed to measure allergen-specific IgE antibodies. Radioimmunoassay is a technique ideally suited to a clinical situation where a specific assay of high sensitivity is required for the routine estimation of an antigen in a large number of test specimens. Therefore, it is hardly surprising that the technique is applied to the measurement of IgE and various allergen-specific IgE antibodies. It is our purpose to describe the radioallergosorbent test (RAST) principle and the preparation of RAST reagents and to summarize how a variant of this t e s t - - t h e mixed RAST technique--can be used as a valuable screening test for atopic allergy. R A S T - - A Solid-Phase Sandwich Radioimmunoassay A conventional RIA 2 is one in which the antigen to be assayed competes with a pure radiolabeled preparation of that antigen for a limited number of high avidity antibodies. A conventional RIA can be criticized because (a) it is an indirect (inhibitory) instead of a direct (noninhibitory) method; (b) nonspecific serum effects are often difficult to eradicate; and (c) it cannot distinguish biologically active antigens from inactive fragments that still retain antigenic determinants. Furthermore, a conventional RIA is impractical for the measurement of allergen-specific IgE antibodies because it requires that IgE antibodies of many different specificities be prepared and radiolabeled. It is on acj. Pepys, in "'Clinical A s p e c t s o f I m m u n o l o g y " (P. G. H. (Jell, R. R. A. C o o m b s , and P. J. L a c h m a n n , eds.), p. 877. Blackwell, Oxford, 1975. 2 S. A. Berson, R. S. Yalow, A. B a u m a n , M. A. Rothschild, and K. Newerly, J. Clin. Invest. 35, 170 (1956). 1

METHODS IN ENZYMOLOGY, VOL. 70

Cop.ydght © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-1

[26]

RAST AND MIXED-ALLERGEN RAST

377

NORMAL IMMUNOGLOBULIN LEVELS Class IgG IgA IgM lgD IgE

Serum concentration (mg/ml) 12.5 2.2 1.1 0.02 0.00004

count of these disadvantages inherent in conventional RIA that the sandwich type of disequilibrium assay a may be preferred. The radioallergosorbent test (RAST) 4 is a solid-phase sandwich RIA and does not suffer from these general disadvantages: that is, it is a direct assay, eliminates nonspecific serum effects, and measures biologically active allergen-specific IgE sandwiched between allergen and Fc-specific anti-IgE antibodies. In principle, the RAST is similar to the red-cell-linked antigen-antiglobulin reaction (RCLAAR) s and the test sequence is summarized as follows: 1. An excess of insolubilized allergen is incubated with the test serum, and will bind allergen-specific antibodies including those of the IgE class. 2. The presence of specific IgE antibodies complexed with allergen is tested by incubating 12SI-labeled, immunosorbent-purified anti-IgE with the washed complex. 3. The radioactivity associated with the complex is determined after a further washing step and is compared with results obtained using reference data (Fig. 1). An excess of allergen and labeled anti-IgE is used in the test in order to minimize interference by non-IgE antibodies. It should be remembered that the test is semiquantitative because the composition of allergen extracts can vary from batch to batch and test sera can contain IgE antibodies with a range of titers and avidities. Similar tests to the RAST have emPloyed radiolabeled allergens and solid-phase antibodies, 6 but these are best restricted to certain well chara L. Wide, in "'Radioimmunoassay Methods" (K. E. Kirkham and W. M. Hunter, eds.), p. 405. Churchill-Livingstone, London, 1971. 4 L. Wide, H. Bennich, and S. G. O. Johansson, Lancet 2, 1105 (1967). 5 R. R. A. Coombs, A. N. Howard, and L. S. Mynors, Br. J. Exp. Pathol. 31, 525 (1953). C. R. Zeiss, J. J. Pruzansky, R. G. Patterson, and M. Roberts, Eur. J. Immunol. 110, 414 (1973).

378

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

:>-+

~

+

[26]

O 0 ----)

OO

SP IGE ALLG ANTI= HI/LO • +DISC" - - - b COMPLEX + I G E , k - ~ . SCORE

:>'+

)

+

O0 O0

)

FIG. 1. The radioaUergosorbent test (RAST). See text for details.

acterized allergens, since the heterogeneity of allergen extracts--even relatively simple pollens7--will raise doubts as to whether or not biologically relevant allergens have been labeled. By comparison, the RAST has the very important practical advantage that only one radiolabeled substance (anti-IgE) is required for the measurement of specific IgE antibody levels to a variety of allergens. R A S T Reagents Although the technology for estimating IgE levels was available in the 1950s, 2 IgE was not reported until 1966, s and sufficient IgE reagents for the technique to be used by clinical allergists awaited the discovery of an IgE myeloma protein) About one myeloma patient with very high circulating levels of IgE has since been discovered each year (currently, 15 cases have been reported), and the purified protein has been used for radiolabeling and to raise Fc-specific anti-IgE. A high degree of immunochemical expertise is required in the preparation of RAST reagents, and so their commercial availability (Pharmacia Diagnostics AB, Uppsala, Sweden) is to be welcomed. Solid Phases

Cyanogen bromide (CNBr)-activated insoluble matrices, such as microcrystalline cellulose particles, 1° or paper disks, 1~ can be chemically M. D. Topping, J. Brostoff, W. D. Brighton, J. Danks, and M. Minnis, Clin. Allergy 8, 33 (1978). s K. Ishizaka, T. Ishizaka, and M. H. Hornbrook. J. Immunol. 97, 65 (1966). 9 S. G. O. Johansson and H. Bennich, Immunology 13, 81 (1967). ~0 L. Wide, in "Immunoassay of Gonadotrophins" (E. Diczfalusy, ed.), Acta Endocrinol. 63, Suppl. 142, 207 (1969). 11 M. Ceska and U. Lundkvist, Immunochemistry 9, 1021 (1972).

[26]

RAST AND MIXED-ALLERGENRAST

379

linked to amino groups of allergens under mild conditions. An alternative matrix, which requires no preactivation, is polymaleic anhydride. TM

CNBr-Activated MicrocrystaiHne Cellulose Particles Reagents Microcrystalline cellulose (Sigma Chemical Co.) Cyanogen bromide Sodium hydroxide, 1 M Sodium bicarbonate, 5 raM, pH 7-7.2 Distilled water 25%, 50%, and 75% acetone/distilled water (v/v) and acetone Suspend cellulose particles (10 g) in distilled water (100 ml), and dissolve CNBr (10 g) in distilled water (100 ml). Mix the two solutions at 25 ° at pH 4-6. Commence the activation by adding 1 M NaOH (40 ml) dropwise, with constant stirring, and maintain the temperature at 25° and the pH between 10 and 10.5. Transfer the suspension to a Biichner funnel fitted with a sintered glass support (G4) and wash first with cold 5 mM NaHCO3 (3 liters) and then cold distilled water (1 liter), stirring all the time to ensure adequate washing. The particles may then be lyophilized or shrunk by washing successively with 500 ml each of 25%, 50%~, and 75% acetone in water, and finally in 1 liter of acetone. Store at 4 °.

CNBr-Activated Paper Disks Reagents A hard grade of filter paper (e.g., Whatman 2 MM, or Munktells OOH), punched into disks 5 mm in diameter Cyanogen bromide Sodium hydroxide, 1 M Sodium bicarbonate, 5 mM Distilled water 25%, 50%, and 75% acetone/distilled water (v/v) and acetone Suspend 10 g of the paper disks (about 5000 disks) in distilled water (100 ml) for 3 min, then mix with a fresh CNBr solution (10 g per 300 ml of distilled water). Commence activation by dropwise addition of 1 M N a O H (50 ml) and maintain a pH of 10.5 and the temperature at 25 °. Aspirate the liquid and wash the disks 10 times in 500 ml of 5 mM NaHCOa, allowing 2 min between each wash. Similarly, wash disks successively with 500 ml of 25%, 50%, and 75% acetone in water, and then with one liter of acetone. Dry the disks by allowing the remaining acetone to evaporate at room temperature; store at 4 ° . ~ J. Merrett and T. G. Merrett, Clin. Allergy 8, 235 (1978).

380

RADIO1MMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[26]

Polymaleic Anhydride (PMA) Particles Reagents Polymaleic anhydride (British Drug House Ltd.) These particles require no activation, but can be made super-fine by stirring a 10% suspension in 0.1 M NaHCOa, pH 8.5-9, with a high-speed mixer (e.g., Silverson Laboratory Mixer-Emulsifier).

Solid-Phase Allergens Commercially available allergens are compared by the RAST-inhibition technique (see RAST Reagents: l~5I-Labeled Anti-IgE), and the most potent should be selected for insolubilization. Most pollen extracts are satisfactory, but animal danders or epithelia and, particularly, molds have to be carefully selected. Once selected, the allergen should be coupled to the solid-phase support in the ratio of 1 : 100 (w/w), e.g., 10 mg of allergen per gram of solid-phase particles or disks. When the weight of allergen is not certain, assume that 1 ml of skin-prick test solution contains 1 mg, but be prepared to optimize amounts by determining the least allergen concentration that produces the highest RAST results against a panel of allergic sera.

Reagents Allergen to be coupled, dialyzed against 0.1 M NaHCO3 CNBr-activated cellulose particles CNBr-activated paper disks PMA particles NaHCO3, 0.5 M, NaHCO3, 0.1 M Tris, 0.1 M, pH 8.5 Sodium acetate, 0.1 M, pH 4 RAST assay buffer: 50 mM phosphate buffer, pH 7.4, containing 1% Tween 20, 0.3% bovine serum albumin, and 0.05% thiomersal Suspend 1 g of solid phase in 10 ml of the dialyzed allergen extract and vertically rotate for 60 hrs at room temperature. Wash the solid phase with 10 ml of 0.5 M NaHCO3, mix for 3 hr with 1 M Tris, pH 8.5, and then wash twice with 10 ml of RAST assay buffer with continuous mixing for each wash. Finally, store in 100 ml of assay buffer at 4°; this is sufficient for 2000 assay tubes.

12~I-Labeled Anti-IgE It has been suggested that the radiolabeled IgG fraction of anti-human IgE can be used in the RAST, TM but it is our experience that anti-IgE must 1~ j. K. G. Sarsfield and G. Gowland, Clin. Exp. Immunol. 13, 619 (1978).

[26]

RAST AND MIXED-ALLERGEN RAST

381

be immunosorbent purified if falsely positive results are to be avoided. Therefore we describe immunosorbent purification of anti-IgE.

Purification o f lgE Reagents Myeloma IgE serum; conceivably, high titer serum in excess of 100,000 U/ml could be used as an IgE source Phosphate buffer, 10 mM, pH 7.5 Phosphate buffer, 30 mM, pH 7.5 DEAE-cellulose 52 (Whatman Biochemical Co Ltd.) Sephadex G-200. The method is similar to that first described by Nezlin et al. 14 Dialyze myeloma IgE serum against 10 mM phosphate buffer, pH 7.5, and apply 10 mg to a column of DEAE-cellulose 52 equilibrated in the same buffer. Discard the protein removed in this buffer, and collect the bulk of IgE in 30 mM phosphate pH 7.5 buffer wash. Filter this IgE protein through a column (110 × 3 cm) of Ultragel AcA 34 in 50 mM Tris-HC1 buffer with 0.28 M NaC1, pH 8.0. Test the column fraction by RIA, and pool the located pure IgE. Preparing CNBr-activated Sepharose 4B-IgE Immunosorbent Column Reagents Purified IgE Coupling buffer: 0.1 M NaHCO3, pH 8.5, containing 0.5 M NaCI CNBr-activated Sepharose 4B (Pharmacia AB) HC1, 1 mM Tris buffer, 1 M, pH 8.5 Acetate, 0.1 M, pH 4, containing 0.5 M NaC1 Dialyse 1 mg of IgE against the coupling buffer. Reconstitute, and wash 300 mg of freeze-dried CNBr-activated Sepharose 4B on a sintered glass filter (G3) with 70 ml of 1 mM HCI. Suspend the Sepharose in 10 ml of IgE solution, and mix by slow vertical rotation for 60 hr at room temperature. Wash away excess protein with the coupling buffer, and block any remaining active imidocarbonate groups with 1 M Tris buffer, pH 8.5, for 2 hr at room temperature. Finally, wash away excess Tris and adsorbed protein with the coupling buffer, followed by 0.1 M acetate at pH 4 containing 0.5 M NaCI, followed again by coupling buffer. Store at 4° until required. ~4 R. S. Nezlin, Y. A. Zagyansky, A. I. Kaivarainen, and D. V. Stefani, Immunochemistry 10, 681 (1973).

382

R A D I O I M M U N O A S S A Y S AND I M M U N O R A D I O M E T R I C

ASSAYS

[26]

Affinity chromatography--Preparation of lmmunosorbent The immunosorbent-purification of rabbit anti-IgE described is similar to that recorded in detail by Bennich and Johansson, ~ except for the substitution of a commercial anti-IgE produced in rabbit.

Reagents CNBr-activated Sepharose-IgE Rabbit anti-IgE (Behringwerke-Hoechst) NaHCOa, 0.1 M, pH 8.5, containing 0.5 M NaC1 Acetic acid, 0.1 M The procedure should be performed at 4 °. Overnight, stir 20 ml of rabbit antiserum specific for human IgE with an equal volume of SepharoseIgE. Pour into a chromatography column and elute unbound protein with bicarbonate-buffered saline. Desorb the y-globulin fraction of anti-IgE serum with 0.1 M acetic acid; it is now suitable for labeling with 1251.

Labeling lmmunosorbent-Pure Anti-IgE with 1251 This is essentially the chloramine-T procedure first described by Greenwood, Hunter and Glover, ~ and the aim is to label 1 mol of anti-IgE with 1 atom of 1251.

Reagents Immunosorbent-pure anti-IgE Chloramine-T, 2 mg per milliliter of 50 mM phosphate, pH 7.4 12~I, 2 mCi per 10/zl of 50 mM phosphate, pH 7.4 Sodium metabisulfite, 4 mg per milliliter of 50 mM phosphate, pH 7.4 RAST assay buffer: 50 mM phosphate buffer, pH 7.4, with 1% Tween 20, 0.3% bovine serum albumin and 0.05% thiomersal Sephadex G-200 equilibrated in albumin buffer Perform the reaction in a well-ventilated fume-chamber in an ice-water bath. Use a micropipette to add 2 mCi of 125I (10/~1) to 10/.d of anti-IgE (10/.~g) in a 55 × 12 mm polystyrene tube and then in quick succession, with rota-mixing, add 10/zl of chloramine-T (20/zg), 10 txl of sodium metabisulfite (40/zg), and 1 ml of albumin buffer. Separate the radiolabeled anti-IgE from denatured products and free iodide by filtration through Sephadex G-200 with albumin buffer. Store at 4 °. 15 H. Bennich and S. G. O. Johansson, Adv. l m m u n o l . 13, 1 (1971). ~e F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J., 89, 114 (1963).

[26]

RAST AND MIXED-ALLERGEN RAST

383

R A S T Procedure Paper Disks Reagents Allergen coupled to paper disks Test and reference sera Immunosorbent-pure [lzSI]anti-IgE RAST assay buffer: 50 mM phosphate, pH 7.4, with 1% Tween 20, 0.3% bovine serum albumin, and 0.05% thiomersal Saline, 0.9% Polystyrene tubes (55 × 12) mm Dilute 50/zl of test (or reference) serum to 150/zl with incubation buffer; incubate overnight with the appropriate allergen-paper disk. Stop the reaction by washing the disk three times with 2 ml of 0.9% saline, and then "develop" the disk by adding 17 nCi of [125I]anti-IgE in 100/~1 of incubation buffer; again incubate overnight at room temperature. Finally, remove the unbound radioactivity by again thoroughly washing the disk with normal saline. Measure the bound radioactivity in a gamma scintillation spectrometer. The amount of specific IgE in the test sample is compared with a set of four reference sera and assigned a RAST grade based on the commercially developed scoring system (Pharmacia, Phadebas RAST): zero is negative; 1 is equivocal; 2, 3, and 4 are increasingly positive. The test time can be reduced by half, at the expense of using three times as much labeled anti-IgE, using undiluted test serum (50/.d), and reducing the first incubation period from overnight to 3 hr. Cellulose or PMA Particles The RAST can be performed using allergens linked to particles instead of to paper disks. Reagents Allergen particles Test and reference sera Immunosorbent-pure [x25I]anti-IgE RAST assay buffer: 50 mM phosphate, pH 7.4, with 1% Tween 20, 0.3% bovine serum albumin, and 0.05% thiomersal Decanting aid: 1% Tween 20 in 0.15 M NaCI NaCI, 0.15 M Allow the test serum (100/~1) to react with a suspension of allergen particles (50 ~1) on a horizontal shaker for 3 hr at room temperature. Stop

384

RADIOIMMUNOASSAYS AND I M M U N O R A D I O M E T R I C ASSAYS I*

~

I~ = i~+~._ I~ ~

Io-

OO

~ l, i k . ~ _ + l ~ l l ~

TEST+Si:~.IGE ALLG.

[26]

+ ALLG.DiSC

.a.~.+OO ~ ~ ~ OO OO

I~

+ ANTIIGEA"

FIG. 2. Reverse RAST. See text for details,

the primary reaction by adding 2 ml of decanting aid, centrifuge the assay tubes at 2000 g for 1 min, and decant the supernatant solution. Mix 17 nCi of xzsI-labeled anti-IgE (100/zl) with the particles, and allow to stand overnight at room temperature. Finally, thoroughly wash the particles three times with 2 ml of 0.9% saline and measure the bound radioactivity in a gamma scintillation spectrometer. Multiple suction devices (Garner Engineering Co. Ltd., Sherborne, England) are very useful in speeding the washing and decanting steps. R A S T Inhibition Technique

The RAST principle can be used for the assay of allergens by either direct or indirect techniques, ar The indirect method has been commonly used and depends on the ability of the test allergen to inhibit the RAST result of a known positive reference serum. (Fig. 2). Reagents (In addition to those necessary for the RAST Technique, see p. 383. Reference serum pool, from patients with grade 4 RAST scores to the allergen being tested. Patients should not have received specific immunotherapy. Test allergen Prepare serial threefold dilutions of the test allergen in RAST assay buffer; the number of dilutions depends on the strength of the allergenic extract. To aliquots (100/xl) of the serial dilutions add 50/~1 of reference serum and incubate the mixture overnight at room temperature. Add the solid phase allergen (disk, or 50/xl of particles) and perform the RAST in the usual manner. x7 L. Yman, G. Ponterius, and R. Brandt, Dev. Biol. Standard. 29, 151 (1975).

[26]

RAST AND MIXED-ALLERGEN RAST

385

Mixed-Allergen H A S T Allergen extracts coupled to disks or particles usually contain several allergens, each of which may have one or more allergenic determinants. Consequently, a specific IgE reference serum probably has a spectrum of IgE ~intibodies directed to the extract. The spectrum will vary among patients, and for this reason an allergen-independent reference system was developed based on a direct, sandwich RIA for total IgE. TM This assay is similar to the RAST, except that anti-IgE instead of allergen is coupled to either disks or particles. It is simpler to standardize allergen particles for the mixed-allergen RAST, la and so we have limited this part of the text to cellulose particles.

Total IgE Reference Curve Reagents Rabbit anti-IgE serum (Behringwerke-Hoechst) CNBr-activated microcrystalline cellulose particles (see RAST Reagents: Solid Phases: CNBr Activated Microcrystalline Cellulose Particles RAST assay buffer: 50 mM phosphate, pH 7.4, with 1% Tween 20, 0.3% bovine serum albumin, and 0.05% thiomersal Sodium Sulfate, 18% NaHCO3, 0.1 M Decanting aid: 1% Tween 20 in 0.15 M NaCI IgE reference serum dilutions: 0.5, 2, 5, 10, 20, 50, and 100 U/ml in 50% horse serum/RAST assay buffer Immunosorbent-pure [125I]anti-IgE Precipitate the T-globulin fraction from 1 ml of anti-IgE serum by mixing 1 ml of serum with 0.18 g of anhydrous sodium sulfate and 1 ml of 18% sodium sulfate. Allow to stand overnight at room temperature. Wash the precipitate twice with 2 ml of 18% sodium sulfate, reconstitute in 1 ml of 0.1 M NaHCO3, and covalently link to 100 mg of CNBr-activated microcrystalline cellulose particles (see RAST Reagents: Solid Phase Allergens). Incubate 50/~1 of anti-IgE particles for 3 hr at room temperature with dilutions of an IgE reference serum previously calibrated against an international standard (e.g., WHO 69/204). Stop the reaction by adding 2 ml of is U. Lundkvist, in "Advances in Diagnosis of Allergy: RAST" (R. Evans, III, ed.), p. 85. Symposia Specialists, Miami, Florida, 1975. la J. Merrett and T. G. Merrett, Clin. Allergy 8, 235, (1978).

386

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC

ASSAYS

[26]

decanting aid, centrifuge the assay tubes at 2000 g for 1 min, and decant the supernatant solution; add 17 nCi of immunosorbent-pure [12~I]anti-IgE in 100/.d of RAST assay buffer, mix, and allow to stand overnight at room temperature. Finally, wash the particles three times with 0.9% saline (2 ml) in the usual way, and measure the radioactivity associated with the particles.

Estimating the Potency of Allergen Particles Individual particle RASTs must be related to the total IgE reference curve. Reagents [In addition to those necessary for the IgE reference curve (see Mixed Allergen RAST) and the cellulose particle RAST (see RAST Procedure)]. Allergen-specific IgE reference pools: for inclusion in these pools, a serum must have a RAST score of 4 to selected allergen, but no more than 2 to other common allergens Low IgE serum pool: composed of sera from nonatopic patients with total IgE levels below 20 U/ml The object is to determine the amount of allergen particles that, when made to react with a ¼ dilution of the appropriate reference serum pool, produces a count rate in the particle RAST equal to that produced by 20 U/ml in the IgE reference curve. Perform total IgE and particle RAST assays at the same time; it is usually sufficient to use doubling dilutions of allergen particles ranging from x (i.e., undiluted) to ~ in a volume of 50 t~l; the more potent the allergen, the fewer particles will be required. Having determined the optimal concentration of allergen particles, first check that the nonspecific binding is minimal; i.e., when 50 Izl of the titrated amount of particles are reacting with 100 ~1 of the low IgE serum pool, the count rate should be less than that produced by 0.5 U/ml in the IgE reference curve. If the nonspecific binding is high, then a more pure allergenic extract should be used to prepare the allergen particles. Finally, dilute the reference serum pool with serum from the low IgE pool and check that the RAST curve is approximately parallel to the IgE reference curve, and that the particles are unsaturated at a reference serum pool dilution of one-third. Generally, the serum dilutions equivalent to RAST scores of 4, 3, 2, and 1 produce radioactive counts equivalent to 20, 5, 1.2, and 0.6 U/ml when read off the IgE reference curve (Fig. 3).

Mixed-Particle RAST The mixed-particle RAST is performed identically to the particle RAST, except that the 50 tzl suspension will contain more than one vari-

[26]

RAST AND MIXED-ALLERGEN RAST

387

/ 4000

TOTAL IGE

/

GRASS POLLEN-SPECIFICIGE

3000 •

,t/4)

D--

OC W O. u') I.Z 0

2000

I000

i iiiiii

i

I

1

~ iiiill

t0 UNITS IGE PER ML

i

i illlll

1

I00

I

I

1/100 Ii10 SERUMDILUTIONS

I

I

FIG. 3. Estimating the potency of allergen particles. IGE, immunoglobulin E; SP, serum pool. See text for details.

ety of allergen particle. For example, instead of 0.5 mg of grass pollen, the 50 ~l might contain in addition 0.5 mg ofDermatophagoides pteronyssinus and 1 mg of cat epithelial particles--that is to say, 2 mg of particles instead of 0.5 mg. It is likely that the nonspecific binding will increase slightly with the use of more particles, and so a "nonatopic serum" blank should always be run with each assay. This is sometimes more of a problem with PMA than with cellulose particles. Returning to the immunological definition of the atopic person as being one who readily makes IgE antibodies to common environmental allergens, ~ we should like to emphasize the great potential of a mixed-particle RAST screen in any preliminary allergy investigation. For example, in the United Kingdom three common allergens are grass pollen, D. pteronyssinus, and cat epithelium, and we believe that a mixed-particle RAST using these three allergens will detect at least 90% of the atopic population. In other climates, of course, different allergens may be more important, but the principle remains the same: the mixed-allergen RAST technique can be a very efficient screening test for atopic allergy.

388

RADIOIMMUNOASSAYS

[27] S e m i a u t o m a t i o n Magnetic

AND

IMMUNORADIOMETRIC

of Immunoassays Transfer Devices

ASSAYS

[27]

by Use of

B y K E N D A L L O . S M I T H a n d WARREN D . G E H L E

This chapter is designed to review some of the approaches now used for automating immunoassays, specifically the radioimmunoassay (RIA) and the enzyme-linked immunosorbent assay (ELISA), and to describe the use of magnetic devices for processing solid phase elements in these assays. The RIA and ELISA fields, though relatively new, are broad, and newly reported technique variations are accumulating very rapidly. Since the first reports of Berson and Yalow,l'2 radioimmunoassay methods have been used as powerful analytical and quantitative tools. The value of the RIA in biology and medicine is suggested by the fact that Dr. Yalow was awarded a Nobel prize for her pioneering work in this area. The ELISA technique, originally described by Engvall and Perlmann 3 in 1972 for quantitation of antibodies and antigens, has likewise been widely exploited, particularly in the area of rapid serodiagnosis. Several excellent reviews of RIA and ELISA techniques have appeared in the last few years that describe in detail the chemical and physical principles involved, the myriad of technical variations that have been devised, some of the sources for components required for setting up the techniques, and the tremendous variety of applications in which RIA and ELISA have been used. 4-a It would be helpful to the reader to consider from the outset that the RIA and ELISA techniques are similar in many respects, differing principally in the fact that the label in one case is an isotope (e.g., 125I), while in the other case it is an enzyme (e.g., horseradish peroxidase). As will be indicated later, results with one system are often comparable or identical with results with the other, assuming that the immune reagents used are 1 S. A. Berson and R. S. Yalow, J. Clin. Invest. 38, 1996 (1959). R. S. Yalow and S. A. Berson, J. Clin. Invest. 39, 1157 (1960). 3 E. Engvall and P. Perlmann, J. lmmunol. 109, 129 (1972). 4 A. J. Moss, G. V. Dalrymple, and C. M. Boyd, "Practical Radioimmunoassay.'" Mosby, St. Louis, Missouri, 1976. 5 D. S. Skelley, L. P. Brown, and P. K. Besch, Clin. Chem. 19, 146 (1973). e j. p. Felber, Adv. Clin. Chem. 20, 129 (1978). 7 F. W. Spierto and W. Shaw, Crit. Rev. Clin. Lab. Sci. 7, 365 (1977). s j. L. Sever and D. L. Madden, J. Infect. Dis. Suppl. 136, 257 (1977). 9 A. Voller, A. Barlett, D. Bidwell, M. Clark, and A. Adams, J. Gen. Virol. 33, 165 (1976). METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form r~served. ISBN Ikl2-181970-1

[27]

MAGNETIC DEVICES FOR USE IN IMMUNOASSAYS

389

the same except for the tag. Mechanical methods that were first used for RIA work or for ELISA work can frequently be used easily and effectively with the other technique. The quantitation of radiation (counts per minute) or the intensity of color development at the end of the two procedures constitutes the main difference in their mechanics.

A Case for Using a Solid Phase Leiva 1° contrasted and compared the many separatory techniques currently employed in various RIA systems. He observed that the choice of the separation technique to use in any assay is of prime importance because that choice will largely govern the kind of equipment required, the maximum feasible batch size in an assay run, the manipulations required for extractions and/or washings, the reproducibility of the test, and the sensitivity of the test. Separatory techniques now employed include antibody precipitation (second antibody precipitation of antigen-antibody complexes), nonspecific adsorption of immune complexes (e.g., with charcoal, talc, dextran), selective precipitation of immune complexes by salting-out or partitioning (e.g., with ammonium sulfate, organic solvents, or polyethylene glycol), ion exchange techniques, molecular sieving and

solid phase systems. A strong case can be made for using solid phase methods for separating bound from unbound reagents on the basis of greater technical simplicity. A solid phase can be separated from various reagents by simply aspirating, decanting, or mechanically moving by magnetic force. Perhaps the most attractive point is that washing immune complexes attached to a solid phase is extremely efficient. The simplicity, reproducibility, and sensitivity of most existing solid phase assays are such as to make conversion from more cumbersome separatory methods to solid phase methodology very tempting. Nonspecific binding to the solid phase can be greatly reduced by use of purified labeled reagents and by blockage of nonspecific binding sites with immunologically "indifferent" proteins (such as bovine serum albumin). Centrifugation, a time-consuming and awkward procedure required in some separatory techniques, is not required in most solid phase systems. Very tiny amounts of antigen or antibody are required for coating solid phase units, so thousands of units can be coated with the appropriate reagent, dried, and stored indefinitely in a highly stable state. For example, we have coated plastic beads with herpes simplex virus antigen, soaked 10 B. Leiva, Am. J. Med. Technol. 43(1), 41 (1977).

390

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them in 1% bovine serum albumin, dried them, and found no change in antigenic potency after storage for 6 months at - 70 °, - 24 °, or + 5°. Solid Phase Materials The primary requirement for a solid phase material is either that it be capable of nonspecifically adsorbing useful amounts of the desired reagent (usually a protein) or that it be possible to chemically link the solid phase to the reagent. Since the solid phase in an immunoassay is usually used only once and discarded, it is also desirable that the material be relatively inexpensive and obtainable in quantity. Ready availability of large, uniform lots of solid phase material (e.g., plastic) is important for test reproducibility. 1°-12 Uniformity in the total surface area on which the immune reactions take place is important for precision; thus the shape of the solid phase is important. Solid phases that have been used for RIA or ELISA include the following ones: Plastic test tubes 13--16 Plastic microtiter plates 17-20 Plastic disks or beads 21-26 Plastic filters or disks 27,2s Glass fiber filters 29 n p . Atanasiu, S. Avrameas, J. Beale, P. S. Gardner, M. Grandien, K. Mclntosh, D. M. McLean, A. Schuurs, O. Sobeslavsky, and A. Voller, Bull. WHO 56, 241 (1978). lz B. S. Chessum and J. R. Denmark, Lancet 1, 161 (1978). la K. J. Catt and G. W. Tregear, Science 158, 1570 (1967). 14 S. E. Salmon, G. Mackey, and H. H. Fudenberg, J. lmmunol. 103, 129 (1969). 15 H. Daugharty, D. T. Wartield, and M. L. Davis, Appl. Microbiol. 23(2), 360 (1972). le M. Ceska, F. Grossmfiller, and F. Effenberger, Infect. Imrnun. 19, 347 (1978). 17 j. D. Rosenthal, K. Hayashi, and A. L. Notkins, J. Immunol. 109, 171 (1972). is R. H. Prucell, D. C. Wong, Y. Moritsugu, J. L. Dienstag, J. A. Routenberg, and J. D. Boggs, J. Immunol. 116, 349 (1976). 19 A. R. Kalica, R. H. Purcell, M. M. Sereno, R. G. Wyatt, H. W. Kim, R. M. Chanock, and A. Z. Kapikian, J. lmmunol. 118, 1275 (1977). z0 E. Reiss, H. Hutchinson, L. Pine, D. W. Ziegler, and L. Kaufman, J. Clin. Microbiol. 6, 598 (1977). 21 K. J. Catt, H. D. Niall, and G. W. Tregear, Aust. J. Exp. Biol. Med. Sci. 45, 703 (1967). 22 K. O. Smith, W. D. Gehle, and A. W. McCracken, J. Immunol. Methods 5, 337 (1974). 23 K. O. K. Kalimo, R. J. Marttila, K. Granfors, and M. K. Viljanen, Infect. Immun. 15, 883 (1977). 2~ B. R. Ziola, M.-T. Matikainen, and A. Salmi, J. lmmunol. Methods 17, 309 (1977). 25 O. H. Meurman, M. K. Viljanen, and K. Granfors, J. Clin. Microbiol. 5, 257 (1977). 2e j. j. Langone, M. D. P. Boyle, and T. Borsos, J. Imrnunol. Methods 18, 281 (1977). 27 p. Davis, A. S. Russell, and J. S. Percy, Am. J. Clin. Pathol. 67, 374 (1977). 38 S. P. Halbert and M. Anken, J. Infect. Dis. 136, Suppl., $318 (1977). 39 E. D. Sevier and R. A. Reisfeld, Immunochemistry 13, 35 (1976).

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Porous glass 30 Paper disks or filters 31.32 Infected cells adherent to culture vessels 22,33-36 Sepharose (agarose) beads 37,38 Bentonite (clay) 39 Polyacrylamide gel 4o Staphylococcal protein A 41,42 Magnetically attractable cellulose 43 Magnetically attractable polyacrylamide-agarose beads 44 Magnetically attractable ferric oxide particles 45-47 Magnetically attractable plastic coated beads 4s A finely divided solid phase (employing small particles) has the advantage of (a) high efficiency in surface:volume ratio; (b) capability for being dispersed throughout the reaction mixture, allowing immune reactions to proceed to completion very quickly. However, single-piece solid phases have the distinct advantage of being easier to manipulate and process. Moreover, many immunoassays employing single-piece solid phases are sufficiently sensitive that it is not necessary to allow the immune reactions to go to completion in order to obtain good dose-response quantitation. Dilution of the unknown sample and the labeled reagenta, as well as incubation temperature, can be adjusted upward or downward to give sufficiently sensitive immunoassay results within the time frames required for most laboratory testing. a0 H. H. Weetall, Biochem. J. 117, 257 (1970). 31 R. Pauwels, H. Bazin, B. Platteau, and M. van der Straeten, J. lmmunol. Methods 18, 133 (1977). 32 H. Watanabe and I. H. Holmes, J. Clin. Microbiol. 6, 319 (1977). 33 K. Hayashi, D. Lodmeli, J. Rosenthal, and A. L. Notkins, J. lmmunol. 110, 316 (1973)." 34 N. H. Levitt, H. V. Miller, and G. A. Eddy, J. Clin. Microbiol. 4, 382 (1976). 35 B. Forghani, N. J. Schmidt, and E. H. Lennette, J. Clin. Microbiol. 4, 470 (1976). 30 M. A. Jankowski, E. E. Petersen, and J. F. B6cker, Acta Virol. 21, 405 (1977). 37 y . S. Choi, Immunochemistry 14, 53 (1977). 38 A. A. A. Ismail, P. M. West, and D. J. Goldie, Clin. Chem. 24, 571 (1978). 39 W. C. Cheng and D. W. Taimage, J. Immunol. 103, 1385 (1969). 40 G. D61ken and G. Klein, J. Natl. Cancer Inst. 58, 1239 (1977). 41 M. E. Soergel, F. L. Schaffer, J. C. Sawyer, and C. M. Prato, Arch. Virol. 57(3), 271 (1978). 42 p. B. Jahrling, R. A. Hesse, and J. F. Metzger, J. Clin. Microbiol. 8, 54 (1978). 43 M. J. Anderson, Med. Lab. Sci. 35(2), 173 (1978). 44 j. L. Guesdon and S. Avrameas, Immunochemistry 14, 443 (1977). 45 L. S. Hersh and S. Yaverbaum, Clin. Chim. Acta 63, 69 (1975). 4s L. Nye, G. C. Forrest, H. Greenwood, J. S. Gardner, R. Jay, J. R. Roberts, and J. Landon. Clin. Chim. Acta 69, 387 (1976). 47 N. Gochman and L. J. Bowie, Anal. Chem. 49, 1183A (1977). 4s K. O. Smith and W. D. Gehle, J. Infect. Dis. 136, Suppl., $329 (1977).

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Automated Processing It is important that mechanical processing of solid phase units during an immunoassay be efficient and highly uniform. In fact, the mechanical facility with which the solid phase can be manipulated during the assay procedure is sufficiently important that it probably should be a primary determining factor in the choice of the assay system. M a n u a l manipulation of each solid phase unit, particularly during the multiple washing steps (e.g., handling units one at a time with forceps), severely restricts the number of tests that can be accomplished within an acceptable time frame. Operator boredom and consequent fatigue are significant negative factors in highly repetitious operations such as immunoassays. Moreover, reproducibility is adversely affected when time-consuming manual manipulations are required, particularly in assays involving large numbers of specimens and short incubation periods (the first and last units can hardly be equivalent in treatment if each unit must be handled separately by a technician). Major manual steps involve pipetting and diluting specimens, dispensing reagents, incubation, multiple rinsings, gamma counting or spectrophotometry, and data-reduction interpretation. Efficient use of technician time is difficult if these steps are all done manually. For these reasons there has been considerable interest in automated systems for RIA and ELISA; consequently, several more or less automated systems have been devised. The review by Gochman and Bowie 47 compared the following commercially available (or soon to become available) systems: (a) the Union Carbide "Centria" (a solid phase is not used); (b) the Micromedics "Concept 4," which uses antibody-coated tubes as a solid phase; (c) the Becton-Dickinson "Aria II," which is designed around a flow-through, • reusable solid phase chamber; (d) the Technicon automated RIA system, which employs antibody-coated ferric oxide particles as a solid phase. 4a Sevier and Reisfeld 29 described a semiautomated RIA processor that utilizes a solid phase double-antibody procedure and glass fiber filters. Brooker e t al. 49 devised an automated system called "Gamma-flow" which uses column separation of reactants instead of a solid phase adsorbent. Ismail e t al. 3~ designed a fully automated, continuous flow system employing agarose (Sepharose) beads as the solid phase. The Gilford Corporation has developed an automated system for accomplishing ELISAs; it uses plastic cuvettes as the solid phase, automatic spectrophotometry, and computerized reduction of data. Cordis Laboratories, Inc. has marketed an ELISA system employing plastic-coated disks (composition is proprietary information), which are processed with mechanical devices 49 G. Breoker, W. L. Terasaki, and M. G. Price, Science 194, 270 (1976).

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that they provide. Organon Diagnostics has devised a semiautomated solid phase ELISA for detecting viral antigens in clinical specimens. 5° Leinikki and Passila, 51 in cooperation with Finnipipette-Labsystems, developed a semiautomated ELISA for viral antibodies employing disposable polystyrene cuvettes with optically clear bottoms. The capacity of most of these automated systems for handling specimens is probably in the range of I0-40 test samples per hour. 38,47Claims for greater capacity have been made, but such claims seem to ignore the substantial time required for specimen dilution, instrumental reading of the results, and data analysis. Galen, ~2 speaking from the position of a hospital pathologist, expressed the view that "industry efforts directed toward total automation of RIA procedures have been numbingly earnest in their intentions and crushingly dull in their execution." A Case for Choosing Semiautomation Judicious use of labor saving devices obviously increases work efficiency by decreasing the boredom and personal fatigue that result from excessively repetitious manual operations. Moreover, mechanical devices obviously perform some tasks more precisely and economically than technicians can. Fully automated systems, however, can be prohibitively expensive as capital investments, trouble-prone, expensive to maintain, and highly restrictive in how they can be used (e.g., they may be inflexible in the kinds of tests they can perform). The manufacturers of automated equipment may deliberately design components of their system so that they will be the sole source of these components (e.g., proprietary reagents and odd-shaped tubes) and this can be a big disadvantage. Between the extremes of n o automation and c o m p l e t e automation there is an area of semiautomation that most laboratories may wish to consider because of cost and flexibility considerations. The marketplace is becoming crowded with devices designed to accomplish the following major steps in solid phase immunoassays: sample pipetting, diluting, dispensing diluted samples into appropriate tubes or wells, dispensing reagents, adding the solid phases to appropriate tubes or wells (or the reagents to the solid phase tube or well), processing the solid phases through various washes and specific reagents and, finally, measurement of the resulting color (in the ELISA) or radioactivity (in the RIA). Automated pipetting-diluting stations are commercially available in 5oQ. R. Miranda, G. D. Bailey,A. S. Fraser, and H. I. Tenoso,J. Infect. Dis. 136, Suppl., $304 (1977). s~ F. Leinikki and S. P/isillfi,J. Infect. Dis. Suppl., 136, 294 (1977). 5~R. S. Galen, Human Pathol. 8(4), 359 (1977).

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configurations that accommodate various numbers and sizes of test tubes or wells, including 96-well Microtiter plates. Either serial dilutions or single, screening dilutions of a sample can be accomplished with some of these devices. Gamma counter manufacturers are marketing pipetter-diluters and computer data processors as ancillary equipment for immunoassay work. Most gamma counters are automated in that they provide automatic sequencing of 300 or more samples per load and a printout of counts per minute on tapes. Reduction and processing of this kind of data can be accomplished with relatively simple, programmable desk top computers by methods such as those described by Grotjan and Steinberger sa and Naus et al.S4 A spectrophotometric system has recently been offered commercially that allows measurement of substrate color development in each of the 96 wells of a Microtiter plate within 60 sec and performs a number of dataprocessing functions, s4a Both RIA and ELISA techniques have been steadily miniaturized; the Microtiter plate level of mechanical miniaturizing may be a reasonable compromise in size for test accuracy and precision, and still provide good economy of reagents (volumes of 100/zl or less are required for tests). The Gilford Processor/Reader 54b uses 300/~1 volumes and allows optional manual control or automation in the washing, reagent-dispensing, and data collection steps of the ELISA. Flexibility in immunoassay design is important because the state of the RIA and ELISA art is changing so rapidly that new improvements must be taken into account continually. Accepting rigidity in assay design options by choosing complete, "hands-off' automation may, therefore, be unwise at this particular time. Technical flexibility is highly desirable, but is possible only if each of the major steps in the immunoassay can be independently modified to fit specific needs of the laboratory. Thus, a strong case can be made at the present time for semiautomation, which sufficiently minimizes technical drudgery, yet gives the needed flexibility for optimizing the immunoassay through modification of any one or a combination of technical steps. Most of the labor-saving components are now commercially available for assembling semiautomated systems for either the RIA or ELISA, tailored exactly to the needs of a particular laboratory. H. E. Grotjan and E. Steinberger, Comput. Biol. Med. 7, 159 (1977). A. J. Naus, P. S. Kuppens, and A. Borst, Clin. Chem. 23, 1624 (1977). u~ Titertek Multiskan Plate Reader, Flow Laboratories, 1710 Chapman Avenue, Rockville, Maryland 20852. ~b Gilford Instrument Laboratories, Inc,, Oberlin, Ohio 44074.

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An Example of Semiautomation: Magnetic Transfer Devices for Processing One of the primary purposes of this chapter is to present a detailed description of magnetic transfer devices for simultaneously processing solid phase units (beads) in either RIA or ELISA systems. It is understood that automatic pipetting devices, automatic instrumentation for reading v-radiation or color development, and programmable calculators could, at the discretion of the investigator, be used in conjunction with the magnetic processing devices described below. We have already described the design and use of 24-place and 60-place devices for use in solid phase RIA or ELISA techniques. 48 Since that time, we have designed modifications that have increased the numbers of specimens handled simultaneously to 400 and have miniaturized the me-

FIG. 1. Crosman BB shot " b e a d s " plated with "bright nickel" and "black nickel" (for color coding purposes), then coated with polycarbonate by immersing in 5% solution of methylene chloride, scattering on slick paper, and allowing to dry. Various antigens or antibodies are adsorbed onto this plastic surface; the beads are soaked in a 1% solution of bovine serum albumin in phosphate-buffered saline, drained, air dried, and stored below 0° until needed.

FIG. 2. Device " A " consists of 96 soft iron rods embedded in a I-inch Lucite plastic sheet; the rods protrude about ~ inch on one side and are flush with the reverse side of the plastic sheet (outer dimensions are 123 m m × 80 mm).

FIG. 3. Device "B'" is a powerful permanent ceramic magnet (obtained from Texas Magnetics Corp., Garland, Texas); the outer dimensions are the same as those of device " A . "

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FIG. 4. Test tube rack containing small (6 mm x 50 mm) test tubes (also see Fig. 11); foreground; U-bottom Microtiter plates (Linbro S-MRC 96).

chanical system so as to allow use of smaller volumes of reagents (70/zl, in Microtiter plates). For several reasons the Microtiter configuration may be ideal for immunoassay work (much ancillary equipment has already been designed and marketed for Microtiter work). Therefore, the following description is aimed toward accommodating the Microtiter design. The apparatus required for performing an RIA or ELISA with this particular magnetic processing system includes: plastic-coated steel beads (see legend of Fig. 1 for details of preparation); a 96-place device ( " A " ) consisting of short iron rods embedded in a Lucite plastic sheet (Fig. 2); a permanent magnet ( " B " ) of similar dimensions (Fig. 3); two or three disposable-type Microtiter plates (depending upon whether the system is RIA or ELISA); and a test tube rack that holds small test tubes in the same spatial configuration as the Microtiter plate wells (Fig. 4). Each piece of apparatus is designed to facilitate the simultaneous processing of from 1 to 96 solid phase units (plastic-coated steel beads) through the vail-

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RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

PLASTIC

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PLASTIC CLASSSPECIFIC

COATED VIRAL HUMANANTI-HUMAN,,~ COATED ANTI-HUMAN HUMAN ANTI-HUMANjq::,~. BEAD ANTIGEN ANTIBODYANTIBODYt~I~ BEAD GLOBULIN GLOBULIN GLOBULIN~ r

(~= IzsI LABEL

BOVINESERUMALBUMIN

SERUMALBUMIN

FIG. 5. (A) Diagram showing sequence of the "indirect" or labeled second antibody reaction (left to right): plastic-coated bead is coated with viral antigen, soaked in bovine serum albumin, dried, stored, withdrawn, placed in a 1 : 100 dilution of human serum, then placed in enzyme-labeled antihuman globulin. The final step (see text) is development of a color reaction by dropping the bead-antigen-antibody complex into wells containing 2,2'-azinodi(3-ethylbenzthiazoline) sulfonate. (ABTS). (B) Diagram showing sequences for quantitating class-specific human globulin (left to right): plastic-coated bead is coated with purified antihuman globulin, soaked in bovine serum albumin, dried, stored, withdrawn, placed in serial dilutions of serum (also tears, saliva, etc.), then placed in ~"I-labeled antihuman globulin. The final step is reading counts per minute in a gamma counter. These results are plotted on semilog graph paper and compared with similar results with human globulin of known concentration (standard curve).

ous reaction mixtures and rinsing baths to the point in the procedure at which test tubes containing the beads are placed either in a gamma counter (for RIA) or, alternatively, the beads are dropped into the wells of a Microtiter plate containing an appropriate enzyme substrate (for ELISA). The processing sequence can be varied with complete freedom in order to accomplish different purposes in an assay (i.e., for either RIA or ELISA; for either antigen or antibody detection; by either the "direct" or the "indirect" techniques). As an example, the following sequence of steps is appropriate for ELISA detection of human antibody to a specific viral antigen, by the "indirect" (labeled second antibody) technique (Fig. 5A shows diagrammatically how this can be accomplished). Step 1. Antigen-coated beads are placed, with plastic tipped forceps, onto the magnetic terminals of the 96-place transfer device (as in Fig. 6); all beads are dropped simultaneously into wells of a Microtiter plate containing either serial serum dilutions or a screening dilution of many differ-

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FIG. 6. Placing of antigen-coated beads onto iron terminals of device " A , " preparatory to dropping the beads (simultaneously) into a Microtiter plate whose wells contain diluted human sera. Dropping of beads into the wells is accomplished by simply removing the magnet (device " B " ) from the iron terminals of device " A . "

ent human sera (1:50 - 1 : 100 dilution is frequently a good choice of a screening dilution). Dropping of the beads is accomplished by simply removing device " B " (the magnet) from the top of device " A " (the soft iron terminals). Step 2. Beads are incubated for 1 hr at room temperature, then removed simultaneously by placing device " A " over the plate and placing the magnet ( " B " ) over the top of device " A " (see Fig. 7); beads then " j u m p " by magnetic attraction from the bottom of the microtiter wells to the iron terminals of device " A " and are held there very firmly (the magnet is powerful) (see Fig. 8). Step 3. Beads are washed sequentially in two separate water baths by dipping them six times in and out of each bath; washing is more efficient if beads are positioned upward (Fig. 9). Step 4. By removing magnet " B " from device " A , " beads are simultaneously dropped into a second Microtiter plate, each of whose wells contain 70/zl of horseradish peroxidase linked (labeled) antihuman globulin.

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FIG. 7. Placing of magnet (device " B " ) over iron terminals of device " A , " so as to cause beads at bottom of Microtiter plate wells to jump to the terminals and be held there securely (beads on right have jumped; those on left have not).

Step 5. Incubation and bead removal (step 2) is repeated. Step 6. Washing (step 3) is repeated. Step 7. Step 4 is repeated (dropping of beads into wells), except that beads are simultaneously dropped into a third Microtiter plate whose wells contain a solution of the HRP substrate 2,2'-azino-di(3-ethylbenzthiazoline) sulfonate (ABTS). Step 8. Beads are incubated for 5-20 min at room temperature, or until the positive serum controls turn dark blue-green but before the negative serum controls develop a detectable color (Fig. 10). Color development in the substrate wells can be quantitated by spectrophotometry or, less precisely, by eye. If spectrophotometry is to be done directly in the Microtiter plate wells, plates with fiat-bottomed wells are used and the beads are removed so as to allow an unobstructed beam of light to pass through the colored substrate in each well (as with the Flow Laboratories plate reader). The standard curve principle can be used, as for RIA quantitation) 5 If the human eye is used for reading color 55 W. R. Patterson and K. O. Smith, J. Clin. Microbiol. 2, 130 (1975).

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FIG. 8. Devices " A " and " B " are in place, with all beads adhering securely to the iron terminals.

reactions, visual reference needs to be made to positive and negative controls in order to evaluate each unknown. The standard curve principle can be used effectively to obtain reasonably good quantitation by visually comparing the color intensity of an unknown at a single dilution with the nearest-match color intensity in a serially diluted known positive sample (standard). Reproducibility of this method (visually matching unknowns with dilutions of a standard) is usually within one twofold dilution, which is adequate for many purposes. The main problem with this visual method of quantitation is that a very strong positive unknown has such a dark color that it is difficult to compare with similar dark colors in a standard sample. Therefore, an unknown sample must be diluted to a low level to determine whether it is positive and to a higher level for adequate visual quantitation (the eye seems to " s e e " only so much color intensity, and no more). As already indicated, the steps used for magnetically processing plastic-coated beads can be varied easily to accomplish different tasks. A further example is given below, which illustrates RIA quantitation of an "antigen" in human sera or saliva; the "antigen," in this particular case, is either IgG, IgA, or IgM (see Fig. 5B for a diagrammatic explanation of

402

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS

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FIG. 9. Rinsing of beads by immersion of device " A " into a water bath. The inverted, diagonal position is preferable. Alternate total removal and total submersion gives efficient washing. Alternatively, this washing step may be accomplished by serial transfer of beads from one Microtiter plate to another (the wells simply containing water), or by holding the beads under a moving stream of water.

this assay; the solid phase bead is coated with either anti-IgG, anti-IgA, or anti-IgM; antiglobulins are prepared in goats or rabbits and are heavychain specific). Step 1. Antibody-coated beads are placed onto the magnetic terminals of the transfer device (see Fig. 6) then dropped simultaneously into wells of a Microtiter plate (Fig. 7) containing appropriately diluted specimens (serum or saliva). Step 2. Beads are incubated for 1 hr at room temperature, then removed simultaneously by magnetic force (see Fig. 8). Step 3. Beads are washed sequentially in two separate wash baths (see Fig. 9). Step 4. Beads are simultaneously dropped into a second Microtiter plate whose wells contain l~H-labeled antihuman globulin (prepared against the Fc portion). Step 5. Incubation and transfer (step 2) is repeated.

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FIG. 10. Appearance of a completed ELISA for toxoplasma antibodies. Row 1 contained a serially diluted, positive control, human serum (1 : 100-12,800). Row 2 contained a similarly diluted negative control human serum (no color developed). All remaining wells contained 1 : 100 dilutions of " u n k n o w n " human sera being tested for toxoplasma antibodies.

Step 6. Washing (step 3) is repeated. Step 7. Beads are simultaneously dropped into small test tubes (see Fig. 11), then placed in a gamma counter. Step 8. Beads (in the tubes) are read in the gamma counter. Dose-response curves are plotted for the unknown specimens (dilution factor vs counts per minute) and for serially diluted standards (containing known amounts of IgG, IgA, and IgM). From these curves, one can calculate the concentration of immunoglobulin in each of the unknown specimens. Advantages in using a magnetic processing system of this kind for immunoassays are summarized as follows: 1. The equipment can be used either for RIA or ELISA equally well; the sensitivity of the two systems is about the same4S; also see Section VIII.

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FIG. 11. Beads that have been completely processed in the radioimmunoassay procedure and are about to be dropped into small test tubes, then placed in a gamma counter for reading (the last step in the assay).

2. Any number of beads (up to 96) can be processed as quickly and easily as a single bead; antigen-sensitized beads and expensive reagents are not wasted because only the number of beads required for a particular test run are used (when the Microtiter well is sensitized and used as the solid phase, an entire plate of 96 sensitized wells must be used if one test is done). 3. Beads are dropped into reaction mixtures simultaneously and are later removed and washed simultaneously, so that large number of antigen-antibody reactions are instantly started and stopped. Thus, highly reproducible results are obtainable when large numbers of assays are performed in a short time period (when unknown samples are added to 96 sensitized wells one at a time, the first and last samples cannot be incubated identically). 4. In the ELISA technique, beads can be removed simultaneously from the substrate-containing wells, thereby instantly stopping further color development due to specific enzyme-substrate turnover. In techniques where the wall of the Microtiter plate well is the solid phase, simultaneously stopping development of the color reactions in all wells is difficult or impossible. 5. Steel BB shot are uniform in diameter (see Table I), inexpensive,

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TABLE I UNIFORMITY OF BB SHOT FROM THREE DIFFERENT SOURCES

Manufacturer o f BB shot Crosman P o w e r Line Daisy

Mean Diameter (inches) a

Standard deviation o f diameter (%)

Standard deviation o f calculated surface area ~ (%)

0.1723 0.1741 0.1702

0.21 0.27 2.59

0.59 0.78 7.7

a Eleven different shot, from each o f three different commercial sources, were m e a s u r e d with a m i c r o m e t e r caliper. E a c h shot was m e a s u r e d in three different directions, and the m e a n diameter m e a s u r e m e n t was calculated. T h e m e a n diameter o f 11 different shot was then calculated and is s h o w n in the chart. b Surface area = ~'r a.

and commercially available in very large lots. They can be coated with almost any plastic (including polycarbonate), are magnetically attractable, and thus provide an almost ideal solid phase surface upon which immune reactions can take place. The small size of the beads allows scaling down reagent volumes to very small proportions and, therefore, permits substantial conservation of precious reagent materials. If larger wells and volumes are used, several color-coded beads can be incubated in the same well at the same time, each bead coated with a different antigen or antibody, and all beads processed simultaneously) e Color coding of beads can help avoid errors in usage, several different antigens can be used for simultaneous use in one 96-well plate, and there is complete flexibility in choice of combinations. 6. Reproducibility of immunoassay results can be adversely affected by variations in the chemical formulation of different lots of plastics used as the solid phase (see section on Solid Phase Materials). Therefore, use of a thin coating of one lot of plastic over a material like metal for millions of tests could be important in standardizing immunoassay techniques. Extremely small amounts of polycarbonate (or other plastics) are required for putting a thin coating on steel beads. Therefore, one small commercial lot of plastic (e.g., one 212 pound 4 x 8 foot sheet of polycarbonate) would be sufficient to prepare beads for over 100 million tests. Plastic solid phases employing Microtiter plates, test tubes, and polystyrene beads require relatively large quantities of plastic for fabrication and, cons6 W. D. Gehle, K. O. Smith, D. A. Fuccillio, A. Perry and A. P. A n d r e s e , J. Immunol. Methods 26, 381 (1979).

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sequently, the lots of plastic change frequently. Such changes in the formulation of plastic lots might cause devastating variations in immunoassay results. Reagents

Antibodies The quality of immune reagents are of at least equal importance to the choice of the mechanics for solid phase immunoassays. The entire globulin or y-globulin fraction from hyperimmune animals can be labeled and used successfullyg; however, the specific antibody playing a useful part in the immunoassay is a very small fraction of the total labeled proteins. Therefore, immune affinity chromatography is highly recommended for purifying the appropriate specific antibody fraction prior to labeling. Several good methods are available for this purpose (Cuatrecasas and Anfinsen57; Pharmacia Fine Chemicals Monograph, 1974). The main benefit in labeling only the desired, specific antibody fraction is that the background ("noise") in the assay is diminished because labeled nonspecific proteins inevitably adsorb to some extent to solid phase surfaces, in spite of prior "blocking" of the solid phase surface with nonspecific proteins such as BSA. Moreover, there is a large savings in the quantitiy of the ~25Ior enzyme label required for labeling, since only specific antibody is labeled. Labeling antibody with 1251can be accomplished in several different ways (see the review by Skelley et al.5); among the commonly used methods are (a) various modifications of Hunter and Greenwood's 58 chloramine-T oxidation method, including that of Bolton and Hunter; 59 (b) the use of catalytic amounts of lactoperoxidase for iodination, s° It is important that unbound ~25Ibe thoroughly removed from the reaction mixture, after labeling, by chromatography with an anion exchange resin, ~5 or by dialysis through a cellophane membrane. Otherwise, unlabeled ~5I may adsorb nonspecifically to the solid phase and increase background noise in the assay. Labeling antibody with an enzyme can be accomplished rapidly and efficiently by the gluteraldehyde linkage method. 9"6~ Other widely used methods for labeling antibodies with enzymes include those of Nakane and Kawaoi e2 and Engvall and Perlmann. 3 Volleff has described in techni5~ p. Cuatrecasas and C. B. Anfinsen, Annu. Rev. Biochem. 40, 259 (1971). W. M. Hunter and F. C. Greenwood, Nature (London) 194, 495 (1962). 5g A. E. Bolton and W. M. Hunter, Biochem. J. 133, 529 (1973). ~o j. j. Marchalonis, Biochem J. 113, 299 (1969). 61 S. Avrameas, lmmunochemistry 6, 43 (1969). ~2 p. K. Nakane and A. Kawaoi, J. Histochem. Cytochem. 22, 1084 (1974).

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MAGNETIC DEVICES FOR USE IN IMMUNOASSAYS

407

PLASTIC COATED VIRAL HUMAN SIMIANANTIBEAD ANTIGEN ANTBODY ENZYME

ANTI-PRIMATE ~ELANT/BODY BOVINSERUM E ALBUMIN FIG. 12. Diagram showing sequence for detecting viral antibody by the peroxidase-antiperoxidase (PAP) method of Sternberger et al. ~ This method uses a "bridging" antibody (anti-primate antibody) between the human and simian antibody. Artificial (chemical) linkage of enzyme with antibody is replaced by an antigen-antibody reaction.

cal detail a "double antibody sandwich" method for detection of a n t i g e n and a simple method for detecting a n t i b o d y , using an "indirect" ELISA in Microtiter plates. This general approach to doing ELISAs (equivalent to methods we and others have described for the RIA) is now in general use (the inner wall of the Microtiter plate well can be used as the solid phase). Alternatively, the "indirect" ELISA method of Sternberger et al. ~3 or the modification of this approach by Butler et al. ~ can be used; this involves the production of soluble immune complexes of antienzyme globulin and enzyme (see diagram in Fig. 12). The Sternberger-Butler method, called by Butler the "amplified E L I S A , " is interesting because affinity chromatographed, purified antiglobulins are n o t required for enzyme labeling; artificial linkage by chemical (covalent) bonding is completely circumvented. The Organon Corporation holds patents on processes for covalent linkage of enzymes to antibodies and use of these reagents in the ELISA (enforcibility of these patents remains to be established in a court case, now pending, against one of the many commercial sources of ELISA reagents). No patents now exist on the Sternberger-Butler approach to producing labeled antibody, i.e., use of an enzyme-antibody reagent resulting from coupling by an i m m u n e reaction.

sa L. A. Sternberger, P. H. Hardy, Jr., J. J. Cuculis, anti H. G. Meyer, J. Histochem. Cytochem. 18, 315 (1970). J. E. Butler, P. L. McGivern, and P. Swanson, J. Immunol. M e t h o d s 20, 365 (1978).

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Protein A Protein A, a structural component of certain strains of staphylococci, has been used successfully in the place o f the second antibody in radioimmunoassay techniques, ~6 as well as the solid phase adsorbent of antibody (mentioned in the section Solid Phase Materials). This protein can be heavily labeled with ~25Iand is, therefore, a reasonable alternative for the use of labeled antiglobulins in immunoassays.

Antigens Immunoassays can be accomplished in systems where an unknown antigen (rather than antibody) competes with labeled antigen for specific antibody adsorbed onto the surface of a solid phase (such as the lower inner wall of a plastic tube). This method can be used effectively for measuring serum antibody levels (Clinical Assays Division of Travenol Laboratories, 620 Memorial Drive, Cambridge, Massachusetts, 02139); the more highly purified the labeled antigen, the less background noise there is likely to be in the assay system.

Enzyme-Substrate Combinations Judicious choice of an enzyme-substrate combination is important if optimal ELISA results are to be obtained. For example, the relative merits of peroxidase, alkaline phosphate, and glucose oxidase in the ELISA have been studied, 44 and substantial differences in both dose-response kinetics and assay reproducibility have been demonstrated. Solubility of the colored substrate derivative and the manner in which the color reaction is measured may also influence the investigator's choice of an enzyme-substrate combination. Some investigators may read ELISA results by eye rather than by spectrophotometer, therefore an easily visualized color reaction should be chosen. Optimal lighting conditions should be arranged for reading the results, and colors apt to cause visual perception problems in partially color-blind people should be avoided. Two combinations that are now widely accepted for ELISA work are (a) alkaline phosphatase-p-nitrophenyl phosphate (PNPP) substrate; (b) horseradish peroxidase-ABTS substrate. The former gives a bright yellow reaction product; the latter gives a bluish green color; both colors can be visualized easily or quantitated spectrophotometrically. Differences between shades of very dark colors are hard to distinguish by eye, therefore lighter shades are preferable if visual quantitation is desired.

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409

A Case for Selecting an E n z y m e R a t h e r T h a n a Radioisotope for Labeling Most RIA methods arc highly sensitive and reproducible, but there are several distinct disadvantages associated with isotopic labeling. 28 Probabl~, the greatest of these is the short half-life of the most frequently used isotopes (only 8 days for 131I and 60 days for 1251), which drastically reduces the useful shelf life of labeled reagents. Since it is impractical to make large lots of such reagents, reagents must be labeled frequently and proper standardization requires careful quality control on each lot of labeled reagents. Other problems are the licensing requirement for use of radioisotopes, special training requirements for personnel, health risks, disposal of radioactive waste, and the requirement for relatively expensive equipment (gamma counters) for readout. In contrast, use of an enzyme label has several major advantages: labeled reagents are stable on storage (shelf life of lyophilized reagents is probably infinite), disposal of labeled waste is not a problem, no special licensing is required for handling enzymes, and spectrophotometric readout equipment required for ELISA techniques is usually less expensive than gamma counters. Moreover, many workers prefer to read ELISA end points by eye, so no readout equipment is required. RIA and ELISA techniques are probably about equal in sensitivity, 4s although ELISA methods theoretically could be more sensitive. In experiments we have done comparing the relative sensitivity of the two methods by serial dilution-end point assays, 29 of 30 sera gave RIA and ELISA end points within twofold of each other when titrated for herpes simplex type 1 antibody. Similar comparisons of titrations for human cytomegalovirus (CMV) and toxoplasma antibody showed good correlation between the two methods; 26 of 27 and 27 of 29 sera showed titers within twofold of each other (CMV and toxoplasma assays, respectively) with the RIA. In all of these comparisons, ELISA results were read by eye, not with a spectrophotometer. There is a large advantage associated with the ELISA for screening specimens visually for the rare positive or negative (for example, assaying for hepatitis B antigen in human sera). A visual glance at a panel of 96 wells will establish the identity of one or two positive specimens within a few seconds. Close to 2 hr are required to cycle 96 specimens through a gamma counter to find these positive specimens, assuming 1 min counting time and time for mechanical movement of tubes. Therefore, counting tubes in a gamma counter can actually require as much time as the rest of the RIA procedure. ELISA systems employing visual readings seem ideally suited to situations involving an occasional or rare outcome, whether the result be positive or negative.

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Application of the Solid Phase (Bead) Magnetic Processing System; Some Examples Until recently most of our own serologic work has utilized isotopic labeling and the RIA, but we are convinced that ELISA methodology will gradually replace the RIA for much immunoassay work. The technical transition from RIA to ELISA is easy, especially when steel beads are used as solid phase units. Differences in the sequential procedure merely involve use of enzyme-labeled reagents, placing the bead (with the enzyme-labeled immune complex) into a suitable substrate solution (instead of the gamma counter), and estimating color development in the substrate solution rather than measuring gamma radiation. Examples of how we have used the magnetic processing system in immunoassays follows.

Correlation Studies between RIA and Other Serologic Methods Sera were obtained from several different sources in order to compare the RIA with the serologic assays commonly used in the past in clinical laboratories for detecting antibody to members of the TORCH (toxoplasma, rubella, cytomegalovirus, and herpesvirus) group of agents. Ninety-eight sera were obtained under code from source A. Most of these sera had been previously tested by older methods for antibody to toxoplasma, rubella virus, cytomegalovirus (CMV), and herpes simplex virus type 1 (HSV-1). One set of sera was chosen to represent a cross section of the normal population; that is, the proportion of positive and negative sera in this sample was similar to the proportion likely to be seen in the clinical laboratory setting. Because these sera represented a cross section of the normal population, there were relatively few sera with antibody to toxoplasma and only a few without antibody to rubella virus. To ensure that the RIA could detect antibody to toxoplasma, 28 additional toxoplasma-positive sera were obtained from source B. To ensure that the RIA did not give false positive reactions in the rubella test, 20 additional rubella-negative sera were obtained from source C. Besides this large number of sera, 43 sera from source D and 27 sera from source E were obtained under code. Most of these sera had been previously tested by older methods for antibody to either CMV, HSV-1, or both. Not all sera were used from each group for all tests because of limited quantities available. Toxoplasma. The commonly used serologic tests for antibody to toxoplasma are the fluorescent antibody (FA) test and the indirect hemagglutination (IHA) test. Table II shows a comparison of the RIA with these two tests. The most important point to be seen is the agreement between the RIA and the other tests in distinguishing positive and negative sera.

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411

TABLE II m COMPARISON OF TOXOPLASMA SEROLOGIC ASSAYS WITH THE MAGNETIC

RADIOIMMUNOASSAY (RIA) Correlation with the RIA Source of sera

Type of test used ~

No. of negative sera

No. of positive sera

Negative (%)

Positive (%)

A B

IHA FA

82 --

16 28

~- (98%) --

~ (100%) ~ (96%)

a IHA, indirect hemagglutination; FA, fluorescent antibody.

This correlation was excellent, approaching an overall agreement of approximately 98%. The 28 toxoplasma-positive sera that had been titrated for antibody by FA were titrated by RIA, and the titers of the two tests were correlated, using Spearman's rank-order correlation test. The correlation coefficient was 0.68, and probability that this value differs from zero (no correlation) by chance alone was <0.001. Thus there was reasonably good agreement between the titers obtained in the two tests. Rubella Virus. Antibody to rubella virus is currently detected in most laboratories by the hemagglutination inhibition (HI) test. Since there are relatively few people in the normal population who do not have antibody to this virus, 20 sera which were negative by HI were tested by RIA, in addition to the 100 sera from source A. A comparison between the HI and RIA showed almost complete agreement in distinguishing positive from negative sera (99%; see Table III). A comparison of the titers obtained by these two tests gave a correlation coefficient of 0.30 and p<0.05 that this coefficient differs from zero by chance alone. One reason for the poorer correlation between the titers of these two tests is that they may be deTABLE III A COMPARISON OF RUBELLA VIRUS HEMAGGLUTINATION INHIBITION (HI) AND MAGNETIC RADIOIMMUNOASSAY (RIA) TESTS Correlation with the RIA Source of sera

Type of test used

No. of negative sera

No. of positive sera

Negative (%)

Positive (%)

A C

HI HI

8 20

93 --

~ (100%) ~ (95%)

~] (100%) --

412

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RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS TABLE IV m COMPARISON OF CYTOMEGALOVIRUS SEROLOGIC ASSAYS WITH MAGNETIC RADIOIMMUNOASSAY (RIA)

Correlation with the RIA Source of sera

Type of test used "

No. of negative sera

No. of positive sera

Negative (%)

A D D E

IHA IHA CF CF

39 12 12 --

45 32 31 19

~ (97%) ~ (100%) ~ (100%) --

Positive (%) ~ ~ ~ ~

(100%) (100%) (100%) (89%)

a IHA, indirect hemagglutination; CF, complement fixation.

tecting different antibodies. This should not present an interpretation problem in the clinical laboratory since there is no significant disagreement between these tests as to whether or not the patient has been infected with rubella virus and has antibody. Cytomegalovirus (CMV). The RIA was compared to two standard serologic tests for antibody to CMV: the IHA test and the complement fixation (CF) test. Table IV shows the results obtained with sera from three different sources. The overall agreement in distinguishing between positive and negative sera was 98%. The titers we obtained with the RIA were compared with titers obtained in other laboratories using the IHA test. The coefficients of correlation were 0.85 (p<0.001) and 0.77 (p<0.001), respectively. This excellent agreement between titers indicates that the two tests probably measure the same antibody. The correlation between the CF test and the RIA was not as good, however. The correlation coefficient was 0.35 (p >/0.05), indicating that the CF test probably measures an antibody different from that measured by either RIA or the IHA test. It should be noted, however, that all three tests gave similar results as to the presence or the absence of specific CMV antibody in these sera. Herpes Simplex Virus Type 1 (HSV-1). The RIA was compared to two standard serologic tests for detecting antibody to HSV-I: the IHA test and the CF test. Table V shows the results obtained with sera from three different sources. The overall agreement in distinguishing between positive and negative sera was 98%. The titers we obtained with the RIA were compared with the titers obtained by other laboratories, using the IHA test. The coefficients of correlation were 0.60 (p<0.001) and 0.55 (p<0.01), respectively. The correlation between the RIA and the CF test was also in good agreement, 0.56

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413

MAGNETIC DEVICES FOR USE IN IMMUNOASSAYS TABLE V m COMPARISON OF HERPES SIMPLEX VIRUS TYPE 1 SEROLOGIC ASSAYS WITH THE MAGNETIC RADIOIMMUNOASSAY (RIA)

Correlation with the RIA Source of sera

Type of test used a

No. of negative sera

No. of positive sera

Negative (%)

Positive (%)

A D D E

IHA IHA CF CF

35 13 13 --

51 31 29 8

~ (100%) ~ (100%) ~ (100%) --

~ (98%) ~ (97%) ~ (100%) ~ (88%)

IHA, indirect hemagglutination; CF, complement fixation.

TABLE VI THE PRECISION OF THE MAGNETIC RADIOIMMUNOASSAY FOR DETECTING ANTIBODY TO HERPES SIMPLEX VIRUS TYPE 1 (HSV-I)

Serum SAT 6

Mean (cpm)

Standard deviation (cpm)

Control beads HSV-I beads HSV- l-specific cpm a

754 1268 514

74.7 73.4 714

Coefficient of variation (%)

9.9 5.7 13.9

a HSV-1 cpm minus control cpm.

(p<0.01). The reason for this good correlation is probably that HSV-1 infection causes recurrent disease so that all types of antibodies to HSV-1 remain at relatively high levels and one type or another is sure to be detected by a given test. The precision of the RIA was determined by assaying one serum 15 different times (in triplicate) for antibody to HSV-1. The results are shown in Table VI. The coefficient of variation was 13.9%. In the group of 100 sera from one source, several sera were aliquoted, coded, randomized and assayed as separate samples. A comparison of these values also gives a measure of the precision of the RIA. Table VII shows the reproducibility of the titers of a single serum that was assayed for antibody to either CMV or toxoplasma. The coefficients of variation were 14% and 15%, respectively, which is similar to the result with HSV-1. The coefficient of variation (CV) allows an estimate of the variability that might be expected for any given serum titer. For example, if a serum

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TABLE VII THE PRECISION OF THE MAGNETIC RAD1OIMMUNOASSAYFOR ANTIBODY TO CYTOMEGALOVIRUS(CMV) AND TOXOPLASMA Serum codea

CMV titer

Serum code a

Toxoplasma titer

18 19 20 118 119 120

3400 3300 2500 3700 3300 3800 2 = 3330 s = 459 CV= 14%

46 47 48 146 147 148

320 340 370 460 330 330 2 = 360 s = 53 CV = 15%

Serum samples from the same individual. h a s a t o x o p l a s m a t i t e r o f 1000, it w o u l d b e e x p e c t e d t h a t t h e t i t e r w o u l d r a n g e b e t w e e n 700 a n d 1300 95% o f t h e t i m e . ~ T h i s is c o n s i d e r a b l y b e t t e r t h a n t h e m i n i m u m ___t w o f o l d r a n g e ( 5 0 0 - 2 0 0 0 ) e x p e c t e d f o r m o s t o t h e r serologic tests.

RIA Precision between Laboratories T h e " b e t w e e n - l a b o r a t o r y " r e p r o d u c i b i l i t y o f the s o l i d p h a s e R I A h a s been evaluated. Four laboratories (one of which had never done RIA w o r k p r e v i o u s l y ) t e s t e d 15 d i f f e r e n t s e r a (5 high p o s i t i v e s , 5 l o w p o s i t i v e s , a n d 5 n e g a t i v e s , w h e r e p o s s i b l e ) a g a i n s t a v a r i e t y o f a n t i g e n s (see T a b l e V I I I ) . O n e o f t h e s e l a b o r a t o r i e s w a s u s e d as a " s t a n d a r d " a g a i n s t w h i c h the results of the other laboratories were compared. The overall agreem e n t in d e t e r m i n i n g p r e s e n c e o r a b s e n c e o f a n t i b o d y w a s g r e a t e r t h a n 85%. I n t h o s e i n s t a n c e s w h e r e t h e r e w a s n o t c o m p l e t e a g r e e m e n t , m o s t sera were missed by only one laboratory, not by the others. Summary

Solid p h a s e i m m u n o a s s a y s a r e p a r t i c u l a r l y a t t r a c t i v e b e c a u s e s e p a r a tion of reactants and processing of immune complexes are technically s i m p l e . T h e f a v o r a b l e s u r f a c e - v o l u m e r a t i o a n d c a s e w i t h w h i c h spherical beads c a n b e h a n d l e d m e c h a n i c a l l y m a k e t h e m a g o o d c h o i c e a s a s o l i d This range is determined as follows: (a) 95% of the area under a normal distribution curve lies between the mean -4- two standard deviations (2 -- 2 S); (b) i f 2 = 1000 and the CV [s/x = 100] = 15, t h e n S - 115(1000)/(1000)= 150, a n d 2 _ 2S = 1000_ 300; and (c) ff the serum is titrated 100 different times, 95 of those titers will fall between 700 and 1300.

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415

TABLE VIII MAGNETIC RADIOIMMUNOASSAYPRECISION BETWEEN LABORATORIES

Antigens

Precision: No. of sera in agreement/no. of sera tested

Toxoplasrna gondii Rubella virus Cytomegalovirus Herpesvirus I IgG, IgA, IgM Hepatitis B antigen Antinuclear antibody Varicella zoster virus Epstein- Barr virus Measles virus Mumps virus Influenza virus

~, ~,

jq

Escherichia coli

phase. Plastics (particularly polycarbonate because of its toughness in thin layers) are excellent adsorbents for protein-containing reagents and are, therefore, useful as the outer covering of the solid phase. A thin layer of plastic on the surface of another material is advantageous because one commercial lot (production batch) of plastic can be used for over 100 million tests; this eliminates a very real problem, that of variations in plastic formulations, already found to be a significant factor in ELISA test reproducibility. Iron is a good choice for the core of beads because iron can be moved conveniently from one place to another (processed) by magnetic force. The size of the solid phase bead should be sufficiently large to allow convenient mechanical movement of single units and give sufficient surface area upon which measurable immune reactions can take place. Ideally, beads should be small enough (miniaturized sufficiently) to allow maximum conservation of immune reagents, which are usually expensive, through use of small volumes. The 0.173 inch (diameter) precision-made BB is almost ideal for this purpose, is already commercially available, is uniform, is inexpensive, and, fortuitously, fits almost perfectly into an already existing serologic technology, the Microtiter tray system. Since Microtiter methodology has been in worldwide use for many years, ancillary, labor-saving equipment has already been developed and is commercially available (mechanical pipetters, diluters, spectrophotometric readers, computerized data processors, etc.).

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The magnetic ELISA and RIA processing system we described in this paper allows a high degree of automation, yet total flexibility in the mechanics of processing. Ninety-six tests can be performed at one time; starting and stopping the immune reactions is instantaneous and, therefore, simultaneous for each of the 96 specimens in one immunoassay run. Investment in processing equipment is relatively small because no electronics or sophisticated mechanics are involved in the design of the transfer devices. The sensitivities of the RIA and ELISA techniques are so high that it is not necessary to force immune reactions to completion in order to obtain satisfactory results. Therefore, many viral antibody titrations can be obtained within a period of about 2 hr, and the results can be expressed quantitatively in terms of positive and negative reference standards (longer incubation times obviously increase the sensitivity of the assays but are probably not required). This magnetic processing system for performing ELISAs or RIAs is probably adaptable for any immunoassay in which reagents will adsorb to the polycarbonate surface of the iron bead (presumably, all protein-containing materials). It compares well with other automated systems in simplicity, flexibility of application, mechanical reliability, precision, and economy.

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ENZYME IMMUNOASSAY: E L I S A

[28] E n z y m e

Immunoassay

AND E M I T

ELISA

419

and EMIT

B y E V A ENGVALL

Enzyme immunoassays today can be classified into two fundamentally different types of assays. For want of better nomenclature, these are at present referred to as heterogeneous and homogeneous enzyme immunoassays (EIA). The heterogeneous enzyme immunoassays, which include the enzyme-linked immunosorbent assay (ELISA), l-a are based on the same principles as are used in radioimmunoassays (RIA). In short, after incubation of antigen and antibodies, the antigen-antibody complexes formed are separated from free antigen and antibody by one of a number of different techniques, and the activity in one or both of the fractions is determined. In the homogeneous enzyme immunoassay4 (EMIT = enzyme multiplied immunoassay technique), no such separation is necessary. The principle of EMIT is similar to the modified bacteriophage technique? Antigen-coupled enzyme (or bacteriophage) will show a change in activity (infectivity) upon incubation with antibody. This change is inhibited when the binding of antibody to the antigen-coupled enzyme (bacteriophage) is prevented by addition of free antigen. ELISA is generally applicable to the measurement of almost any antigen. The usefulness of EMIT will probably remain limited to assay of low molecular weight haptens. In ELISA, the enzyme is a passive passenger through the actual immunoassay. In EMIT, the enzyme plays a key role throughout the assay process. ELISA requires very little knowledge of enzyme technology, whereas enzymology is the key to success in EMIT.

i E. Engvall and P. Perimann, Immunochemistry 8, 871 (1971). E. Engvall, K. Jonsson, and P. Perlmann, Biochim. Biophys. Acta 251, 427 (1971). a E. Engvall and P. Perlmann, J. Immunol. 109, 129 (1972). 4 K. E. Rubenstein, R. S. Schneider, and E. F. Ullman, Biochem. Biophys. Res. Commun. 47, 846 (1972). J. Haimovich, E. Hurwitz, N. Novik, and M. Sela, Biochim. Biophys. Acta 207, 125 (1970).

METHODS IN ENZYMOLOGY, VOL. 70

Copyrisht© 1980by AcademicPress, Inc. All fightsof reproductionin any formreserved. ISBN 0-12-181970-1

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IMMUNOASSAYS

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Classification of E I A Techniques A brief description of techniques used in EIA follows. The various assays have been classified as either competitive or noncompetitive assays, depending on whether or not the technique involves a reaction step in which unlabeled and labeled antigen compete for a limited number of antibody sites (competitive assay) or whether the antigen (or antibody) to be measured is first allowed to react with antibody (antigen) on a solid phase followed by measurement of the binding of enzyme-labeled immune reactant (noncompetitive assay)

Competitive Assays ELISA Using Antigen-Enzyme Conjugate. Assays for IgG 1 and HCG, 6 respectively, were the first two examples of quantitative enzyme immunoassay. Both were based on competition of enzyme-labeled antigen with antigen from either standard or unknown sample for the binding to a limited amount of antibody coupled to a solid phase. Figure 1 illustrates the features of the assay. In this type of assay, the first operation is the physical or chemical attachment of an appropriate amount of antibody to a solid phase. This is then incubated with a solution containing a fixed concentration of enzyme-labeled antigen, and no unlabeled antigen, a known but variable concentration of standard antigen, or an unknown concentration of test antigen from a sample is included. The reaction mixture is then incubated until the antigen-antibody reaction attains equilibrium. After washing, the enzyme activity on the solid phase is determined, usually by incubation with substrate buffer for a certain time period. The product concentrations are inversely proportional to the concentrations of standard or test antigen added. EMIT Using Hapten-Enzyme Conjugate. In this assay, a fixed amount of hapten-enzyme conjugate is incubated with a fixed amount of antibody to the hapten together with a variable amount of free hapten. 7 The antibody will cause a change in enzymic activity upon reaction with one or more haptens on the enzyme. In this assay, equilibrium does not necessary have to be attained before enzyme measurement, no separation of free from antibody-bound hapten is required, and the rate of enzyme reaction is measured, rather than an end point. ELISA Using Enzyme-Labeled Antibody. Another type of competitive ELISA employs enzyme-labeled antibody with the antigen attached to a solid phase. In this technique, the binding of enzyme-labeled antibody to e B. van Wcemen and A. H. W. M. Schuurs, FEBS Lett. 15, 232 (1971). K. E. Rubenstein, Scand. J. Immunol. 8, Suppl. 7, 57 (1978).

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ENZYME IMMUNOASSAY: E L I S A Competitive

ELISA

for

AND E M I T

421

measuring antigen

1. Attach antibody to solid phase

2. Incubate with enzyme-labeled antigen in presence or absence of standard antigen or unknown sample

5. Incubate with enzyme substrate

FIc. 1. Scheme for a competitive solid phase enzyme immunoassay. From Engvall and Ruoslahti. 2~ Reproduced with permission.

immobilized antigen is competitively inhibited by an added standard or test antigen. As is the case in the competitive ELISA with enzyme-labeled antigen, the product concentrations measured at the end are inversely proportional to the concentrations of the standard or test antigen in the incubation solutions. In its most versatile and convenient form, this assay is performed as a two-step assay with enzyme-labeled anti-immunoglobulin, s Antigen on the solid phase is incubated with a high dilution of antiserum with or without addition of standard antigen or unknown samples. After washing, the antibodies bound to the solid phase are detected using enzyme-labeled anti-immunoglobulin. Semipurified antigen works satisfactorily in this assay, and the antiserum can be of a fairly low titer. The enzyme-labeled anti-immunoglobulin can be readily prepared using conventional procedures. The same conjugate can be used for the assay of several different antigens, provided that the primary antisera have been prepared in the same species. s E. Engvall and P. Perlmann, in "Automation in Microbiology and Immunology" (C.-G. Heden and T. Illeni, eds.), p. 529. Wiley, New York, 1975.

422

IMMUNOASSAYS

[28]

Noncompetitive Assays "Sandwich" Assay. Immobilized antibody in excess is incubated with standard or test antigen (Fig. 2). After washing, the immobilized antibody -antigen complex is incubated with an excess of enzyme-labeled antibody which binds to one or more remaining antigenic sites. 9 Alternatively, the second antibody may be unlabeled, and the procedure is expanded to include an incubation with excess enzyme-labeled third antibody specific for IgG of the animal species in which the second antibody is elicited? ° In the latter case, the immobilized and second antibodies must be obtained from different animal species in order to prevent the binding of enzymelabeled third antibody directly to the immobilized antibody. In both variants, the concentration of the product from the enzyme reaction is directly proportional to the concentration of standard or test antigen. Assays for Measuring Antibody. Another type of noncompetitive Sandwich ELISA 1. Attach antibody to solid phase

2. Incubate with sample

©0

5. Incubate with enzyme-labeled antibody

FIG. 2. Scheme for a noncompetitive solid phase enzyme immunoassay. From Engvall and Ruoslahti, z~ Reproduced with permission. 9 L. Belanger, C. Sylvestre, and D. Dufour, Clin. Chim. Acta 48, 15 (1973). 10 A. R. Frackeiton, Jr., R. P. Szaro, and J. K. Weltman, Cancer Res. 36, 2845 (1976).

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AND E M I T

423

ELISA is the indirect method for measuring antibody concentration. This procedure employs immobilized antigen and enzyme-labeled second antibody against IgG of the species in which the test antibody has been elicited. This method has been used to measure antibodies to a variety of antigens.Z,lld 2

Factors Involved in the Choice of Assay Design Although competitive ELISA techniques are specific and easy to execute, they also suffer from several disadvantages. To perform a competitive ELISA using enzyme-labeled antigen, purified antigen in relatively large amounts is required for preparation of the enzyme-antigen conjugate. In cases where purified antigen is not available, a variant of the competitive ELISA method employing enzyme-labeled antibody or anti-immunoglobulin can be used. A more serious problem in the application of the competitive ELISA relates to the difficulties caused by the need to incubate enzyme-labeled antigens or antibodies with biological fluids such as serum, urine, or tissue extract. These fluids contain proteases, and noncompetitive enzyme inhibitors may also be present. Such substances, when present, may alter the activity of the enzyme used as label. This difficulty is avoided in the noncompetitive ELISA techniques in which the incubation with test samples is carried out separately from the incubation with enzyme-labeled antigen or antibody. The noncompetitive ELISA offers additional advantages. Since most of such assays employ enzyme-labeled antibodies, the purification and specific enzyme-labeling of individual antigens is not necessary. Thus, the same enzyme-labeling procedure and solid phase attachment method can be used for different antibodies. Another advantage of the noncompetitive ELISA is the possibility of binding several enzyme-labeled antibody molecules to a single polyvalent antigen molecule, thus providing an element of amplification. This may be an advantage in procedures in which the ultimate sensitivity has not been attained, i.e., the sensitivity limit is not set by the affinity between the antigen and antibody.

Preparation of Enzyme Conjugate Selection of Enzyme for ELISA. In the following, some characteristics of an enzyme suitable for enzyme immunoassay are discussed and attention is focused on the characteristics of enzyme that will influence the sensitivity, practicality, and cost of the assays. 11 H. E. Carlsson and A. A. Lindberg, Scand. J. lmmunol. 8, Suppl. 7, 97 (1978). 12 A. Voller, A. Bartlett, and D. E. Bidwell, Scand. J. lmmunol. 8, Suppl. 7, 125 (1978).

424

IMMUNOASSAYS

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First, the enzyme should be conveniently detectable at or below the nanogram level. This not only means that the enzyme should have a high specific activity, i.e., convert a high number of substrate molecules into product molecules per time unit, but also requires that the product be detectable with high sensitivity. An important consideration in the choice of enzyme is also that the samples to be measured should not contain substances that could interfere with the activity of the enzyme or its measurement. This applies primarily but not exclusively to assays in which the enzyme-labeled compound is incubated together with the sample. Some obvious examples include the need to avoid samples containing EDTA if the enzyme utilized requires metals for activity. Similarly, oxidoreductases cannot be used when the samples contain some commonly used preservatives. Endogenous enzyme inhibitors or other substances interfering with the activity or stability of the enzymes may also be present in the samples to be assayed. Endogenous enzymic activity will probably not interfere in a solid phase enzyme immunoassay where the activity bound to the solid phase is measured. It is evident that endogenous enzymes can interfere in EMITtype assays. Other trivial but nonetheless important aspects in the choice of enzymes include availability, cost, and shelf life. Choice of Enzyme for EMIT. Since EMIT does not include any separation of immune reactants from sample before enzyme assay, the choice of enzyme becomes restricted to a much less variable group than those that can be used for ELISA. It is of prime importance in EMIT that an enzyme be chosen that is not present in the test sample. Furthermore, the test sample must not contain inhibitors of the enzyme chosen, nor appreciable amounts of enzyme substrate. In addition, the enzyme must change its catalytic properties in a measurable way after interaction with antibodies. Most enzymes are not inhibited by antibodies. However, enzymes acting on high molecular weight substrates, can be sterically hindered in their activity by antibodies. This is the basis for the use of lysozyme in homogeneous enzyme immunoassays. 4 The situation is more complex in the case of two other enzymes, malate dehydrogenase and glucose-6-phosphate dehydrogenase, which are also extensively used in EMIT-type assays. Substitution of unique residues in the molecule with hapten apparently makes the enzyme susceptible to conformational changes upon reaction with antibody, and this leads to inhibition of enzyme activity. ~a 13 G. L. Rowley, K. E. Rubenst¢in, J. Huisjen, and E. F. Ullman, J. Biol. Chem. 250, 3759 (1975).

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REAGENTS FOR COUPLING ENZYMES TO PROTEINS

Compound Glutaraldehyde

Toluene diisocyanate

p,p'-Difluoro-m,m'odinitrophenyl sulfone Carbodiimides

Reacting group in protein

Reference

--NH~ --NHz --NH~ --COOH

a, b c, d e f, g

--NH2 p-Benzoquinone N,N'-o-Phenylenedimaleimide m-Periodate

--NHz --SH --SH --NH~ --CHOH

h i j, k

a S. Avrameas, Immunochemistry 6, 43 (1969). b S. Avrameas and T. Ternynck, lmmunochemistry 8, 1175 (1971). c A. F. Schick and S. J. Singer, J. Biol. Chem. 236, 2477 (1961). a R. R. Modesto and A. J. Pesce, Biochim. Biophys. Acta 229, 384 (1971). e S. S. Tawde and J. S. Ram, Arch. Biochem. Biophys. 97, 429 (1962). sS. Avrameas and J. Uriel, C. R. Acad. Sci. 262, 2543 (1966). g P. K. Nakane, J. S. Ram, and G. B. Pierce, J. Histochem. Cytochem. 14, 789 (1966). n T. Ternynck and S. Avrameas, lmmunochemistry 14, 767 (1977). K. Kato, Y. Hamaguchi, H. Fukui, and E. Ishikawa, J. Biochem. 78, 235 (1975); Eur. J. Biochem. 62, 285 (1976). J P. K. Nakane and A. Kawaoi, J. Histochem. Cytochem. 22, 1084 (1974). A. Murayama, K. Shimada, and T. Yamamoto, lmmunochemistry 15, 523 (1978).

An interesting exception to this generalization is the increased activity exhibited by malate dehydrogenase which has been coupled to thyroxine, in the presence of antibodies to thyroxine. 7 Labeling of Antigens and Antibodies with Enzyme. Many methods exist for coupling haptens, proteins, and carbohydrates to proteins. Some of the most commonly used are summarized in the table together with some selected references. An extensive review on protein-protein coupling reactions has recently appeared 14 and could be used as a source of additional chemical details on the coupling methods. Reagents for protein-protein coupling are generally nonspecific in that they react with functional groups that are common to all proteins. The extent of intermolecular as opposed to intramolecular cross-linking will depend on the relative number and availability of such functional ~4 j. H. Kennedy, L. J. Kricka, and P. Wilding, Clin. Chim. Acta 70, l (1976).

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groups in the two proteins to be conjugated. The number of total functional groups present in any given protein can be determined, but whether these are available for intermolecular cross-linking to another protein is not so easily tested. The optimal conditions for conjugating two proteins to each other will, therefore, have to be determined by trial and error. Glutaraldehyde as a Cross-Linking Agent. Glutaraldehyde (GDA) is the cross-linking agent used most extensively in enzyme immunoassay. 15 It is a dialdehyde, and theoretically could cross-link two proteins via the C-amino groups of lysine by formation of a Schiff's base. However, the observed stability of proteins cross-linked with GDA does not agree with the known reversibility of Schiff's base formation. The mechanism for GDA cross-linking involving polymerization products of GDA has been proposed by Richards and Knowles, 16 and agrees better with the efficiency of coupling and the stability of the resulting conjugates. We have always used technical grade GDA, which is presumably extensively polymerized, for conjugations and have obtained good and reproducible results. Others have reported good results with the use of GDA purified by distillation. However, I have talked to several investigators who have been unable to obtain cross-linking in spite of adherence to published procedures, and it has, without exception, become evident that they have been using highly purified GDA. Cross-Linking via Carbohydrate Moiety by Using Periodate. An interesting and potentially very useful method for coupling peroxidase to antigens and antibodies in EIA was introduced by Nakane and Kawaoi. 17The carbohydrate moiety of horseradish peroxidase was oxidized with sodium periodate, and the resulting aldehyde groups were allowed to react with amino groups of antibody. The reverse approach was explored by conjugating peroxidase and lysozyme to periodate-oxidized antibody. 18 The advantage of utilizing the carbohydrate moiety of antibodies and enzymes is obvious, considering that carbohydrate is not essential for either the immunological activity of antibodies or the catalytic property of enzymes. Characteristics of Enzyme Conjugates. Most conjugation procedures give rise to products heterogeneous in size and composition. After conjugation of antigen (or antibody) to an enzyme, the reaction mixture may thus be composed of free antigen (antibody), free enzyme, and one or more antigen (antibody) molecules linked to one or more enzyme molecules. The presence of free antigen (or antibody) will lower the specific activt5 S. Avrameas, Immunochemistry 6, 43 (1969). ~6 F. M. Richards and J. R. Knowles, J. Mol. Biol. 37, 231 (1968). lr p. K. Nakane and A. Kawaoi, J. Histochim. Cytochem. 22, 1084 (1974). 18 A. Murayama, K. Shimada, and T. Yamamoto, Immunochemistry 15, 523 (1978).

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427

ity of the conjugate and should be minimized by design of the coupling procedure and by purification of the conjugate. For instance, if the enzyme itself or the conjugate are of higher molecular weight than the antigen, elimination of unconjugated antigen can be accomplished fairly easily by gel filtration on an appropriate matrix. Another way, which to my knowledge has not yet been tried, would be by affinity chromatography or immunochromatography specific for the enzyme. Free enzyme in the conjugate will interfere mainly in the methods where the activity of the "free" fraction is measured. In most cases, however, the activity is measured in the " b o u n d " fraction, and the presence of free enzyme will cause only minor interference in the form of increased background. If the enzyme is smaller than the labeled compound, free enzyme can be eliminated by gel filtration as above. Immunoadsorbent purification could be useful in separating enzyme-labeled (and free) antigen (antibody) from free enzyme, provided that an elution buffer can be found that does not inactivate the enzyme label. As already mentioned, most conjugation methods in use give rise to high molecular weight complexes, each containing several enzyme molecules bound to several antigen molecules. In a solid phase assay, only one of the antigen molecules in such a complex will be capable of reacting with an antibody. Since this antigen in the conjugate is associated with several enzyme molecules, the conjugate will exhibit a high specific activity. On the other hand, since only one out of several antigens will express immunoreactivity at a given time, the yield of antigen after conjugation will seem low. The different nature of the large enzyme-antigen complexes compared to standard antigen does not seem to present any problems in EIA. As in competitive RIA, results from competitive EIA are always obtained by comparing the unknown sample with that of an unlabeled standard antigen. Similarly, in an antibody assay, the results should preferably be compared to a standard antiserum and not expressed as radioactivity or enzyme activity, respectively, which will most likely vary from one laboratory to another, even if the same labeled preparation is used. Immobilization o f Antigen or Antibody. The characteristic that distinguishes ELISA from other EIA is the use of an immune adsorbent to effect a rapid, facile separation of "free" antigen and antibody from antigenantibody complexes. Methods where antibody and antigen are covalently attached to cellulose, agarose, or polyacrylamide have been described. With the exception of the case where such particles have been made magnetic TM and can be separated in a magnetic field, the use of par~8J.-L. Guesdon and S. Avrameas,

Immunochemistry 14, 443 (1977).

428

IMMUNOASSAYS

[28]

ticulate solid phases entails centrifugation in the washing and separation steps. Solid phase carriers, such as large beads, disks, and tubes, facilitate washing and separation steps. Thus, macromolecular antigens and antibodies have been physically adsorbed to plastic carriers (polystyrene, polyvinyl, polypropylene, polycarbonate), and to silicone rubber or treated glass. Indeed, part of the success of ELISA methods arises from the use of disposable polystyrene Microtiter plates ~2 or tubes 2 as the solid phase carriers. Most proteins adsorb to plastic surfaces, probably as a result of hydrophobic interactions between nonpolar protein substructures and the nonpolar plastic matrix. The rate and extent of coating will depend on the diffusion coefficient of the adsorbing molecule, the ratio of the surface area to be coated to the volume of coating solution, the concentration of the adsorbing substances, the temperature, and the duration of the adsorption reaction. Clear polystyrene has been the most widely used support in ELISA methods, and it can be coated easily and reproducibly. However, there are shortcomings in using polystyrene or any other plastic as solid phase. One difficulty is due to the fact that the antigen or antibody is only physically adsorbed, not covalently bound to the solid phase. This type of an immune adsorbent "bleeds" (i.e., loses some of the adsorbed protein during washes and incubations). Furthermore, adsorbed proteins may also undergo denaturation to some extent with loss of immunological activity. The loss of adsorbed antigen or antibody, which amounts to approximately 30% for the time of an assay, lowers the precision of the assay and probably also affects its sensitivity, especially in competitive ELISA techniques. Another disadvantage is that plastic surfaces have a limited capacity of adsorption. However, the ease and rapidity of separation of antigen-antibody complex from "free" antigen and antibody mostly compensates for these drawbacks. The adsorption process, unlike antigen-antibody interactions, is nonspecific. Thus, during the incubation of the immobilized antigen or antibody with enzyme-labeled antigen or antibody, the latter binds specifically to the immobilized immune reactant, but may also be adsorbed directly onto the solid phase. This nonspecific adsorption of enzyme activity can be minimized by inclusion of a nonionic detergent such as Triton X-100 or Tween 20. These do not interfere with the antigen-antibody reaction but prevent formation of new hydrophobic interactions between added proteins and the solid phase without disrupting to any appreciable extent the hydrophobic bonds already formed between the previously adsorbed protein and the plastic surface. The optimal concentration of antigen or antibody for coating is generally between 1 and 10 p.g/ml. Higher concentrations lead to increased

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adsorption, but the percentage adsorbed becomes less. Furthermore, high concentration of protein during coating leads to increased desorption during incubations with immunoreactants. This sometimes gives rise to undesirable prozone phenomena. Enzyme Affinity Assay. The ELISA type assays are also applicable to studies of nonimmunological interactions. Examples of potentially useful applications include assays of various hormone receptors, where radiochemical techniques are currently used to measure the few receptors for which assays are available. To illustrate the possibilities offered by tests that might be called enzyme affinity assays, I will briefly mention here our recent studies on the utilization of such assays to study the interaction of fibronectin with collagen. 2°'~ Fibronectin is a cell surface protein also present in the circulation. It has affinity to collagen, and evidence from in vitro experiments suggests that its function on the cell surface is to attach cells to the extracellular collagenous matrix. The circulating form may enhance opsonization of particulate matter containing collagen and fibrin. Our recent studies on fibronectin have centered around its collagenbinding properties. We originally demonstrated this binding using an

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FIG. 3. Inhibition of binding of fibronectin to Microtiter plates coated with gelatin by collagen type I (O), type II (&), and type III (11) and by AB chains (©). ---, Native proteins; - - , heat-denatured proteins. From Engvall et al. ~ Reproduced with permission. ~0 E. Engvail and E. Ruoslahti, Int. J. Cancer 20, 1 (1977). 2~ E. Engvall, E. Ruoslahti, and E. J. Miller, J. Exp. Med. 147, 1584 (1978).

430

IMMUNOASSAYS

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ELISA. 2° We could demonstrate the binding of fibronectin to collagencoated Microtiter plate wells by detecting the bound fibronectin with enzme-labeled antifibronectin. We have applied this assay to studies on the fibronectin-binding activity of different genetic types of collagens. 21 Microtiter wells were coated with gelatin (denatured collagen). Fibronectin binds to such wells and can be detected using anti-fibronectin labeled with alkaline phosphatase. Collagenous proteins inhibit the binding of fibronectin to the gelatin-coated wells, and this allows measurement of their relative avidities in binding to fibronectin. Figure 3 shows an example of the results obtained. It could be established that of the native collagens, type III collagen was the most active. Denaturation of the collagens increased their activities. Denatured types I, II, and III collagens were equally active, and the collagen containing the A and B chains remained less active than the other types. More recently, we have modified the assay to a simple affinity assay by attaching the enzyme directly to fibronectin. 22 E L I S A in Practice Purification o f lmmunoglobulins and Anti-immunoglobulins. The IgG fraction of an antiserum can be purified according to any of a number of established techniques. If the antiserum was raised in rabbits, the most convenient purification procedure to use is affinity chromatography on protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden). Excellent yield and purity is obtained by following the recommendations of the manufacturer. We usually prepare purified antibodies from anti-immunoglobulin sera rather than use the whole IgG fraction. This can be accomplished by a relatively simple procedure, and the use of purified antibodies results in more efficient conjugates than those prepared from whole IgG, and it also saves enzyme. The procedure we used in isolating antibodies is as follows: An immunoadsorbent can be prepared by coupling IgG (or other immunoglobulin) to Sepharose. Before use, the immunosorbent is washed with the buffer used later for eluting the antibodies followed by washing with phosphate-buffered saline (PBS), pH 7.2. The anti-immunoglobulin serum of the proper specificity (heavy chain-specific or Ig-specific) is inactivated (30 min, 56°) and applied to the column. The column is then washed with PBS until the absorbance at 280 nm is consistently low. The antibodies z2 E. Engvall and E. Ruoslahti, in "Immunoassays in the Clinical Laboratory" (R. M. Nakamura et al., ¢ds.), pp. 89-97. Liss, New York, 1979.

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ENZYME IMMUNOASSAY: ELISA AND EMIT

431

are then recovered from the column by eluting with 0.1 M glycine-HCl buffer at pH 2.6 and collected in fractions. Peak fractions (as measured by absorbance at 280 nm) are pooled, neutralized with solid Tris salt, and dialyzed against PBS or bicarbonate buffer, depending on the purpose (see below). A precipitate will form upon neutralization. This precipitate probably represents antibody complexed with antigen leaking from the column during elution. The precipitate is removed by centrifugation. Coupling of Antibodies to Horseradish Peroxidase (HRP). We use a slight modification of the two-step glutaraldehyde procedure of Avrameas and Ternynck. 2~The HRP (10 mg, Sigma type VI, RZ = 3) is dissolved in 0.2 ml of 1.25% glutaraldehyde (technical grade), in PBS, pH 7.2, and left at room temperature overnight. The reaction mixture is then diluted to 1 ml and dialyzed against 0.1 M carbonate buffer, pH 9.2 (two changes, 1 liter, 4 hr each). IgG or purified antibodies (5 mg) in 0.25 ml of the carbonate buffer is added, and the mixture is incubated again overnight at room temperature. Possible remaining reactive groups are blocked by the addition of 0.1 ml of 0.2 M lysine. The conjugate can be stored in 50% glycerol. The two-step procedure leads to more efficient conjugates than a onestep procedure when peroxidase is used for labeling. The rKason is that peroxidase has few free amino groups available for reaction with glutaraldehyde, whereas IgG has many. If HRP and IgG are incubated together with glutaraldehyde, IgG will effectively compete with HRP for available GDA, leading to extensive intermolecular cross-linking of IgG with little coupling to HRP. Detection of HRP Activity. The substrate for HRP used in assays is H202 or sometimes other peroxides. The cleavage of H202 is coupled to the oxidation of a hydrogen donor (chromogen) and goes through several intermediary steps with different rate constants. There are a variety of chromogens available yielding attractive colors. When choosing a particular color that suits one's taste or matches one's equipment, one should be aware of the fact that recipes for HRP activity measurements in the literature are not always optimal. Other important points to note are that all chromogens are light sensitive and that their colored products tend to stick on surfaces. The following gives, for the convenience of newcomers in the ELISA field, some colorimetric methods for measuring HRP activity. Some of the methods are taken from the literature, and some are our own modifications of published assays.

Chromogen: 2,2'-Azino-di(3-ethyl-benzthiazoline Sulfonic Acid-6) 23 S. Avrameas and T. Ternynck, lmmunochemistry $, 1175 (1971).

432

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Ammonium Salt (ABTS). This method is modified from Saunders. 24 The substrate buffer contains 1 mg of ABTS per milliliter and 0.003% H202 in 0.1 M citrate-phosphate buffer, pH 4.0. The enzyme reaction is stopped by addition o f ] volume of 37 mM NaCN. The green color is measured at 415 nm. NaCN effectively inactivates the enzyme without affecting the color, but it is poisonous and smells bad. However, it has been difficult to find a more suitable reagent that would inactivate the enzyme without affecting the color. Lowering the pH by addition of HCI or H4SO4 increases the color development nonspecifically, whereas elevating the pH by addition of NaOH or inactivation of the enzyme with NaN3 similarly reduces the color. In solid phase assays, where the enzyme activity on the solid phase is measured, the best way to stop further color development is to separate the substrate solution from the enzyme. 2~ Chromogen: o-Phenylenediamine. This method, modified from Wolters et al. ,26 utilizes 0.4 mg ofo-phenylenediamine per milliliter and 0.01% H~O2 in 0.1 M citrate phosphate buffer, pH 5.0. The enzymic reaction is stopped by addition of ½ volume of 4 N H2SO4. The tangerine colored product is completely soluble but somewhat light sensitive. It is measured at 492 nm. Chromogen: o-Dianisidine. This method is modified from Avrameas and Guilbert. 27 To 60 ml of 0.1 M citrate phosphate buffer, pH 5.0, is added 12/~l of 30% HzO2 and 0.5 ml of 10 mg/ml o-dianisidine in methanol. The enzymic reaction is stopped by addition of 50/~l of 5 N HCI per milliliter of enzyme-substrate mixture. The yellow-orange color is measured at 400 nm. It is more stable than the colored product from o-phenylenediamine, but has a tendency to precipitate on solid surfaces. Other Chromogens Used in EIA. These include 5-aminosalicylic acid,28 3,3 '-dimethyloxybenzidine,,9 and p-cresol, 17 which gives a fluorescent product. Labeling o f Antibodies with Alkaline Phosphatase

Alkaline phosphatase from calf intestinal mucosa (Sigma type VII), a 5 mg/ml suspension in 3.4 M (NH4)2SO4, 0.3 ml is added to 0.1 ml of a solution of antibody, 5 mg/ml in PBS. If the antibody solution is available 24G. S. Saunders, Report at the American Societyof ClinicalPathologistWorkshop, Chicago, 1977. 25R. Maloliniand R. Masseyeff,J. Immunol. Methods 8, 223 (1975). 2s G. Wolters, L. J. Kuijpers,J. Ka~aki,and A. Schuurs,J. Clin. Pathol. 29, 873 (1976). 27S. Avrameasand B. Guilbert,Biochimie 54, 837 (1972). 28B. van Weemenand A. H. W. M. Schuurs,FEBS Lett. 15, 232 (1971).

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only in concentrations lower than 5 mg/ml, the enzyme suspension is first centrifuged and the supernatant is replaced by a suitable amount of the antibody solution. The mixture of enzyme and antibody is dialyzed overnight against 1 liter of PBS. Glutaraldehyde in PBS is then added to give a final concentration of 0.2%. 1-3 The conjugation is allowed to proceed for 2 - 3 hr at room temperature, during this time the initially colorless solution becomes pale yellow. The conjugate can then be diluted to any desired volume and dialyzed free of excess GDA. The conjugate is stored in PBS or Tris buffer, pH 8; an unrelated protein (e.g., serum albumin) is added for stabilization, and a preservative (NAN3) to prevent microbial growth. Alkaline phosphatase is highly reactive with GDA and forms crosslinked polymers. However, it is not easily cross-linked to the extent that it will precipitate, as in the case with IgG. Alkaline phosphatase and IgG in a ratio of />3:1 (w/w) will form efficient conjugates with negligible amounts of free IgG. The conjugate will not precipitate out of solution even with increasing amounts of GDA. Alkaline phosphatase can also be coupled to antigens and antibodies using a two-step glutaraldehyde procedure. In the case of coupling to IgG, a two-step procedure does not seem to be of any advantage. However, in the case of labeling of staphylococcal protein A with alkaline phosphatase, a two-step procedure was found to be the only way to obtain workable conjugates, 2a and with fibronectin it was the most efficient way. ~2 Measurement of Alkaline Phosphatase (ALP) Activity. Although a multitude of methods are being used to measure peroxidase, most workers use the following method to measure ALP. The reaction utilizes 1 mg of p-nitrophenylphosphate (Sigma) per milliliter in 1 M diethanolamine buffer, pH 9.8. The reaction is stopped by addition of ¼ volume of 2 M NaOH. The yellow p-nitrophenol is measured at 405 nm.

Stepwise ELISA Assay As one example of how to proceed in setting up ELISA, I have chosen to illustrate a system involving determination of mouse antibodies to human ot-fetoprotein (hAFP). I will also show how this assay can be utilized for the quantitative determination of the antigen, AFP. These assays, which we use to characterize monoclonal hybridoma antibodies to AFP, 29a have been developed with emphasis on simplicity and rapidity rather than sensitivity. Preparation and Testing of Conjugate. First a conjugate, rabbit anti~9 E. Engvall, Scand. J. Immunol. 8, Suppl. 7, 25 (1978). 2~ M. Uotila, E. Engvall, and E. Ruoslahti, Mol. lmmunol., in press.

434

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mouse IgG, is prepared. Purified rabbit antibodies to mouse IgG, 1 mg, are coupled to 3 mg of calf intestinal alkaline phosphatase by a one-step glutaraldehyde procedure as described above. The conjugate is diluted to 2 ml after preparation. The efficiency of the conjugate is tested in microtiter plate wells coated with normal mouse serum in the following way. Mouse serum, 0.2 ml, diluted l:10,000 ( - 1 p,g mouse IgG per milliliter) in 0.1 M NaHCOa is added to each of a number of wells in a Microtiter plate. The plate is incubated in a humid chamber for 3 hr at 37°. The plate is then washed twice with 0.05% Tween 20 in 0.9% NaCl. The washing at this and the following occasions is done so that the plate is turned upside down and the contents of the wells are shaken out, and the plate is hit hard on a paper towel. It is then turned right side up and flushed with washing solution from, e.g., a squeeze bottle. Air bubbles must not be trapped in the wells. The plate with all wells filled with wash solution is left for 2-3 min, after which the procedure is repeated. After the second wash, all wells are filled with 0.2 ml of PBS, pH 7.2, containing 0.05% Tween 20 (incubation buffer). The conjugate is then serially diluted by adding 50/zl to the first well(s), mixing, transferring 50/~l to the next well(s), mixing, etc. (fivefold dilutions). The plate is then incubated in a humid chamber at room temperature overnight. The next morning, the plate is washed three times. The enzyme remaining in individual wells is determined by adding 0.2 ml of the substrate solution (1 mg ofpnitrophenylphosphate per milliliter in 1 M diethanolamine buffer, pH 9.8) to each well. The plate is incubated at room temperature for 15 min. At this time point, chosen arbitrarily, the enzyme is inactivated by the addition of 50/~l of 2 M NaOH. For absorbance measurement, the content of each well is diluted with 0.75 ml of distilled water and then measured at 405 nm. Figure 4 shows the results. For further assays, a conjugate dilution of 1:500 was chosen. Determination o f Antibodies to AFP. Wells in a microtiter plate are coated with 1/~g of hAFP. After washing, antisera and normal sera are serially diluted in the wells, and the plate is incubated at room temperature for 3 hr. The plate is then washed three times and 0.2 ml of the conjugate, diluted 1:500 in the incubation buffer, is added to each well. The plate is again incubated for 3 hr at room temperature, washed three times, and incubated with the substrate solution for 15 min. The enzyme is inactivated with NaOH, and the content of each well is diluted in distilled water before measurement at 405 mm. Figure 5 shows the result of titration of a mouse antiserum to hAFP and of a normal mouse serum. There is a direct relationship between the amount of antibodies in the serum and the absorbance measured. This relationship is linear within a certain

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FIG. 5. Titration of mouse anti-a-fetoprotein (AFP) (O) and normal mouse serum (O) in microtiter wells coated with AFP.

436

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range of antibody concentration. At high concentrations of antiserum, the dose response curve levels off because either the amount of antigen on the solid phase or the amount of antibodies in the conjugate becomes a limiting factor. In general, dose response curves generated from different antisera are parallel. Lack of parallelism can be due to two main effects. 1. The antisera have different specificities. If one serum contains antibodies against a major component of the antigen used for coating, and another contains antibodies against a minor component, the latter is going to give a dose response curve with a plateau level lower than the former. 2. The sera contain antibodies of different immunoglobulin classes, toward which the conjugate reacts with different effectiveness. A certain "background" is always seen with normal sera in this kind of assay. This background may be due to specific or nonspecific binding of IgG in normal serum to the coated plastic and probably to other unknown protein-protein interactions as well. This normal serum background is usually no problem in experimental animal systems since it varies little from animal to animal. This in turn is probably a consequence of the relative genetic and environmental homogeneity of such species. The "normal" serum background with human sera presents more of a problem because it can be highly variable, and a range has to be established for each type of assay. Determination of Antigen by Two-Step Competitive Assay Antigen can be quantitated by its capacity to inhibit the binding of antibody to the antigen adsorbed on the solid phase. The affinity of the antiserum is the most important factor among variables that determine the sensitivity of the assay. To obtain maximal sensitivity in the assay, the amount of antigen used for coating is decreased as far as practicable and the amount of antibody added should be limited. To determine the concentrations of antigen and antibody to be used in a competitive two-step assay, we make checkerboard titrations of the antigen used for coating and of the antiserum. Figure 6A and B shows the result of an experiment in which various concentrations of hAFP (0.03 /zg/ml to 10/zg/ml) were used for coating. Enzyme activity has been plotted as a function of concentration of the coating antigen at different antibody concentrations (Fig. 6A) and as a function of antiserum concentration at different concentrations of the coating antigen (Fig. 6B). Figure 6A shows that any antigen concentration below 1/zg/ml, which gives a measurable response, can be chosen for coating. We will choose 0.1/zg of hAFP per milliliter for coating. We will then get the antibody concentration from Fig. 6B. This has to be chosen so that it is low enough

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0 •"

1.0

0.5 A ~ L

I

1:15,600 1:3,1~50 1:625

Coating Concentration of Antigen I/.4.g/mll

1:125

1:25

Antiserum Dilution

FIG. 6. Checkerboard titration of the antigen concentration used for coating and of antibody concentration. (A) Enzyme activity as a function of antigen concentration at coating for antiserum diluted 1:25 (O), 1:125 (x), 1:625 (©), and 1:3, 125 (A). (B) Enzyme activity as a function of antiserum dilution in wells coated with a-fetoprotein 1/zg/ml (O), 300 ng/ml (x), 100 ng/ml ( 0 ) , and 30 ng/ml (A).

l.O

0.8

I

0 ku

0.6

O~

02

0.1

10 /.t.g A F P

FIG. 7. Inhibition of binding of mouse anti-~x-fetoprotein (AFP) to Microtiter wells coated with AFP by soluble AFP.

43 8

IMMUNOASSAYS

[28]

to become a limiting factor in the assay. An antiserum dilution of 1:625 fulfills this requirement. The two-step competitive assay for hAFP is then performed in the following way. Wells in a microtiter plate are coated with 0.1/zg of hAFP per milliliter in 0.1 M NaHCOa. The plate is washed twice, and all wells are filled with 100/.d of incubation buffer. Fifty microliters of standard hAFP and unknown samples are added to a set of wells and then titrated by threefold dilutions. One hundred microliters of antiserum diluted 1:300 are then added to each well. The plate is incubated at room temperature for 3 hr. After another three washings, substrate is added and the enzyme is inactivated after 15 min. Figure 7 shows a standard curve for hAFP obtained in this assay. The inhibition curve has all the features of a regular standard curve in a competitive type assay. Factors Involved in the Sensitivity of ELISA. As already mentioned, the experiments described in the previous paragraphs were not designed to obtain assays of highest possible sensitivity. The antiserum to HAFP we used was of relatively low affinity, we used short incubation times, and a short time for color development. To design an assay with optimal sensitivity, it is essential to (a) choose an antiserum of highest possible affinity; (b) use incubation times that allow an equilibrium between antigen and antibody; (c) use lower concentrations of antigen and antibody and longer times for color development. With these factors taken into account, the sensitivity of an ELISA assay is comparable to that of radioimmunoassay. Conclusion Enzyme immunoassays are now firmly established as precise quantitative methods for the determination of various antigenic substances and antibodies. Proceedings of symposia, a°-a2 reviews, aa-ar and monographs a8 30 Immunoenzymatic Techniques, INSERM Symposium No. 2 (G. Feldman, P. Druet, J. Bignon and S. Avrameas, eds.). North-Holland/Elsevier, Amsterdam, 1975. 31 Enzyme-linked Immunosorbent Assay (ELISA) for Infectious Agents (J. L. Sever and D. L. Madden, eds.). J. Infect. Dis. 136, Suppl. (1977). 32 "Enzyme Labeled Immunoassay of Hormones and Drugs" (S. B. Pal, ed.). De Gruyter, New York, 1978. 33 S. L. Scharpe, W. M. Cooreman, W. J. Blomme, and G. M. Laekeman, Clin. Chem. 22, 733 (1976). 34 G. B. Wisdom, Clin. Chem. 22, 1243 (1976). z5 A. H. W. M. Schuurs and B. K. van Weemen, Clin. Chim. Acta 81, 1 (1977). 33 E. Engvall, Med. Biol. 55, 193 (1977). 3~ A. Voller, A. Bartlett, and D. E. Bidwell, J. Clin. Pathol. 31, 507 (1978). 38 "Quantitative Enzyme Immunoassay" (E. Engvall and A. J. Pesce, eds.). Scand. J. lmmunol. Suppl. 7, Vol. 8. Blackwell, Oxford, 1978.

[29]

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

439

are available as sources of detailed descriptions of specific applications as well as general information on methodological aspects. An extension of enzyme immunoassay is the enzyme affinity assay applicable to studies of nonimmunological interactions. This is already exemplified by the measurement of hormone using its receptor a9 and by our studies on the interaction of fibronectin with collagen. ~°-22 Assays of these and similar principles might well become a new area of expression for EIA. Acknowledgment The preparation of this manuscript and parts of the original work described was supported by grants CA 16434, CA 19894, and CA 22108 from the National Cancer Institute, DHEW. 39 F. S. Khan and B. B. Saxena, in "Enzyme Labeled Immunoassay of Hormones and Drugs" (S. B. Pal, ed.), p. 257. De Gruyter, New York, 1978.

[29] E l e c t r o d e - B a s e d E n z y m e I m m u n o a s s a y s Using Urease Conjugates

By M. E. MEYERHOFF and G. A. RECHNITZ The use of enzyme labels in place of radioisotopes for the measurement of antigens, antibodies, and haptens has stimulated the new and expanding field of enzyme immunoassay (EIA). This technique has been the focus of several recent reviews, 1-e and its merits compared to radioimmunoassay (RIA) have been discussed. 7's In many cases, EIA can match RIA in terms of sensitivity and selectivity, yet has advantages of speed, convenience, and reduced cost. EIA sensitivity and simplicity is, however, dependent on the choice of enzyme label. It is the purpose of this work to introduce urease as a new enzyme label and to demonstrate the 1 A. H. W. M. Schuurs and B. K. Van Weemen, Clin. Chim. Acta 81, 1 (1977). S. L. Scharpe, W. M. Cooreman, W. J. BIoome, and G. M. Leekeman, Clin. Chem. 22, 733 (1976). a G. B. Wisdom, Clin. Chem. 22, 1243 (1976). 4 j. Landon, Nature (London) 268, 483 (1977). BA. Voller, D. E. Bidwell, and A. Bartlett, Bull. WHO 53, 55 (1976). e E. Engvail, this volume [28]. M. Numazawa, A. Haryu, K. Kurosaka, and T. Nambera, FEBS Lett. 79, 396 (1975). s F. Dray, J. M. Andrieu, and F. Renaud, Biochim. Biophys. Acta 403, 131 (1975).

METHODS IN ENZYMOLOGY,VOL. 70

Copyright© 1980by Academic Press,Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181970-1

[29]

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

439

are available as sources of detailed descriptions of specific applications as well as general information on methodological aspects. An extension of enzyme immunoassay is the enzyme affinity assay applicable to studies of nonimmunological interactions. This is already exemplified by the measurement of hormone using its receptor a9 and by our studies on the interaction of fibronectin with collagen. ~°-22 Assays of these and similar principles might well become a new area of expression for EIA. Acknowledgment The preparation of this manuscript and parts of the original work described was supported by grants CA 16434, CA 19894, and CA 22108 from the National Cancer Institute, DHEW. 39 F. S. Khan and B. B. Saxena, in "Enzyme Labeled Immunoassay of Hormones and Drugs" (S. B. Pal, ed.), p. 257. De Gruyter, New York, 1978.

[29] E l e c t r o d e - B a s e d E n z y m e I m m u n o a s s a y s Using Urease Conjugates

By M. E. MEYERHOFF and G. A. RECHNITZ The use of enzyme labels in place of radioisotopes for the measurement of antigens, antibodies, and haptens has stimulated the new and expanding field of enzyme immunoassay (EIA). This technique has been the focus of several recent reviews, 1-e and its merits compared to radioimmunoassay (RIA) have been discussed. 7's In many cases, EIA can match RIA in terms of sensitivity and selectivity, yet has advantages of speed, convenience, and reduced cost. EIA sensitivity and simplicity is, however, dependent on the choice of enzyme label. It is the purpose of this work to introduce urease as a new enzyme label and to demonstrate the 1 A. H. W. M. Schuurs and B. K. Van Weemen, Clin. Chim. Acta 81, 1 (1977). S. L. Scharpe, W. M. Cooreman, W. J. BIoome, and G. M. Leekeman, Clin. Chem. 22, 733 (1976). a G. B. Wisdom, Clin. Chem. 22, 1243 (1976). 4 j. Landon, Nature (London) 268, 483 (1977). BA. Voller, D. E. Bidwell, and A. Bartlett, Bull. WHO 53, 55 (1976). e E. Engvail, this volume [28]. M. Numazawa, A. Haryu, K. Kurosaka, and T. Nambera, FEBS Lett. 79, 396 (1975). s F. Dray, J. M. Andrieu, and F. Renaud, Biochim. Biophys. Acta 403, 131 (1975).

METHODS IN ENZYMOLOGY,VOL. 70

Copyright© 1980by Academic Press,Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181970-1

440

IMMUNOASSAYS

[29]

applicability of this label to EIA by developing a solid phase assay system for a model protein antigen, bovine serum albumin (BSA), and for the biologically important nucleotide, adenosine 3',5'-cyclic-monophosphoric acid (cAMP). The basic concepts of competitive-binding solid phase EIA have been described elsewhere. 1"2"6The required separation of antibody from the assay mixture can be accomplished in a variety of ways. Double-antibody techniques are quite popular and involve the use of a second antibody, an antibody to the principal antibody, to induce separation. Cross-linking the second antibody serum 9"1° with ethyl chloroformate forms an insoluble suspension of small particles, which still maintain a high degree of immunoreactivity toward the first antibody. Addition of such particles to an EIA assay mixture pulls the first antibody from solution along with enzyme-labeled molecules bound to the antibody. Such a process is depicted in Fig. 1 and employed for all separations in this work. Measurement of enzyme activity in the bound solid phase is desirable because complete purification of the labeled substance is not necessary. A separation step may not always be necessary when measuring low molecular weight haptens by EIA. In some cases, antibody binding to the enzyme-labeled hapten causes complete inhibition of enzyme activity. This may occur when antibody binding either sterically hinders substrate access to the active site of the enzyme or induces enzyme conformational changes. Whatever the cause, a more convenient homogeneous EIA system can result. H'12 Detection limits in EIA are ultimately determined by how low one can measure the label's concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, 13/3-galactosidase, 15 peroxidase, ~4"~5and glucose oxidase. 14 This is due to the high mo9 E. Engvall and P. Perlman, J. lmmunol. 109, 129 (1972). ~0 E. Engvall, K. Jonsson, and P. Perlman, Biochim. Biophys. Acta 251,427 (1971). " K. F. Rubenstein, R. S. Schneider, and E. F. Ullman, Biochem. Biophys. Res. Commun.37, 846 (1972). 12 G. L. Rowley, K. F. Rubenstein, J. Haisjen, and E. F. Ullman, J. Biol. Chem. 250, 3759 (1975). 13 R. J. Bastiani, R. C. Phillips, R. S. Schneider, and E. F. Ullman, Am. J. Med. Technol. 39, 211 (1973). 14 Sigma Chemical Company Catalog, Sigma Chemical Company, St. Louis, Missouri, 1977. 15 Boehringer Mannheim Biochemical Catalog, Boehfinger Mannheim Biochemicals, Indianapolis, Indiana, 1977.

[29]

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

(~(~

(~.~

EIA assay mixture contains first antibody

:

•

"~

441

•

and

enzyme labeled free molecules

o,

second a n t i b o d y suspension

pellet

insolubilized antiserum particles

supernatant

FIG. 1. Schematic representation of double-antibody solid phase enzyme immunoassay using cross-linked second antibody particles for separation. Encircled E, enzyme label.

lecular weight of urease (480,000), TM and as a result, urease should be a good choice for sensitive immunoassays. Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. 1 Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of ureaselabeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. In this chapter we demonstrate that urease can be used as a label for protein antigen type of molecules by employing a urease-bovine serum albumin (BSA) conjugate to carry out competitive binding EIA for BSA. ~s Biochemica Information II, Publication of Boehringer Mannheim Biochemicals, Indianapolis, Indiana, 1975.

442

IMMUNOASSAYS

[29]

Results here give evidence that urease is a good choice of enzyme label for sensitive and simple assays. We further show that urease can be effectively applied to hapten assays by developing an EIA system for cAMP. Experimental Equipment. All potentiometric measurements were taken on a Coming Model 12 research pH meter in conjunction with a Heath-Schlumberger Model SR-255B strip chart recorder. An Orion Model 95-10 ammonia gas sensing electrode was used for all assays of urease activity. Potentiometric activity measurements were made at 25° in a 10-ml glass thermostatted cell. Reagents. Various lots of urease, type VII (Sigma Chemical Co.) with specific activities ranging from 40,000 to 70,000 units/gram (according to Sigma's assay procedure 14) were used in this work. All nucleotides, including O~'-monosuccinyl adenosine 3'-5' cyclic monophosphoric acid (O2'-monosuccinyl-cAMP), O2'-monosuccinyl guanosine 3'-5' cyclic monophosphoric acid (O2'-monosuccinyl-cGMP), O2'-monosuccinyl inosine 3'-5' cyclic monophosphoric acid (O2'-monosuccinyl-cIMP), adenosine 5'-monophosphoric acid (AMP), guanosine 5'-monophosphoric acid (GMP), guanosine 3'-5' cyclic monophosphoric acid (cGMP), and adenosine 3'-5' cyclic monophosphoric acid (cAMP), were also products of Sigma. Antiserum to BSA (2.4 mg/ml, Miles Laboratories, Inc.) and goat anti-rabbit IGG antiserum (Calbiochem Laboratories) were commercial preparations. A tris(hydroxymethyl)aminomethane hydrochloric acid buffer, pH 7.5, containing 1 mM ethylenediaminetetraacetic acid (Tris-HC1-EDTA) was used as a working buffer throughout this work. The ionic strength of this buffer was 0.1 M. These specific conditions were chosen based on known pH and ionic strength optimums for urease activity. TM EDTA was added because of its stabilizing effect on the enzyme. 17 All other buffers and solutions were prepared with reagent grade chemicals and distilleddeionized water. Preparation of Urease-BSA Conjugate. The method of glutaraldehyde conjugation used was based on the previously reported techniques of Avrameas TM for preparation of enzyme-protein conjugates. Two milligrams of BSA and 2 mg of urease (40,000 U/gram) were dissolved in 1.0 ml of 0.1 M phosphate buffer, pH 6.8, and 0.1 ml of 1% aqueous glutaraldehyde was added slowly to the stirred solution. The reaction mix17D. S. Papastathopoulos and G. A. Rechnitz, Anal. Chim. Acta 79, 17 (1975). is S. Avrameas, lmrnunochemistry 6, 43 (1969).

[29]

443

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

ture was stirred for 2 hr at room temperature, then dialyzed overnight against 3 liters of Tris-HCI-EDTA buffer at 4°. Particulate matter (probably high molecular weight aggregates of urease) were centrifuged out, and the resulting supernatant was partially purified on a Sephadex G-200 column equilibrated with working buffer. The u r e a s e - B S A conjugate appeared in the void volume since its molecular weight is well above 500,000. A second protein fraction of much lower molecular weight appeared later and was assumed to be unconjugated BSA or BSA aggregates. The resulting conjugate could be stored at 4° for several months without significant loss of activity. A 1:10 dilution of this enzyme conjugate was used in the actual assay of BSA.

Preparation of cAMP Antibody and Cyclic-Nucleotide Urease Conjugates. Antibody to cAMP was obtained through the Immunology Department of Roswell Park Memorial Institute, Buffalo, New York. Rabbits were immunized with an O2'-monosuccinyl-cAMP-human serum albumin (HSA) conjugate prepared by the carbodiimide reaction method described by Steiner et al. TM Five milligrams of OV-monosuccinyl-cAMP, 5 mg of 1ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 10 mg of HSA were dissolved in 3 ml of distilled water and the solution was adjusted to pH 5.5 with dilute HCI. This reaction mixture was stirred overnight at room temperature and then extensively dialyzed agamst phosphate-buffered saline (PBS, pH 7.4). The rabbits were injected subcutaneously with 1.5 mg of this conjugate in a 1:1 mixture of PBS and complete Freund's adjuvant. Rabbits were boosted with the same dosage every 2 weeks for the first 2 months, and then monthly after that. The rabbits were usually bled 2 weeks after injections• Production of antibody to cAMP was confirmed by classical immunological techniques (ring test, precipitin test 2°) using a test antigen of O2'-monosuccinyl-cAMP-/3-1ac toglobulin prepared in the same manner as the HSA conjugate. The final antibody solution for the assay proposed was obtained from the ~/-globulin fraction of this serum after 3 × ammonium sulfate fractionation 21 and final dialysis against Tris-HCI-EDTA buffer. This antibody solution contained between 0.5 and 1.0 mg of anti-cAMP antibody per milliliter as determined by precipitin analysis 2° using the/3-1actoglobulin test antigen. Urease-cyclic nucleotide conjugates were not prepared by the carbodiimide method because of high loss of urease activity during the course •

&

.

lg A. L. Steiner, D. M. Kippis, R. Utiger, and C. Parker, Proc. Natl. Acad. Sci. U.S.A. 64, 367 (1969). 20 E. A. Kabat and M. M. Mayer, *'Experimental Immunochemistry." Thomas, Spdngtield, Illinois, 1961. ~1 D. H. Campbell, J. S. Garvey, N. E. Cremer, and D. H. Sussdorf, "Immunology." Benjamin, New York, 1964.

444

!MMUNOASSAYS

[29]

of the reaction. Instead, a modification of the mixed anhydride method used elsewhere to form steroid-protein conjugates was used. 22 Typically, 1-2 mg of the O2'-monosuccinyl derivative of the cyclic nucleotide (free acid) was suspended in 0.15 ml of dioxane and 0.15 ml of dimethylformamide with stirring at 4°. Five microliters of tributylamine, followed by 5/.d of isobutylchloroformate were added to the reaction mixture. The reaction was run for 1 hr at 4°, at which point the mixed anhydride of the succinylated nucleotide had been formed. The approximately 0.3 ml of organic solvent present was then removed by rotoevaporation. Five milliliters of cold urease (0.4 mg/ml) in 0.1 M NaHCOa at pH 9.4 was added to the residue material, and with constant stirring the conjugation reaction proceeded for 2 hr more at 4°. Finally, the urease conjugate was dialyzed extensively against Tris-HCI-EDTA. Contrary to other previously published mixed anhydride procedures using other proteins, it is an absolute requirement in the case of urease that all organic solvent be removed prior to final conjugation, since it was found that exposure of the urease to even a slight amount of hydrophobic solvent renders it irreversibly inactive. Using the above method excellent final yields of enzyme activity are obtained (approximately 80%). Conjugation of the nucleotides to urease was confirmed by ultraviolet (UV) spectra between 300 and 240 nm. Based on the change in the urease spectra before and after conjugation and the known molar extinction coefficients2a of the cyclic nucleotides, an estimate as to the degree of conjugation can be made. Typically, a conjugation between 2 and 7 mol of nucleotide per mole of enzyme is obtained based on a urease molecular weight of 480,000.16 The activity of the urease-cyclic nucleotide conjugates was not significantly inhibited by the presence of a 100-fold excess of anti-cAMP binding sites, with respect to the concentration of enzyme-linked cyclic nucleotide (as determined by spectrophotometric calculations and use of a 0.5 mg/ml antibody solution). This suggests that a homogeneous inhibition of urease-cyclic nucleotide conjugate activity is not possible and all eventual EIA systems involving these conjugates will be of the solid phase type. Procedure f o r Determining Urease Activity B o u n d to Antibody. It has been shown that the activity of enzymes can simply and readily be determined through the use of potentiometric type electrodes. 24-27 In previous 22B. F. Erlanger, F. Borek, S. M. Beiser, and S. Lieberman,J. Biol. Chem. 228, 713 (1957). ~aSchwarz-Mann Radiochemcial-BiochemicalCatalog, Becton, Dickinson and Co., Orangeburg, New York,1977. 24M. Meyerhoffand G. A. Rechnitz,Anal. Chim. Acta 115,277 (1976). ~5R. L. Llenadoand G. A. Rechnitz,Anal. Chem. 44, 468 (1972).

[29]

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

445

work, we have used an ammonia gas sensing electrode to follow kinetically the activity of several enzyme systems including creatininase enzyme. z4 It was found that, in a properly designed cell, activity measurements can be made in volume as small as 0.8-1.0 ml, with stirring, by following the initial rate of potential change over a short period of time. The main advantage of such assays is that they can be performed directly in the enzyme solutions without need for special colorimetric reagents, drastic changes in solution conditions (i.e., pH), etc. In preliminary experiments for this work, a potentiometric assay system for urease was designed to determine the lower limit of urease con-

-I-15ul of 6M u r e a 190 ~ ~ a .

rate portion of c u r v e

180 :>E 170

160

time

[mini

FIG. 2. Typical potential (E) vs time recording obtained for assay of urease with an ammonia gas sensing electrode. Curve shown is for addition of 15 ~1 of 6 M urea to 1 ml of 0.4 nM urease in 0.1M Tris-HCl-I mM EDTA, pH 7.5. " K. Cammann, Fresenius Z. Anal. Chem. 257, 1 (1977). 2~ p. D'Orazio, M. E. Meyerhoff, and G. A. Rechnitz, Anal. Chem. 50, 1531 (1978).

446

IMMUNOASSAYS

[29]

70

,.-50 E E

® t~ 30

10

0.4

0.8 urease

1.2 cone.

1.6

2.0

2.4

IM x1091

FIG. 3. Typical rate of potential change vs urease concentration curve as determined in I ml of u r e a s e - T r i s - H C l - E D T A , p H 7.5.

centration one could measure accurately in a reasonably short assay time (4 min). This lower limit will ultimately determine detection limits for the EIA system using urease. In these experiments, 1 ml of urease buffer solution (prepared from a lot containing 40,000 U/gram) was stirred in a thermostatted cell at 25°. An ammonia gas sensing electrode was placed in the solution, and the potential was allowed to reach a steady state value. Then 15/xl of 6 M urea was added, and potential vs time curves were recorded. A typical recording is shown in Fig. 2. The resulting change in potential arises from the production of ammonia gas according to the following urease-catalyzed reaction: Urea ur©ase ~ CO~ + 2NHa

The "rate portion of curve," expressed in millivolts per minute, can be calculated by graphically extending the straight line portion of the curve. This rate can then be plotted vs urease concentration, and a curve, shown in Fig. 3, is obtained. At lower enzyme concentration the correlation is linear, and at higher levels nonlinearity occurs owing to the time response

[29]

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

447

of the electrode ultimately becoming the rate-limiting step. All EIA work was performed at the lower rate region, and thus the rates seen in the EIA assays are directly proportional to urease activity. Furthermore, reproducible starting potential within _-_1 mV are achieved by dialyzing the electrode back to background ammonia levels in a large volume of buffer for a brief period of time (2-3 min) between each assay. The assay system offers a distinct advantage over the spectrophotometric method for assay of urease in an EIA system. It is most convenient for solid phase EIAs to measure the amount of label bound to the antibody because, as previously mentioned, total purification of the label is therefore not necessary. Spectrophotometric assay of bound enzyme on solid support particles is difficult owing to turbidity problems. Electrode assay of bound enzyme circumvents this problem and allows for a truly kinetic assay of the enzyme (i.e., initial rate method). In this work we employed the insolubilized second antibody approach to separate bound from free label. 2s A 5 ml quantity of goat anti-rabbit 7-globulin serum from Calbiochem (containing 125 units, as 1 unit is defined in the Calbiochem Catalog2a) was reconstituted and dialyzed against 0.15 M NaCI. This antiserum was then polymerized into finely grained immunoreactive particles with ethyl chloroformate according to the method outlined by Avrameas. ~° Five milliliters of 0.2 M acetate buffer were added to the predialyzed serum followed by the gradual addition of 0.3 ml of ethylchloroformate (with stirring). After 20 min 10 ml of 0.2 M acetate buffer was added, and the reaction was allowed to continue for 1 hr. From the initial 5-ml bottle of goat serum (125 units), the resulting particles after thorough washing were resuspended to a final volume of 15 ml with working buffer. It was found that for both BSA and cAMP assays, 300 ~1 of this suspension sufficed to remove all rabbit 3,-globulins from the assay solution. Urease activity on these particles can easily be quantitated by the above electrode method after centrifugation and washing steps. Final urease assays of washed particles are done in 1 ml of 0.1 Tris-HCI-EDTA buffer, pH 7.5. Procedures for EIA Experiments. For both BSA and cAMP systems, all assays were carried out in 3-ml conical centrifuge tubes. A given amount of rabbit antibody, enzyme conjugate, and standard amount of BSA or nucleotide were mixed for 1 hr at 4° (constant shaking action). ~8 B. K. Van Weemen and A. H. W. M. Schuurs, in "'Immunoenzymatic Techniques" (G. Feldman, P. Druet, J. Bignon, and S. Avrameas, eds.), p. 125. American Elsevier, New York, 1976. 2~ Calbiochem Immunochemical Catalog, Calbiochem-Behring Corp., La Jolla, California, 1978. as T. Ternynck and S. Avrameas, Scand. J. lmmunol. Suppl. 3, 29 (1976).

448

IMMUNOASSAYS

[29]

Polymerized goat antirabbit T-globulin, 300/xl, was added followed by further incubation with mixing for 2 hr more. The tubes were centrifuged at 1000 g and the pellet was washed three times with working buffer. Finally, the pellet was resuspended in 1 ml of buffer for urease assay. Calibration curves are expressed as percentage of activity relative to a tube in which plain buffer was substituted for antigen or nucleotide aliquot. This tube's pellet contains maximum urease activity as determined by the change in potential with time (millivolts per minute) by the ammonia electrode method for urease determinations. This rate is designated as 100% activity and is indicative of the amount of urease label bound to the antibody in the absence of competing free antigen or hapten. Presence of standard cAMP or BSA in the respective assay tubes reduces the amount of urease label bound. Nonspecific binding of enzyme label to second antibody particles was very low, usually < 5% of maximum activity bound, as determined by a blank tube, in which all reagents were added except the first antibody and hapten or antigen standard. For BSA assay, to 1.5 ml of buffer-BSA solution, typically 40/zl of a 1:100 dilution of rabbit anti-BSA (2.4 mg/ml) and 40/zl of a 1:10 dilution of urease-BSA conjugate were added. For cAMP assay, 30/zl of a 1:10 dilution of the (NH4)2SO4 fraction of cAMP antiserum (0.5 mg/ml) and 30/zl of a 1:10 dilution of enzyme-cyclic nucleotide conjugate were added to 1.0 ml of buffer or buffer-nucleotide standard. Results and Discussion In order to fully evaluate urease as a potential label for EIA in general, we felt that it was necessary to demonstrate that urease could be covalently coupled to an antigen and still maintain sufficiently high activity for sensitive immunoassay. In addition, the effect of antibody binding to the labeled antigen should be tested to see whether enzyme activity can be homogeneously inhibited by the antibody. Bovine serum albumin was chosen as a model protein antigen and coupled to urease as described in the experimental section. Homogeneous experiments using excess antibody to BSA indicated that no significant inhibition of the conjugate activity took place and that any useful EIA system employing urease-protein conjugates would have to be of the solid phase type. Figure 4 shows the typical decrease in binding of a urease-BSA conjugate to anti-BSA antibody in the presence of increasing amounts of BSA. One hundred percent activity refers to the relative amount ofurease activity present on the insolubilized second antibody particles when no BSA is present. This 100% value was typically 14-15 mV/min using a potentiometric ammonia electrode as described above (activity assay time of

[29]

UREASE CONJUGATES IN ENZYME IMMUNOASSAYS

449

100 8O

.> 60 40 20

i

1()

1() 2 B S A conc.,

103 ng/ml

1() 4

FIG. 4. Calibration curve obtained for bovine serum albumin (BSA), by monitoring amount of urease-BSA conjugate bound to anti-BSA antibody. Data were obtained with tubes containing 1.5 ml of BSA standard, 40/~1 of 1:100 anti-BSA serum, 40/~1 of 1:10 urease-BSA conjugate, and 300/zl of insolubilized goat anti-rabbit y-globulin suspension, all prepared in 10 mM Tris-HCI-EDTA, pH 7.5.

4 min). The limits of detection in this case (< 10 ng/ml or 10 ng of total BSA) are comparable to other antigen assays using different enzyme labels) However, in most of those previous EIA assays, enzyme activity was measured over much longer time periods, usually ranging from 30 min to 2 hr. The results here confirm that the higher activity of urease allows for equal sensitivity at reduced assay times. If urease ammonia production were monitored over a comparable time period (either by rate or fixed-time method) an even more sensitive EIA system would result. The optimum amounts of antibody, urease-conjugate, and insolubilized second antibody used in an assay must be determined from a series of titration experiments. For example, to obtain analytically reproducible antigen inhibition data, it is desirable to have a minumum amount of antibody bind a mimimum amount of enzyme label to produce a sufficiently fast rate at 100% activity conditions. Figure 5 shows a typical series of titrations with rabbit anti-BSA using three different amounts of ureaseBSA conjugate. These experiments were carried out by adding varying amounts of anti-BSA antibody to tubes containing fixed amounts of urease-BSA conjugate in working buffer followed by the normal separation and assay steps. To obtain a sensitive assay system, it is important to

450

IMMUNOASSAYS

[29]

24

"~ 16

O t=

8

o12

' ug

o16 ' antibody

1'.o

FIG. 5. Titration of various amounts of a 1:10 dilution of urease-bovine serum albumin (BSA) conjugate with rabbit antibody to BSA: ©, 25 0d; A, 80 0d; n, 21)0 ~1. Conditions as in Fig. 4, except that 1.5 ml of buffer replaces standards in all tubes.

work at the antibody level at which saturation begins to occur and also have this point correspond to a reasonably fast rate (12-15 mV/min). The same general approach was used to develop an EIA procedure for cAMP. Cyclic AMP was conjugated to urease by the mixed anhydride procedure using the same O2'-monosuccinyl derivative previously employed to prepare the immunizing protein (HSA-cAMP). Preliminary experiments with this u r e a s e - c A M P conjugate and anti-cAMP antibodies (see experimental) indicated that as in the case with the u r e a s e - B S A conjugate, no homogeneous inhibition of the conjugate could take place. This is probably a result of the small size of the substrate, and the inability of antibody binding to sterically hinder urea from getting to the active site of the enzyme. Therefore, the double-antibody solid phase was also used for the cAMP assay. Figure 6 shows a typical calibration curve obtained for the inhibition of binding of a u r e a s e - c A M P conjugate to anti-cAMP antibody as determined by the ammonia electrode. One hundred percent of activity refers to blank tubes, which had rates of 11-12 mV/min in the absence of cAMP. Selectivity of the assay over structurally similar cGMP is also shown in Fig. 6. It takes approximately 1000 times more cGMP than

[29]

UREASE CONJUGATES IN ENZYME 1MMUNOASSAYS

100

L

•

>'- 60

=

451

cyclic

;

cyclic

~

N 40

20

i

10-8

h

10-7

i

i

10-6 10-5 nucleotide c o n c . , M

i

10-4

=

10-3

FIG. 6. Calibration curves obtained for c A M P and cGMP using a urease-cAMP conjugate and cAMP antibody. Data obtained in 0.1 M Tris-HCI-EDTA, pH 7.5, using 1.0 ml of

nucleotide standard, 30/zl of 1:10 rabbit anti-cAMP antibody, 30 ttl of 1:10 uretase- cAMP conjugate, and 300 ttl of second antibody suspension.

cAMP to get equal inhibition, indicating the high selectivity of the antibody. However, the relative insensitivity (> 10-7 M) toward cAMP presented a problem if one wanted eventually to use such a system in physiological samples where cAMP levels are quite low (10 -a to 10-e M). Van Weemen and Schuurs 28 demonstrated that for EIA-hapten systems, the nature of the hapten linked to the enzyme can have a profound influence on sensitivity. They found that if the hapten of interest is linked to the enzyme in the same manner as the hapten was linked to the immunizing protein, a relatively insensitive assay may result because antibody production is elicited to the bridging group as well as the rest of the haptenic structure. Evidence of this type of antibody specificity in cAMP antibodies has been shown by RIA methods. Immunization with the 0 2'monosuccinyl derivative gives rise to antibody production with strong recognition of the ester linkage at the 0 2' position of the ribose ring. Initial acetylation of cAMP samples (at the 0 2' position) has brought forth increased sensitivity in the RIA method 31,~2 as a result of stronger affinity between antibody and the acetylated free cAMP. To increase sensitivity al j. E. Harper and G. Brooker, J. Cyclic Nucleotide Res. 1,207 (1975). 32 M. L. Goldberg, Clin. Chem. 23, 576 (1977).

452

1MMUNOASSAYS

100

[29]

AMP, G M P

80 ~_

~

cyclic

•-> 60 ~ cyclic

"X,,.

40

20

i

10 -9

i

i

10-8

10-7

i

10-6 n u c l e o t i d e conc., M

i

10-5

10-4

FIG. 7. Calibration curves obtained from inhibition response to cAMP, cGMP, AMP, and GMP, using a urease-cGMP conjugate and cAMP antibody. Data were obtained as for Fig. 6.

for EIA systems, Van Weemen and Schuurs showed that one could alter the site of hapten attachment to the enzyme, change the nature of the briding group (i.e., succinyl to glutaryl), or use a hapten structurally similar to that to be measured. This last approach was pursued here in an attempt to improve the sensitivity of the cAMP assay. Figure 7 shows a typical calibration curve for cAMP when using a u r e a s e - c G M P conjugate with cAMP antibody in the electrode-based system. Comparison with Fig. 6 illustrates the dramatic improvement in sensitivity obtained. Inhibition of label binding begins to occur at less than 10-9 M cAMP. The u r e a s e - c G M P conjugate is prepared with the O vmonosuccinyl derivative of cGMP and differs from the initial ureasecAMP conjugate and immunogen only by a guanine instead of adenosine moiety in the haptenic structure. This substitution effectively decreases the binding constant between the label and the cAMP antibody, thus allowing free cAMP to inhibit at lower concentrations. Direct comparison of the two conjugates is possible because final activity and degree of nucleotide conjugation were very similar for both. Figure 7 also shows the selectivity of this assay system over cGMP, GMP, and AMP. As expected, in switching to a u r e a s e - c G M P conjugate, selectivity over cGMP itself is reduced, but it still takes 20 times more cGMP than cAMP to produce the same amount of inhibition. This again

[29]

UREASE C O N J U G A T E S IN E N Z Y M E I M M U N O A S S A Y S

453

can be explained by the relative affinity of cAMP antibody for free cGMP vs cGMP conjugated to the enzyme. The succinyl group present in the enzyme conjugate causes greater affinity for enzyme-linked cGMP than for free cGMP. The system is highly selective over the corresponding noncyclic nucleotides AMP and GMP. This result agrees well with previous RIA systems, indicating that the cAMP antibodies have strong recognition of the cyclic phosphate ring. 19 Along this same line, a urease-cIMP conjugate was prepared and tested in the EIA system for cAMP. Figure 8 shows the resulting inhibition curves obtained for both cAMP and cGMP. Sensitivity for cAMP is not as good as when using the urease-cGMP conjugate and selectivity over cGMP also appears to be somewhere between that obtained with the other conjugates (approximately 80 times). These results seem appropriate in view of the fact that cIMP is more similar in structure to cAMP than to cGMP. It is important to note here that cIMP was not tested for inhibition because it has not yet been found to exist in physiological fluids at detectable levels. Cyclic GMP itself is present in serum and urine, but at levels 10 times lower than cAMP. 32 Furthermore, the antiserum to cAMP used throughout this work was taken from a single rabbit and there was no attempt to produce higher quality antisera in other rabbits, which, if obtainable, could lead to even more sensitive and selective assays.

• •

100

cyclic

•> 60

~

cyclic

4C

2C

i

10 -9

i

i

i

10-6

10-7

10-6

nucleotide

i

10-5

i

10-4

conc. , M

FIG. 8. Calibration curves obtained from inhibition response to c A M P and c G M P using a urcasc-clMP conjugate and c A M P antibody. Data were obtained as f o r Figs. 6 and 7.

454

IMMUNOASSAYS

[29]

REPRODUCIBILITY OF cAMP CALIBRATION CURVES

Percentage of activity 'z cAMP conc. (M)

Day 1

Day 2

Day 3

Day 4

Mean percentage of activity

2.5 × 10-s 2.5 × 10-7 2.5 × 10-e

66.1 34.8 8.7

67.7 33.1 8.5

64.0 30.1 10.0

69.0 33.0 8.0

66.7 32.8 8.8

Rates of 100% activity tubes were 11.8, 11.5, 10.8, and 11.5 mV/min for days 1-4; average: 11.5; relative standard deviation: 3.7%

Analytical precision of an EIA method is ultimately limited by how reproducibly one can assay enzyme concentration via activity determinations. Previous potentiometric activity determinations have resulted in excellent relative standard deviations of 10% or less. za'25"27In the preliminary part of this work, analysis of urease by our potentiometric system yielded similar precision, with maximum standard deviations at low urease levels (i.e., < 10-a M). Typical reproducibility of subsequent EIA calibration curves from day to day is summarized in the table. These data represent the relative percentage of activity values for three different cAMP concentrations when using a u r e a s e - c G M P conjugate (as in Fig. 6) over a 4-day period. It can be seen that excellent reproducibility is observed, indicating the good precision of the activity assay as well as the time stability of the reagents involved. In fact, all urease conjugates prepared in this work can be stored for at least 4 months without significant loss of immuno or enzymic activity. Moreover, working buffer conditions have been chosen so that possible inhibitors of urease that may be present in real samples (e.g., heavy metals) would not be expected to create a problem with the assays (EDTA preserves full activity of urease under physiological conditions). 17 We have developed in this work the techniques necessary to utilize urease as a label for EIA of both protein type antigens and low molecular weight haptens through the use of an ammonia electrode to measure bound enzyme. Furthermore, the first EIA procedure for cAMP has been demonstrated using urease-cyclic nucleotide conjugates. In view of the great interest in measuring cAMP in physiological samples, the EIA system described here should provide the basis for developing an attractive alternative to traditional cAMP assay procedures.

[30]

PASSIVE HEMAGGLUTINATION [30] Passive Hemagglutination and Hemolysis Estimation of Antigens and Antibodies

By

FRANK

L. ADLER

and

LOUISE

455 for

T. ADLER

Serological techniques provide specific and sensitive means for the detection and measurement of antibodies and antigenic substances. Among the many procedures that are available, some are more suitable for measuring antibody, others are better adapted for the assay of antigens. Passive hemagglutination (HA) and hemagglutination inhibition (HI) are here described and discussed as highly versatile techniques that require no specialized or expensive equipment and provide semiquantitative answers rapidly. A few remarks concerning the specificity and sensitivity of serological tests seem appropriate to assist the reader in the judicious application and interpretation of the procedures to be described. It is well to remember that the reaction between antibodies and antigenic substances is mediated by the specific binding of antibody to exposed antigenic determinants on the antigen molecule. The specificity of the antibody is for such determinants as, in native proteins, are thought to have the dimensions of tetrapeptides or, in substituted proteins, such as dinitrophenylated ovalbumin, may be as small as the substituent. Some unanticipated reactions of antibodies, superficially suggestive of lacking specificity, can often be traced to the sharing of antigenic determinants by the "cross-reactive" antigens. Antisera usually contain a mixture of antibodies differing from each other with regard to specificity as well as biological and other properties. Immunization with a highly purified antigen does not, in itself, assure the production of highly specific antibodies, and it is not unusual that a trace contaminant may evoke a disproportional amount of antibody. Since tests for the specificity of antisera are often based on precipitin procedures, such as Ouchterlony tests or immunoelectrophoresis, it is important to keep in mind that HA tests are 100-1000 times more sensitive in the detection of antibodies than are precipitin tests. Thus it is obvious that specificity must be ascertained by tests of appropriate sensitivity. Finally, in view of the advent of monoclonal antibodies produced in vitro or in vivo by cloned hybrids of myeloma and antibody producing cells, one should keep in mind that, while the specificity of such antibodies will be restricted to a single determinant, they will still react with diverse antigens that possess this determinant.

METHODS IN ENZYMOLOGY, VOL. 70

Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181970-I

456 Passive

IMMUNOASSAYS

[30]

Hemagglutinafion

Principles In passive hemagglutination (HA) erythrocytes serve as the inert carriers of suitably affixed extraneous antigens, and the clumping Of such indicator cells by antibody specific for the coating antigen is a sensitive test for antibody. Under appropriate conditions the test measures antibody in concentrations of as little as 1-10 ng/ml. Since reasonably potent immune sera will contain at least 1 mg of antibody per milliliter it is apparent that their HA titer, defined as the highest dilution that will bring about agglutination, is often greater than 1:100,000. Since dilutions of the antisera to be tested are usually made in twofold steps, one deals with a semiquantitative test, and because antibodies sharing the same specificity but belonging to different classes, such as IgM or IgG, may differ in their HA activities, the test is more accurately viewed as one for activity rather than amount of antibody. Nevertheless, when applied to antisera obtained after intensive immunization and thus containing mostly IgG antibodies, the HA test results generally correlate reasonably well with those of binding assays for antibody. Soluble antigens can be attached to erythrocytes either through adsorption or by covalent linkage, often through the use of bifunctional agents. Whatever the procedure used, it is evident that firm attachment is required since antigen leakage from the erythrocyte would result in the binding and diversion of antibody and result in the specific inhibition of HA. This, indeed, is the principle of the hemagglutination-inhibition (HI) test to be described later. Some antigens adhere to red cells with sufficient tenacity and in adequate amounts to obviate the need for anything more elaborate than incubation of the cells with the antigen. Among them are many polysaccharides of microbial or protozoan origins. 1 Other antigens, such as most proteins, require treatments that either increase their ability to bind the antigen or their susceptibility to agglutination by antibodies against the coating antigen. The prototype for such procedures is the treatment of red cells with tannic acid, 2 which is to be described in detail below. Some antigenic determinants, such as dinitrophenyl groups, can either be attached to red cells directly3 or can be introduced into an antigenically irrelevant protein, which is then affixed to the erythrocytes.4 A typical method for 1 E. Neter, Bacteriol. Rev. 20, 166 (1956). 2 S. V. Boyden, J. Exp. Med. 93, 107 (1951). 3 M. B. Rittenberg and K. L. Pratt, Proc. Soc. Exp. Biol. Med. 132, 575 (1969). 4 F. L. Adler and C.-T. Liu, J. Immunol. 106, 1684 (1971).

[30]

PASSIVE HEMAGGLUTINATION

457

the covalent linkage of protein antigens to red cells is that employing bisdiazobenzidine, which is to be described in detail below. Procedures that have been successfully employed by various workers also include the use of chromic chloride, 5 water-soluble carbodiimide, e difluorodinitrobenzene, 7 toluene 2,4-diisocyanate, 8 glutaraldehyde, 9 and others. The choice is often determined by preferences of the investigator, but always requires selection of a procedure that will not block or destroy essential determinants and will yield cells of acceptable stability and sensitivity.

Materials and Reagents The HA test can be done in tubes (10 × 75) or in plastic hemagglutination trays. The latter are available either as permanent (Lucite) or disposable units. Those made of rigid plastic and with cone-shaped ( " V " plates) wells are preferred. When trays are used one can supplement them with other components of the Microtiter series, which include diluting devices to replace conventional pipettes and calibrated droppers that deliver 25or 50-/.d amounts. Alternatively, dilutions can be prepared in test tubes and aliquots can then be transferred into the wells of the trays with disposable Pasteur type pipettes of standard bore size that deliver approximately 30-/zl drops. When trays are used, static electricity can lead to difficulties; it should be reduced by wiping the trays with a moist tov~el or by use of an t~-particle emitting Staticmaster. An assortment of graduated conical (15 ml) and plain round-bottom tubes (12-15 ml), serological pipettes, flasks, and a bench-type centrifuge with a swinging bucket head complete the equipment needs. Antisera to be used in HA must be free of bacterial contamination and other foreign matter. It is also important that harvesting of the sera from the blood clot be postponed until the clotting is complete, since residual fibrin or fibrinogen can seriously interfere in HA. The use of preservatives, such as 0.01% sodium azide, is recommended, as is storage of aliquots of the sera at - 20°. Prior to this, sera should be heated at 56° for 30 min, and antibodies reactive with the erythrocytes to be used should be removed by three successive absorptions with washed, packed red cells. Generally 3 × 1 volume of packed cells for 9 volumes of serum with 30 min incubation at room temperature should suffice. Diluted antisera should not be stored because deterioration of HA activity may occur. 5 j. W. Goding, J. Immunol. Methods 10, 61 (1976). e E. S. Golub, R. K. Mishell, W. O. Weigle, and R. W. Dutton,J. lmmunol. 100, 133 (1968). r N. R. Ling, Immunology 4, 49 (1961). 8 D. E. Mahan and R. L. Copeland, Jr., J. lmmunol. Methods 19, 217 (1978). S. Lemieux, S. Avrameas, and A. E. Bussard, Immunochemistry 11, 261 (1974).

458

IMMUNOASSAYS

[30]

Dilutions of the antisera are generally made in 2-fold steps. If the initial dilution to be tested is less than 1:1000 it is advisable to use separate pipettes for each step because antibody from the more concentrated serum carried on the outside of the pipette can introduce serious errors. An initial 1:1000 dilution is most readily made in three successive steps of 10-fold dilutions. For the procedures to be described, phosphate-buffered saline (PBS) consisting of equal volumes of 0.15 M NaC1 and 0.15 M phosphate buffer, pH 7.2-7.3, is satisfactory as the basic diluent or as washing and suspending medium for the cells. It is used without additives in the washing of the erythrocytes and in preparing the original 5% suspension. For antiserum dilutions and for washing and suspending the coated cells the PBS is enriched by the addition of 1.0 ml of heated (56° for 30 min) and absorbed (red cells) normal rabbit serum to 100 ml of PBS. Fetal calf serum can also be used provided that it does not react with the antibody or the coating antigen. The serum addition is required for the maintenance of optimal suspension stability of the cells, which is a critical factor in the test because insufficient stability causes agglutination in controls and excessive stability reduces the sensitivity of the procedure. None of the various substitutes that have been tried have proved to be equal to normal serum. It is sometimes convenient to add some dye, such as Evans' blue, to give a slight tint to the diluent. This helps in differentiating empty from filled wells in the tray, especially when the trays sit on a nonglossy bright surface. Usually sheep red cells (SRBC) are used; human type O cells are equally suitable. Pooled SRBC are better than cells from single animals; they are available commercially in the form of whole pooled blood in modified citrate solution containing about 25% red cells. Such blood samples are stable at 4° for at least 1 month; they should be discarded at the first sign of spontaneous lysis or lysis during washing. Coated cells, prepared by either of the two procedures to be described, are stable for 1 week at 4° .

Procedure Preparation and Coating of Tanned SRBC. Citrated sheep blood is centrifuged in conical graduated tubes for 15 rain at 750 g. The supernatant is removed by suction, and the sedimented cells are thoroughly resuspended in the original volume of PBS. In this and all subsequent steps resuspension is best accomplished by first adding 1 ml of suspending fluid, suspending cells in this small volume with the aid of a Pasteur pipette with attached 2-ml bulb, and then adding the remainder of the suspending fluid.

[30]

PASSIVE HEMAGGLUTINATION

459

The cells are washed at least three times; the supernatant should be free of protein, which is detectable by adding a drop of nitric acid to a drop of supernatant. After the last wash the cells are suspended in PBS to yield a 5% (v/v) suspension. A freshly prepared solution of tannic acid, diluted 1:20,000 (w/v) in PBS is mixed with an equal volume of 5% SRBC. After 15 min at 37° the cells are harvested in round-bottom tubes by centrifugation for 8 min at 750 g. The supernatant is decanted by partial inversion of the tubes and by blotting the tube lips with filter paper. Properly tanned cells are sufficiently sticky to permit this procedure. After the tanned cells have been made into a 5% suspension in PBS, they are coated by mixing 1 volume of cell suspension with 4 volumes of antigen solution (in PBS) and incubating the mixture 15 min at 37° with occasional agitation. The cells are then harvested by centrifugation as described above, washed three times with PBS containing 1% normal serum, and then made into a 5% suspension in this medium. Coated cells stored at 4° should be washed once just before use on subsequent days. The concentration of antigen optimal for coating is the minimal concentration that will yield cells that provide the highest sensitivity in the HA test. It will vary with the nature and degree of aggregation of the antigen and, to some extent, with the avidity of the antisera to be tested. '° For most proteins the optimum will be in the range of 10-100 p,g/ml. Since the amount of antigen removed from the coating solution is minute, it is possible to reutilize coating antigen several times for the coating of successive batches of tanned cells provided dilution is avoided through addition of coating antigen to appropriate amounts of packed tanned cells. The coated cells should be kept at 4° until they are used in the test. Bisdiazobenzidine Procedure. To prepare bisdiazobenzidine (BDB) one dissolves 0.23 g of benzidine (a carcinogen) in 45 ml of 0.2 N HCI. The solution is chilled to 0° in an i c e - w a t e r - s a l t bath, and 0.175 g of NaNO~ in 5 ml of chilled water is added rapidly. The reaction is allowed to proceed for 30 min at 0°, with occasional mixing. A suitable preparation should, upon 15-fold dilution of a sample in PBS, turn brown and turbid in 90 sec at room temperature. If this occurs too fast, addition of a trace of 1% NaNO~ will correct it; if too slow it can be adjusted by addition of a trace of benzidine. The BDB solution is then distributed in 2-ml amounts into plastic tubes, which are then capped and rapidly frozen in a Dry Ice-ethanol bath. The reagent is stored at - 20° or - 70° and remains stable for up to 6 months. When red cells are to be coated, a 5% suspension is prepared exactly ,o G. Wolberg, C.-T. Liu, and F. L. Adler, J. Immunol. 103, 879 (1969).

460

IMMUNOASSAYS

[30]

as described for tanning. To 24 ml of a protein solution in PBS, add 12 ml of the cell suspension and 6.0 ml of BDB thawed and diluted 15-fold in PBS just before use. Rapid and thorough mixing of the reagents is essential. After 15 min at room temperature the cells are harvested, washed, and resuspended to a 5% suspension exactly as described for tanned cells. Here also PBS containing 1% heated normal serum is used. The spontaneous lysis of cells prepared by the BDB procedure, noted by earlier investigators, can readily be prevented either through the use of lesser amounts of BDB or through an increase in the concentration of the coating protein. To attain the desired protein concentration of 18 mg in the 24 ml of coating solution it is often desirable to use some protein that will not participate in the antigen-antibody reaction as a "filler." For example, in studies involving y-globulin as coating antigen we have used 12 mg of y-globulin (antigen) and 6 mg of bovine serum albumin in 24 ml. Coated cells remain in usable condition for at least 5-7 days at 4° . The useful life-span of either tanned or BDB cells can be extended indefinitely by mild fixation with formaldehyde, glutaraldehyde, or other agents.11 Freezing or lyophilization of such preparations is possible. Since these procedures entail some disadvantages and may be of more restricted utility, they will not be described here. Tube Assay. It is convenient to use 10 x 75 mm disposable tubes and 96-place racks to support these tubes. Serial 2-fold dilutions of the sera to be tested should be prepared in PBS containing 1% normal serum, leaving 0.5 ml amounts in each tube. As previously noted, separate pipettes should be used until a dilution of about 1:1,000 is reached. One then adds 0.05 ml amounts of a 2.5% suspension of the coated red cells, mixes the contents of the tubes thoroughly by vigorous shaking of the rack, and incubates for 2 hr at room temperature. One basic control to be included in the assay is a tube containing diluent and test cells (negative control); another is a tube containing antiserum in the highest concentration and cells coated with an irrelevant antigen (negative control). Another desirable control is discussed later as part of the description of the HI test. It is also advisable to select an antiserum to be used as the standard that will indicate variations in the sensitivity of different batches of coated cells (positive control). Hemagglutination patterns are read from below. Absence of agglutination is indicated by the formation of a compact button or a sediment resembling a doughnut, with smooth and regular edges. Agglutination, in contrast, is indicated by the presence of a mat of cells that covers the bottom. If agglutination is very strong this mat develops folds and contracts, leading to a highly irregular shape. 11T. Suzuki, S. Tanaka, and Y. Kawanishi,lrnmunochernistry 11, 391 (1974).

[30]

PASSIVE HEMAGGLUTINATION

461

Plate Assay. Dilutions of the antiserum can be prepared in the wells of the plates if the required spiral or loop diluting devices are available; otherwise they can be prepared in tubes as described above and 0.05-0.06 ml amounts can be transferred to wells using calibrated dropper pipettes (0.05 ml), automatic pipetting devices, or 2 drops from disposable Pasteur pipettes (0.06 ml). One then adds to each well a matching volume of a 0.5% suspension of coated erythrocytes in PBS containing 1% normal serum. The contents of the wells are mixed by vigorous rotating of the tray while it rests on the laboratory bench. The results are read after 1,52 hr of incubation at room temperature. In the V-shaped cups nonagglutinated cells slide to the bottom (tip of the cone) and, as seen from above, appear as a compact button. Agglutinated cells adhere to each other and to the sides of the wells, forming a matlike pattern. Controls and Interpretation It should be stressed again that HA is a semiquantitative test for antibody that, applied to antisera that contain largely IgG antibodies, yields results in relatively good agreement with precipitin assays and binding tests but provides greater sensitivity than the former and greater simplicity than the latter. Essential controls have been described in the preceding paragraphs. One must keep in mind that antigenic impurities in the coating antigen and the presence of corresponding antibodies in antisera may lead to confusion. Since effective coating is generally highly dependent on relatively high concentrations of antigen in the coating solution, we find it advisable to employ the minimal concentration of coating antigen that will assure acceptable, though not necessarily maximal, sensitivity. The use of "filler" protein to meet the requirements of the BDB procedure has been discussed. R e v e r s e Passive Hemagglutination This procedure employs erythrocytes coated with a preparation of purified antibody. It is suitable for the detection of trace amounts of antigen and its semiquantitative measurement. For example, SRBC coated with anti-y-globulin antibody can be used to detect 3,-globulin in concentrations of 1-10 ng/ml. In this reaction the antigen in solution is the agent that links the antibody-coated cells into aggregates. Since most protein antigens are multivalent with respect to their antigenic determinants they are more effective ligands than divalent IgG antibodies. A major shortcoming of the method is the inhibition of agglutination in the presence of excess antigen (prozone) which may occur when cross-linkage is prevented by the excess of antigen binding to all available antibody combining sites on the test cells.

462

IMMUNOASSAYS

[30]

Specific antibody is generally prepared by its absorption to and elution from solid phase immunoadsorbants. Ideally it should be free not only of nonantibody serum proteins but also of antibodies against contaminating antigens. Using the BDB procedure previously described one coats SRBC with a mixture of purified antibody and "filler" protein, such as bovine serum albumin, in a proportion such as 6 mg of antibody plus 12 mg of albumin per 24 ml of coating solution. The optimal amount of antibody must be determined for each preparation. The test itself is done exactly in the same manner as HA. Because the end point in this procedure is not always reached in a single dilution step from complete agglutination to its total absence, it is advisable to include a standard with known concentrations of antigen. It is not difficult to estimate end points by referring to the standard. Hemagglutination Inhibition

Principles The hemagglutination-inhibition test detects and measures antigen with extremely high sensitivity. It is based on the principle of competition for a finite and limiting amount of antibody by antigen in two forms: that coating the indicator red cells and that present in a solution that is to be tested for its antigen content. The sensitivity of the test for protein antigens of molecular weights 50,000 to 200,000 is generally about 0.1 to 1.0 /zg/ml. For smaller molecules, such as morphine (M = 285) it is about 100 to 1000 times more sensitive. The reason for this difference stems from the fact that 2 mol of antigen are required to saturate 1 mol of divalent antibody; thus the relative inhibitory efficiency of antigens (haptens) are inversely proportional to their molecular weights.

Procedure A preliminary HA test is required to determine the HA titer of the antiserum that is to be used. It is advisable to use in the HI test a dilution of the antiserum that is 4-8 times less than that corresponding to its HA titer. An amount of this dilution sufficient for a day's work is prepared in PBS containing 1% normal rabbit serum. It is kept cold until use, and it is not recommended to store it for use on subsequent days. If the tube assay is to be employed, 0.25 ml amounts of serially diluted antigen, made in the same diluent, and 0.25 ml amounts of the selected antiserum dilution are mixed and incubated for 30 min at room temperature. The test cells (0.05 ml of 2.5% suspension) are added, the tubes are shaken virogously, and the agglutination patterns are read after 1.5-2 hr

[30]

PASSIVE HEMAGGLUTINATION

463

of incubation at room temperature. In the plate assay 0.025 ml or 0.03 ml amounts of antigen dilutions and of antiserum, respectively, are used and 0.05 ml of 0.5% coated cells are used. A series of dilutions of antigen of known concentration serve as control and standard and are to be included in each day's titrations. The end point is the highest dilution of the antigen solution that causes complete inhibition of HA. The minimal amount of antigen required for total inhibiton is indicated by the standard. Controls should be included to rule out agglutination of the coated cells by the antigen solution (omit the antiserum), and to rule out inhibition of agglutination by nonimmunological activities of the test solution, such as proteolysis. For this purpose test cells coated with an unrelated antigen and the corresponding antiserum can be used. Optimal conditions for HI call for the use of (a) highly purified antigen in the coating of the red cells; (b) antiserum that in the dilution in which it is to be used is free of effective (agglutinating) amounts of antibodies against residual antigenic contaminants of the coating solution; and (c) dilution of the antiserum to near its agglutination end point to assure both maximal specificity and sensitivity. With these precautions it is generally possible to test for the presence and amount of a given antigen in crude preparations, and thus to have a convenient assay for a given antigen in biological fluids or to monitor progress toward purification of an antigen. Passive Hemolysis The passive hemolytic (PH) test is an extension of HA in which an additional reagent, complement, causes lysis of test cells that is mediated by antibody against the coating antigen. In some instances this modification leads to further enhancement of sensitivity, but this advantage is balanced by a number of complicating factors. Dominant among these is the introduction of complement as an additional variable. Choice of the hemolytic test also imposes limits on the method for linking antigen to the red cells, since some procedures enhance and others diminish the susceptibility of erythrocytes to lysis. Although PH is not widely used in assays for serum antibodies it provides a most useful method for the enumeration and identification of lymphoid cells that produce and secrete antibodies against a given antigen. In a further extension, as passive reverse hemolysis, it is a test applicable to enumeration, identification, and isolation of cells that secrete a given antigen. The basic principle common to the several forms of the test calls for mixing a suspension of cells to be tested with a suitable excess of coated red cells (targets), to incubate the mixture under conditions that prevent movement of the cells, and to score circular clear (hemolytic)

464

IMMUNOASSAYS

[30]

plaques surrounding individual test cells. The complement is added in some procedures to the initial incubation mixture; in others it is added after an initial incubation period. The plaque assay, originally described for cells secreting antibodies against erythrocytes, lz has been modified by numerous workers to allow observations on cells that secrete antibodies specific for antigens that can be effectively attached to red cells. One method and one example only will be described here, namely, an assay for spleen cells making antibody against the trinitrophenyl determinant. Alternate procedures are well described elsewhere. 13

Materials and Reagents Trinitrophenylated SRBC are prepared as originally described? In brief, 20 mg of trinitrobenzene sulfonate are dissolved in 7.0 ml of 0.28 M cacodylate buffer, pH 6.9; 2.0 ml of washed 50% SRBC in complement buffer (5,5-diethylbarbituric acid, 0.575 g; sodium 5,5-diethylbarbiturate, 0.375 g; CaCI~, 0.017 g; MgClz, 0.048 g; NaCI, 8.5 g; distilled water to 1 liter) are added while the mixture is gently stirred. After 10 min at room temperature one adds 6.0 ml of cold complement buffer, harvests the cells by centrifugation at 750 g for 10 min, suspends them in 12 ml of complement buffer containing 7.5 mg of glycylglycine, and washes them three additional times in complement buffer. A 10% suspension is then made in the same buffer. It should be remembered that the reaction and product are sensitive to light. The assay can be conveniently done in glass or plastic petri plates (60 mm) containing about 4 ml of 0.8% agarose in complement buffer. Guinea pig complement is readily available commercially and generally does not require absorption. Lymphoid cells are prepared by teasing spleens or lymph nodes in cold tissue culture medium buffered with Hepes. If the "indirect" plaque assay is to be done, an antiserum specific for IgG of the antibody-secreting cells is also required.

Procedure The freshly teased and uniformly suspended lymphoid cells are adjusted to some convenient starting concentration, such as 107 viable cells per milliliter. A series of dilutions are made, and 0.1 ml amounts are transfered into 10 × 75 mm tubes containing 0.8 ml of 0.8% agarose at 12 N. K. Jerne, C. Henry, A. A. Nordin, H. Fuji, A. M. C. Koros, and I. Lefkovits, Transplant Rev. 18, 130 (1974). in W. J. Herbert, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 2nd ed., Vol. 1, Ch. 20, Blackwell, Oxford, 1973.

[30]

PASSIVE HEMAGGLUTINATION

465

45 °. Aliquots of 0.1 ml of 10% coated erythrocytes are quickly added, and the contents of the tubes are poured over the bottom layer of agarose in the prepared plates. Incubation for 1 hr at 37° in a tissue culture incubator is followed, in the "direct" assay, by a second incubation for 1 hr at 37°, in air, after the addition of 1 ml of guinea pig complement diluted 1:10 in complement buffer. This procedure develops plaques surrounding cells that make hemolytically efficient antibodies, such as IgM. For more comprehensive count of antibody-secreting cells, an incubation for 1 hr at 37° in air is interpolated between the two incubations mentioned. In this additional step 1 ml of a suitable dilution of an anti-IgG serum is placed on the top layer of agarose; it is removed prior to the addition of complement. This assay yields an estimate of total antibody-secreting cells. Plaques are counted under low magnification under a dissecting microscope, and results are expressed as the number or proportion of secreting cells per total cells applied. An essential control is one for the specificity of plaque formation. In the example cited this consists of plates in which trinitrophenylated serum albumin (100 ~g/ml) is incorporated into the top layer agarose as a specific inhibitor. Plaques forming in these plates are presumed to be antiSRBC, and correction is made by subtracting their number. Another essential control omits complement. This description of the plaque assay is lacking in some details but it will suffice to introduce the reverse hemolytic plaque assay, which probably will be more relevant to the readers' interests. Reverse Hemolytic Plaque Assay This procedure is of great potential utility for the identification and enumeration of cells that secrete an antigenic product. Patterned after the assay just described, it employs similar techniques with two major differences: The coating for the indicator red cells consists of purified antibody, as in the reverse passive hemagglutination previously described~ and an antiserum against the secretion product is used as the developing agent. 14,15 In an application of the method to the detection of murine hepatocytes that secrete albumin, the authors 14 employed SRBC coated with purified antibody against mouse serum albumin and plated such cells together with varying numbers of teased liver cells exactly as described for the hemolytic plaque assay. After the initial incubation, anti-mouse serum albumin x4G. A. Molinaro,E. Maron, W. C. Eby, and S. Dray,Eur. J. lmmunol. 5, 771 (1975). 15W. C. Eby, C. A. Chong, S. Dray, and G. A. Molinaro,J. Immunol. 115, 1700(1975).

466

IMMUNOASSAYS

[30]

antiserum in a suitable dilution was added for 1 hr at 37° in air. This reagent was decanted, and incubation at 37° in air was continued for another hour in the presence of 1.0 ml of guinea pig complement 1:10. The resulting plaques are counted, and the results are expressed in terms of number of secreting cells per number of viable cells. Rosette Test for Cell M e m b r a n e Antigens In contrast to the method just described, the rosette test is applicable to antigens that are membrane-bound or integral components of the cell membrane. It has been used to detect membrane-bound Ig on lymphocytes and will be described in terms of this model, but has unquestionably wider applicability. The principle of the test is based on the specific binding of antibody-coated erythrocytes to the corresponding antigens on the cell membrane as observed by the specific adherence of such indicator cells. In general, adherence of at least three red cells is the minimal criterion for a rosette. Cells that actively secrete the antigen in question may not form rosettes and, in fact, may inhibit rosetting through specific binding of the antibody on the red cells by the secreted antigen. It is believed that rosetting matches or exceeds fluorescent antibody techniques in sensitivity for the detection of membrane antigens. The antibody-coated red cells are prepared as previously described. It is particularly important for this procedure that the indicator red cells are absolutely free of clumps. The sensitivity of coated cells can be assayed by reverse passive hemagglutination if, as in the model under consideration, the antigen is available in soluble form. The cells under study are washed in suitable tissue culture medium or other buffered solution and suspended at a concentration of 107 per milliliter in the same diluent to which serum has been added (usually 1% fetal calf serum). A small volume (50-100/zl) of the cell suspension is placed in a 10 x 75 mm disposable tube. The addition of an equal volume of 1% coated red cells results in a mixture that contains about 25 red cells per lymphocyte. Linkage of antibody on the red cells to the corresponding antigen determinant on the surface of the lymphocyte results in the formation of a "rosette" or lymphocyte surrounded by red cells. The mixing of cells and incubation for at least 1 hr are done in an ice bath. The tubes are then centrifuged very briefly (1 min at 1000 g), and a drop of dye is added to tint the lymphocytes (e.g., crystal violet or brilliant cresyl blue). The mixture is then aspirated four or five times with a Pasteur pipette and examined in a hemacytometer chamber at about 400 x. A cell is scored as a rosette if it is surrounded by three or more adherent erythrocytes, and usually 300 cells are counted.

[31]

MICRO COMPLEMENT FIXATION

467

[31] Quantitative Micro Complement Fixation: Serologic Properties of Pig Liver Carboxylesterase

By

LAWRENCE LEVINE, AUGUSTIN BAER, and WILLIAM P. JENCKS

Changes in quaternary structure of macromolecules are sometimes accompanied by changes in their serologic properties. Some of these altered serologic activities reflect spatial rearrangement of the antigenic determinants whose conformations depend on the structural integrity of other parts of the molecule, or changes in density of such antigenic determinants. 1,2 Quantitative micro complement fixation 3 is a most sensitive serologic method for detection of conformational changes in macromolecules. Denaturation of D N A and polyribonucleotides, 4-s altered conformation of lactic dehydrogenases, ° hemoglobin, 1°-12 myoglobin, 1° collagen, 13 lysozyme, 14 aspartate transcarbamylase, 3,15 pepsinogen and pepsin, ~e-~a carboxypeptidase, 2° and S-100 brain protein zl,zz have been detected and 1 M. Reichlin, M. Hay, and L. Levine, lmmunochemistry 1, 21 (1964). z M. R. Bethell, R. von Fellenberg, M. E. Jones, and L. Levine, Biochemistry 7, 4315 (1%8). 3 E. Wasserman and L. Levine, J. Immunol. 87, 290 (1961). 4 L. Levine, E. Wasserman, and W. T. Murakami, lmmunochemistry 3, 41 (1966). 5 W. T. Murakami, H. Van Vunakis, L. Grossman, and L. Levine, Virology 14, 190 (1%1). 6 D. Stollar, L. Levine, H. I. Lehrer, and H. Van Vunakis, Proc. Natl. Acad. Sci. U.S.A. 48, 874 (1%2). r L. Levine, Fed. Proc. 21, 711 (1%2). a B. D. Stollar, in "The Antigens" (M. Sela, ed.), Vol. 1, pp. 1-85. Academic Press, New York, 1973. 9 R. D. Cahn, N. O. Kaplan, L. Levine, and E. Zwilling, Science 136, 962 (1962). 10 M. Reichlin, M. Hay, and L. Levine, Biochemistry 2, 971 (1%3). 11 M. Reichlin, Adv. lmmunol. 20, 71 (1975). 12 M. Reichlin, M. Hay, and L. Levine, Immunochemistry 2, 337 (1965). lap. F. Davison, L. Levine, M. P. Drake, A. A. Rubin, and S. Bump, J. Exp. Med. 126, 331 (1%7). 14 R. von Fellenberg and L. Levine, Immunochemistry 4, 363 (1967). 15 R. von Fellenberg, M. R. BetheU, M. E. Jones, and L. Levine, Biochemistry 7, 4322 (1968). 16 H. Van Vunakis, H. I. Lehrer, W. Allison, and L. Levine, J. Gen. Physiol. 46, 589(1%3). lr j. Gerstein, H. Van Vunakis, and L. Levine, Biochemistry 2, 971 (1%3). is j. Gerstein, L. Levine, and H. Van Vunakis, Immunochemistry 1, 3 (1964). 19 T. G. Merrett, L. Levine, and H. Van Vunakis, Immunochemistry 8, 201 (1971). 2o H. I. Lehrer and H. Van Vunakis, Immunochemistry 2, 255 (1965). 21 D. Kessler, L. Levine, and G. D. Fasman, Biochemistry 7, 758 (1968). 22 p. S. Dannies and L. Levine, J. Biol. Chem. 246, 6276 (1971).

METHODS IN ENZYMOLOGY,VOL. 70

Copyright© 1960by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181970-1

468

IMMUNOASSAYS

[31]

I00

80

60

2O

0.01

0.05

0.1

0.2

/u.g Carboxylesterose FIG. 1. Complement (C) fixing activity of native and dissociated molecules mixed in varying proportions: 100% dissociated ([~); 80% dissociated, 20% native (A); 60% dissociated, 40% native (©); 40% dissociated, 60% native (11); 20% dissociated, 80% native (A); 100% native (@). [From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission.]

quantified by micro complement fixation. In addition, the extreme sensitivity of the method has been exploited to measure molecular changes induced by evolutionary processes. ~-z7 The principle and the procedures for performing the micro complement fixation test have been presented in detail previously, z8"20Here, we record the use of micro complement fixation to measure the rates of dissociation and the equilibrium constants for dissociation of pig liver carboxylesterase (EC 3.1.1.1) as a function of pH and salt concentration. The pig liver carboxylesterase preparation and some of its physical properties have been described by Barker and Jencks. 3° Antibodies to the carboxylesterase preparation were obtained by immunization of rabbits via the toe pads and muscles with 2 mg of enzyme emulsified in complete 23 A. C. Wilson, N. O. Kaplan, L. Levine, A. Pesce, M. Reichlin, and W. S. Allison, Fed. Proc. 23, 1258 (1964). 24 A. H. Tashjian, Jr., L. Levine, and A. E. Wilhelmi, Endocrinology 77, 563 (1965). 25 L. Nonno, H. Herschman, and L. Levine, Arch. Biochem. Biophys. 136, 361 (1969). E. M. Prager and A. C. Wilson, J. Biol. Chem. 246, 7010 (1971). 27 N. Arnheim, in "The Antigens" (M. Sela, ed.), Vol. 1, p. 377. Academic Press, New York, 1973. 2s L. Levine and H. Van Vunakis, this series, Vol. 35, p. 928. 29 L. Levine, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), p.22.1. Blackwell, Oxford, 1973. 30 D. L. Barker and W. P. Jencks, Biochemistry 8, 3879 (1969).

[31]

MICRO COMPLEMENT FIXATION

469

90 8O

g

70

-.,7.

u_

60

¢.)

E

5O

.E K O

40 3O 20 0

I 20

I 40

I 60

I

I

I

I

I

I

I00

80

60

40

20

0

%

%

I 80

I I00

Notive

Dissocioted

FiG. 2. Maximum complement (C) fixing activity of native and dissociated ~holecules mixed in varying proportions. [From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission.]

• •

8

x~x=

I00

=

o

75

"2_

N gl ILl

0

o

6

50 .-

O9

5

Z

25 ~.

0 3

I 2

I 3

I 4

I 5

I 6

I 7

I 8

pH FIG. 3. Complement-fixing activity (×) after incubation of esterase (0.1 mg/ml) for 40 min at indicated pH. The sedimentation coefficient (s=o) as a function of pH for 2 mg/ml esterase. [From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission.]

470

IMMUNOASSAYS

[31]

Freund's adjuvant. At monthly intervals, the rabbits were boosted intramuscularly with 1 mg of enzyme, again emulsified in complete Freund's adjuvant. Immune sera were collected 1 week after each booster injection. The antiserum used in the immunochemical application described here was collected 1 week after the sixth boost. Estimation of Native Carboxylesterase Molecules Varying quantities of carboxylesterase were incubated with a constant amount of rabbit anti-carboxylesterase (1.0 ml of a 1:7000 dilution) and a constant level of complement to give a complete complement fixation curve and a maximum of 80% complement fixed with 0.05/zg of carboxylesterase. Pig liver carboxylesterase (2 mg/ml) undergoes a decrease in sedimentation rate over a relatively narrow pH range (pH 4-5) that reflects dissociation of the whole molecule into subunits. The serologic activities of these whole and dissociated molecules are changed. Whereas 80% of the complement is fixed with the whole molecules, only about 20% is fixed with the dissociated molecules. Maximum complement fixation is still observed with about 0.05/zg of the dissociated protein. A decrease in complement fixing properties accompanies dissociation. In order to relate dissociation and decreased complement fixing activity, dissociated molecules were mixed with the whole molecules in varying proportions, and complement-fixing activities of the admixtures were determined (Fig. 1). The whole molecules reacted more effectively than mixtures of the dissociated and native molecules. Moreover, the decrement of maximum complement fixation with the several mixtures of whole and dissociated molecules reflected their percentage, in weight. This decrement probably results from inhibition by dissociated molecules of the complement fixed with whole molecule s; i.e., the antigenic determinants of the dissociated molecules are recognized by the antibodies, but lattice formation requisite for complement fixation zl is affected by the changed density of these antigenic determinants. With all these mixtures, maximum complement fixation is still obtained with about 0.05/zg of esterase. Maximum complement fixation, as a function of the percentage of whole and dissociated molecules, is shown in Fig. 2. Such a calibration curve was used to estimate the number of native molecules in unknown carboxylesterase solutions. The sedimentation coefficients and the percentage of native molecules, as measured serologically, remaining in carboxylesterase preparations incubated for 30 min at 20° at various pH values are shown in Fig. 3. 31 A. G. Osier and B. M. Hill, J. Immunol. 75, 137 (1955).

IxlO - 9

I x l 0 -10

Ixl0 -u

o/

/

I xl0 -i2 N ~E Cr Y

o I x l 0 -13

I xlO-14

I x l 0 -15 0

I xlO -16

4

0

0

0

I

I

i

I

I

I

I

5

6

7

8

9

10

II

pH FIG. 4. Equilibrium constants, as measured by complement (C) fixation, f o r dissociation o f pig liver carboxylcstcrasc as a function o f pH. [From L. Levinc, A. Baer, and W. P.

Jcncks, Arch. Biochem. Biophys., in press (1980), with permission.]

472

IMMUNOASSAYS

[31]

There is a sharp decrease in sedimentation properties between pH 4 and 5. There is also a sharp decrease in the content of native molecules over this pH range. In this experiment and in all subsequent experiments on dissociation of carboxylesterase, further dissociation or association during the course of serologic analysis was stopped, or at least minimized, by dilution of the reaction mixtures into ice-chilled Tris buffer (10 mM Tris pH 7.4, 0.14 M NaCI containing 0.1% gelatin) to give a carboxylesterase concentration of 0.1 ~g/ml (in some cases to 0.05 ~g/ml), and immediate addition of the diluted enzyme solution into the antiserum and complement for the complement fixation assay. The combination of dilution to pH 7.4 at low temperature and the interaction of the antibodies with the carboxylesterase, which is very rapid, effectively prevents continuous dissociation. While this is difficult to prove unequivocally, the following considerations attest to its validity. Antibodies to pig liver carboxylesterase quantitatively precipitate the enzyme in the regions of the equivalence and excess antibody zones of the precipitin curve. All the catalytic activity of the carboxylesterase is recovered in these immune precipitates, suggesting that the enzyme is catalytically active in the associated state. In our complement fixation procedure, in which the concentrations of antibodies and carboxylesterase are around 10-l° M , the reaction of antibody with the associated molecule would be sufficiently rapid to inhibit continuous dissociation of the carboxylesterase. It should be recalled that complement fixation analyses are performed at pH 7.2-7.4 where the associated state is most stable, and at 2-4 °, where the rate of dissociation is also relatively slow. Determination of Equilibrium Constants Studies by Junge and Krisch 32 and by AuneY reported in 1973, of the subunits of pig liver carboxylesterase suggest that the whole enzyme is composed of three subunits of about 60,000 molecular weight. Thus, our values for the equilibrium constants for dissociation were calculated according to the equation K~q = [T]3/[W] where T and W refer to the molar concentrations of third and whole molecules. In experiments designed to estimate equilibrium constants, varying quantities of carboxylesterase were incubated at specified conditions for 24 and 48 hr. At the time of measurement, the carboxylesterase was di32 W. Junge and K. Krisch, Mol. Cell. Biochem. 1, 41 (1973). aa K. Aune, Arch. Biochem. Biophys. 156, 115 (1973).

[31]

MICRO COMPLEMENT

FIXATION

473

TABLE I PERCENTAGE OF COMPLEMENT FIXATION AND EQUILIBRIUM CONSTANTS FOR DISSOCIATION 24 AND 48 HR AFTER INCUBATION IN 10 m/~ TRIS, p H 7.2, AT 35 °a

% Complement fixation

% Whole molecules

Keq (10-1° M 2) W ~ 3T

Carboxylesterase (/,~g/ml)

24 Hr ~

48 Hr ~

24 H r b

48 Hr e

24 Hr

48 Hr

1.0 2.0 3.0

43 60 59

49 60 63

33 54 53

35 49 53

8.4 6.9 18.2

7.6 13.5 18.0

a From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission. b 76% maximumcomplement fixation with untreated carboxylesterase in these analyses. c 79% maximumcomplement fixation with untreated carboxylesterase in these analyses.

luted into ice-chilled Tris buffer (pH 7.4, 10 m M Tris, 0.14 M NaC1 containing 0.1% gelatin) to contain 0.15, 0.1, or 0.05/zg of esterase per milliliter and assayed for serologic activity. E v e n at a p H around neutrality and at low ionic strength, equilibrium has been reached by 24 hr, so that in most of our experiments only one time o f incubation (24 hr) was used. To illustrate the data generated in these complement fixation experiments and to measure equilibria after 24 and 48 hr of incubation, an experiment was performed to estimate equilibrium constants in 10 m M Tris, pH 7.2, at 35 °. At 24 and 48 hr, aliquots were diluted to contain 0.1/zg/ml, and complete complement fixation curves, similar to those shown in Fig. 2, were obtained. At the same time, the complement fixation o f a carboxylesterase solution not previously incubated was obtained in order to normalize day-to-day variation in the technique. The percentage of native molecules in the carboxylesterase preparations was calculated from their normalized maximum complement fixation values and the calibration curve shown in Fig. 2. The equilibrium constants at 24 hr (1.1 x 10-15 M 2) and at 48 hours (1.3 × 10-15 M z) are in good agreement (Table I). The equilibrium constants for dissociation as a function of pH are shown in Fig. 4. R a t e of Dissociation of Pig Liver C a r b o x y l e s t e r a s e As was expected from the experiments in which complement fixation was measured with varying mixtures of whole and dissociated esterase (Fig. 1), a decrease in complement fixation is observed with increased dissociation. Moreover, the point at which maximum complement fixation is obtained, around 0.05/~g, did not change. Such a series o f complement

474

IMMUNOASSAYS

[31]

I00

80

/"~\ //

\\

~ ' \ \ o

u

40

2O

0.01

0.05

0.1

0.2

# g Corboxylesterose

FIG. 5. Complement-fixing activity of carboxylesterase after incubation for varying periods of time in 10 mM sodium acetate buffer (pH 4.5). No incubation (1); 5 min (A); 10 min (11); 20 min (O); 40 min (A); 60 min (D); 120 min (x). [From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission.]

I00 8O 6O

=

40

Q

1~

20

I0

I

0

~l

I

I

20

40

I

I

60 80 Time (Minutes)

I

I00

I

I

120

140

FIG. 6. Dissociation of carboxylesterase at pH 4.0 (©), pH 4.8 (O), and pH 5.5 (&) as measured by complement (C) fixation. [From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1950), with permission.]

[31]

MICRO COMPLEMENT FIXATION

475

1.0

0.5 0.3

0.1

T" .S

0.05

0.03 T

/ 0.01

O.OO5 O.003

0.001 4.0

I

I

I

I

I

I

I

5.0

6.0

7.0

8.0

9.0

I0.0

I1.0

pH

FIG. 7. Effect o f p H on the rates of dissociation of carboxyesterase as measured by complement (C) fixation. [From L. Levine, A. Baer, and W. P. Jencks,Arch. Biochem. Biophys., in press (1980), with permission.]

fixation curves was obtained when carboxylesterase (10/~g/ml) was incubated at pH 4.5 for various periods of time (Fig. 5). Therefore, a procedure for measuring the rate of dissociation by removing 0.05/.~g from the reaction mixture and adding it directly to the complement fixation system was used. This level of esterase gives maximum complement fixation and reflects the percentage of whole molecules remaining. The percentage of complement fixation and the percentage of native molecules calculated from that value during dissociation of carboxylesterase at pH 4.0, 4.8, and 5.5 are shown in Table II. The rates of dissociation are shown in Fig. 6. The rates of dissociation as a function of pH are shown in Fig, 7.

0

,% Z <

@ @ e9

~,

[< <

,4

< ~ [.- ~ < c~ 0 Z

e b<

~5 O

<

i°

476

0

0

a~

477

478

1MMUNOASSAYS

[31]

IxlO-II

ixlO -Iz

ixlO-13 ~E

o" o

IxlO -14

ix j0 -15

ixlO-16

I

0

0.1

I

I

0.2 0.5 Solt M

I

I

0.4

0.5

FIG. 8. Equilibrium constants for dissociation of carboxylesterase as a function of varying concentrations of KBr (O) and LiBr (Q). [From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission.]

Effects of Salts on Dissociation

As can be seen from the data in Fig. 7, carboxylesterase dissociates at a slow rate even at p H 7.4. In the presence o f salt, this dissociation is increased, a° Dissociation in the presence o f salt was also measured by the complement fixation procedure. In Fig. 8 are shown the equilibrium constants for dissociation in varying concentrations of K B r and LiBr at p H 7.2 (10 mM Tris). The effects o f 0.4 M concentrations o f several salts at p H 7.2 on the equilibrium constants for dissociation are shown in Table III.

[31]

MICRO C O M P L E M E N T

FIXATION

479

TABLE III EFFECT OF VARIOUS SALTS ON DISSOCIATION EQUILIBRIA OF CARBOXYLESTERASEa

•Salt, 0.4 M b

Keq (Mz)

KF KCI KBr KI LiBr NaF NaCHsCOO NaCI NaBr NaCIO4 NaI (CHz)4NCI NI-I4CI KCI CsCI NaCI LiCI

4.6 × I0-~4 7.5 × 10-~4 1.2 × 10-~a 7.6 x 10-~a 1.4 x l 0 -~2 1.9 × l0-15 4.4 × 10-14 9.9 × 10-14 1.3 x 10-13 2.6 x 10-13 5.7 x 10-13 3.5 × 10-15 4.3 x 10-14 6.8 × 10-14 8.4 )< 10-14 9.2 x 10-14 2.3 × 10-13

a From L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980), with permission. b In 10 mM Tris, pH 7.2, containing 0.01% bovine albumin. Q u a t e r n a r y S t r u c t u r e of the Pig L i v e r C a r b o x y l e s t e r a s e W e also h a v e u s e d C fixation to m e a s u r e e q u i l i b r i a a n d rates o f dissoc i a t i o n as a f u n c t i o n o f t e m p e r a t u r e . T h e c o n c l u s i o n s r e a c h e d f r o m t h e s e s t u d i e s h a v e b e e n r e p o r t e d .34 T h e d e p e n d e n c e o f the d i s s o c i a t i o n equilibria o n p H w a s c o n s i s t e n t with d i s s o c i a t i o n r e a c t i o n s i n v o l v i n g the addit i o n to t w o p r o t o n s p e r s u b u n i t , a p H - i n d e p e n d e n t d i s s o c i a t i o n , a n d a diss o c i a t i o n u p o n the loss o f o n e p r o t o n p e r s u b u n i t . T h e rate c o n s t a n t s for d i s s o c i a t i o n w e r e c o n s i s t e n t with t e r m s first o r d e r in h y d r o g e n a n d hyd r o x i d e i o n s a n d a p H - i n d e p e n d e n t path. T h e e q u i l i b r i u m c o n s t a n t s in the r a n g e 3 - 3 5 ° at p H 7.2 e x h i b i t e d n o d e p e n d e n c e o n t e m p e r a t u r e ; the assoc i a t i o n r e a c t i o n was e n t r o p y - d r i v e n with AS = 68 cal mo1-1 K -~. T h e rate c o n s t a n t s for the p H - i n d e p e n d e n t d i s s o c i a t i o n f o l l o w e d A H = 6 kcal mo1-1. T h e o r d e r o f e f f e c t i v e n e s s o f c o n c e n t r a t e d salts in p r o m o t i n g d e n a t u r a t i o n w a s c o r r e l a t e d with t h e i r effect o n the a c t i v i t y coefficient o f acet y l t e t r a g l y c i n e e t h y l e s t e r a n d s u g g e s t e d that p e p t i d e g r o u p s b e c a m e m o r e exposed upon dissociation. 34 L. Levine, A. Baer, and W. P. Jencks, Arch. Biochem. Biophys., in press (1980).

PREVIOUSLY PUBLISHED ARTICLES

481

Previously Published Articles from

Methods in Enzymology Related to Sections I - I I I Related to Section I Basic Principles and General Methods Vol. V [3]. Preparative Electrophoresis. M. Bier. Vol. VI [8]. Preparation of Lamb Brain Phospbodiesterase. J. W. Healy, D. Stollar, and L. Levine. Vol. VI [119]. Two-Dimensional Immunodiffusion. D. Stollar and L. Levine. Vol. v m [s]. Immunological Methods for Characterizing Polysaccharides. G. Schiffman. Vol. IX [80]. L-Ribuiokinase. N. L. Lee and E. Engiesberg. Vol. X [57]. Beef Heart TPNH-DPN Pyridine Nucleotide Transhydrogenases. N. O. Kaplan. Vol. X [106]. Preparation and Use of Antisera to Respiratory Chain Components. S. D. Davis, T. D. Mehl, R. J. Wedgewood, and B. Mackler. Vol. X [107]. Antibody against F1. J. M. Fressenden and E. Racker. Vol. XI [73]. Amidination. M. L. Ludwig and M. J. Hunter. Vol. XI [75]. Bifunctional Reagents. F. Wold. Vol. XI [91]. Immunological Techniques (General). R. K. Brown. Vol. XlIB [173]. Purine- and Pyrimidine-protein Conjugates. S. M. Beiser, S. W. Tanenbaum, and B. F. Erlanger. Vol. XIIB [174]. Preparation and Assay of Nucleic Acids as Antigens. O. J. Plescia. Vol. XHB [175]. Preparation of Nucleoside-Specific Synthetic Antigens. M. Sela and H. Ungar-Waron. Vol. XIIB [176]. Immunological Detection of Ribonucleic Acids by Agar Diffusion. F. LaCORr.

Vol. XVHA [50]. L-Tryptophan 2,3-Dioxygenase (Tryptophan Pyrrolase) (Rat Liver). W. E. Knox, A. Yip, and L. Reshef. Vol. XVIHA [93]. Preparation and Properties of Antigenic Vitamin and Coenzyme Derivatives. J.-C. Jaton and H. Ungar-Waron. Vol. XVIIlB [189]. Preparation and Properties of Antigenic Vitamin and Coenzyme Derivatives. J.-C. Jaton and H. Ungar-Waron. Vol. XXI [19]. The Purification o f ~ Protein and Exonuclease Made by Phage h. C. M. Radding. Vol. XXH [22]. Water-Soluble Nonionic Polymers in Protein Purification. M. Fried and P. W. Chun. Vol. XXV [54]. Amidination. M. J. Hunter and M. L. Ludwig. Vol. XXV [57]. Bifunctional Reagents. F. Wold. Vol. XXVIII [16]. Carbohydrate Antigens: Coupling of Carbohydrates to Proteins by Diazonium and Phenylisothiocyanate Reactions. C. R. McBroom, C. H. Samanen, and I. J. Goldstein. Vol. XXVIII [17]. Carbohydrate Antigens: Coupling of Carbohydrates to protein by a Mixed Anhydride Reaction. G. Ashweli.

482

PREVIOUSLY PUBLISHED ARTICLES

Vol. XXVlII [18]. Carbohydrate Antigens: Coupling of Carbohydrates to Proteins by Diazotizing Aminophenylflavazole Derivatives. K. Himmelspach and G. Kleinhammer. VoI. XXIX [14]. Identification of Viral Reverse-Transcriptase. E. M. Scolnick and W. P. Parks. Vol. XXIX [lga]. Purification and Detection of Reverse Transcriptase in Viruses and Cells. D. L. Kacian and S. Spiegelman. Vol. XXX [59]. Immunoadsorption of Ovalbumin Synthesizing Polysomes and Partial Purification of Ovalbumin Messenger RNA. R. T. Schmike, R. Palacios, D. Sullivan, M. L. Kiely, C. Gonzales, and J. M. Taylor. Vol. XXX [61]. DNA- and RNA-Directed Synthesis in Vitro of Phage Enzymes. P. Herdich and M. Schweiger. Vol. XXX [65]. Isolation on Cellulose of Ovalbumin and Globin in mRNA and Their Translation in Ascites Cell-Free System. G. Schutz, M. Beato, and P. Feigelson. Vol. XXXII [6]. Use of Antibodies for Localization of Components on Membranes. W. C. Davis. Voi. XXXIV [2]. General Methods and Coupling Procedures. J. Porath. Vol. XXXIV [7]. Ligand Coupling via the Azo Linkage. L. A. Cohen. Vul. XXXIV [88]. Thyrotropin Receptors and Antibody. R. L. Tate, R. J. Winand, and L. D. Kohn. Vol. XXXIV [90]. Immunoadsorbents. J. B. Robbins and R. Schneerson. Vnl. XXXlV [91]. Immunoaffinity Chromatography of Proteins. D. M. Livingston. Vol. XXXV [35]. Immunology of Prostaglandins. R. M. Gutierrez-Cernosek, L. Levine, and H. Gjika. Vol. XXXVll [9]. Localization of Hormones with the Peroxidase-Labeled Antibody Method. P. K. Nakane. VoI. XLIH [6]. Immunological Techniques for Studying fl-Lactamases. M. H. Richmond and V. Betina. VoI. XLIV [2]. Functional Groups on Enzymes Suitable for Binding to Matrices. P. A. Srere and K. Uydea. Vol. XLVI [53]. Affinity Labeling of Antibody Combining Sites as Illustrated by Anti-Dinitrophenyl Antibodies. D. Givol and M. Wilchek. Vol. XLVI [54]. p-Azobenzenearsonate Antibody. M. J. Ricardo and J. J. Cebra. VoI. XLVI [55]. Affinity Cross-Linking of Heavy and Light Chains. M. Wilchek and D. Givol. Voi. XLVI [56]. Bivalent Affinity Labeling Haptens in the Formation of Model Immune Complexes. P. H. PIotz. Vol. XLVI [58]. Labeling of Antilactose Antibody. P. V. Gopalakrishnan, U. J. Zimmerman, and F. Karush. Vol. XLVIII [13]. The Meaning of Scatchard and Hill Plots. F. W. Dahlquist. Vol. L [5]. Direct Identification of Specific Glycoproteins, Antigens in Sodium Dodecyl Sulfate Gels. K. Burridge. Vol. L [12]. Antibodies to Carbohydrates: Preparation of Antigens by Coupling Carbohydrates to Proteins by Reductive Amination with Cyanoborohydride. G. R. Gray. Vol. L [13]. Carbohydrate Antigens; Coupling Melibionic Acid to Bovine Serum Albumin Using Water-Soluble Carbodiimide. J. L/~nngren and I. J. Goldstein. Vol. L [14]. Carbohydrate Antigens: Coupling of Oligosaccharide-Phenethylamine Derivatives to Edestin by Diazotization and Characterization of Antibody Specificity by Radioimmunoassay. D. A. Zopf, C.-M. Tsai, and V. Ginsburg. Vol. L [15]. CarbGhydrate Antigens: Coupling of Oiigosaccharide Phenethylamine-Isothiocyanate Derivatives to Bovine Serum. D. F. Smith, D. A. Zopf, and V. Ginsburg.

PREVIOUSLY PUBLISHED ARTICLES

483

Vol. L [16]. Affinity Purification of Antibodies Using Oligosaccharide-Phenethylamine Derivatives Coupled to Sepharose. D. A. Zopf, D. F. Smith, Z. Drzeniek, C.-M. Tsai, and V. Ginsburg. Voi. L [33]. Homogeneous Murine Immunoglobulins with Anticarbohydrate Specificity. C. P. J. Glaudemans, M. K. Das, and M. Vrana. Vol. LVI [21]. Use of Antibodies for Studying the Sidedness of Membrane Components. S. H. P. Chan and G. Schatz. Vol. LVI [56]. Chemical Modification of Mitochondria: Cross-Linking Agents. H. M. Tinberg and L. Packer. Voi. LVI [57]. Cleavable Bifunctional Reagents for Studying Near Neighbor Relationships among Mitochondrial Inner Membrane Complexes. R. A. Capaldi, M. M. Briggs, and R. J. Smith. Vol. 62 [57]. Antibodies that Bind Biotin and Inhibit Biotin-Containing Enzymes. M. Berger. Vol. 66 [102]. Preparation of an Antiserum to Sheep Liver Dihydropteridine Reductase. S. Milstein and S. Kaufman. Vol. 68 [30]. In Situ Immunoassays for Translation Products. D. Anderson, L. Shapiro and A. M. Skalka.

Related to Section II Radioimmunoassays and Immunoradiometric Assays for Detection and Estimation of Antigens and Antibodies

Vol. XXH [31]. Affinity Chromatography. P. Cuatrecasas and C. B. Anfinsen. Vol. XXXIV [1]. The Literature on Affinity Chromatography. M. Wiichek and W. B. Jakoby. Vol. XXXVI [1]. Theory of Protein-Ligand Interaction. D. Robard and H. A. Feldman. Vol. XXXVI [2]. Use of Specific Antibodies for Quantification of Steroid Hormones, G. D. Niswender, A. M. Akbar, and T. M. Nett. Vol. XXXVI [4a]. Assays of Cellular Steroid Receptors Using Steroid Antibodies. E. Castafieda and S. Liao. Vol. XXXVH [1]. Statistical Analysis of Radiologand Assay Data. D. Rodbard and G. R. Frazier. Voi. XXXVH [2]. General Considerations for Radioimmunoassay of Peptide Hormones. D. N. Orth. VoL XXXVII [3]. Development and Application of Sequence-Specific Radioimmunoassays for Analysis of the Metabolism of Parathyroid Hormone. G. V. Segre, G. W. Tregear, and J. T. Potts, Jr. Vol. XXXVII [16]. Methods for Assessing Immunologic and Biologic Properties of Iodinated Peptide Hormones. J. Roth. VoI. XXXVH [28]. Methods for the Assessment of Peptide Precursors. Studies on Insulin Biosynthesis. H. S. Tager, A. H. Rubenstein, and D. F. Steiner. Vol. XXXVII [29]. Technique for the Identification of a Biosynthetic Precursor to Parathyroid Hormone. J. F. Hahener and J. T. Potts, Jr. Vol. XXXVI][I [13]. Assay of Cyclic Nucleotides by Radioimmunoassay Methods. A. L. Steiner. Vol. L [14]. Carbohydrate Antigens: Coupling of Oligosaccharide-Phenethylamine Deriva-

484

PREVIOUSLY PUBLISHED ARTICLES

tives to Edestin by Diazotization and Characterization of Antibody Specificity by Radioimmunoassay. D. A. Zopf, C.-M. Tsai, and V. Ginsburg. Vol. 62 [$3d. A Radioimmunoassay for Chicken Avidin. M. S. Kulomaa, H. A. Elo, and P. J. Tuohimaa. Vol. 68 [31]. Selection of Specific Clones from Colony Banks by Screening with Radioactive Antibody, L. Clarke, R. Hitzeman, and J. Carbon. Vol. 68 [32]. Immunological Detection and Characterization of Products Translated from Cloned DNA Fragments. H. A. Erlich, S, N. Cohen, and H. O. McDevitt.

Related to Section III Immunoassays for the Detection and Estimation of Antigens and Antibodies

Vol. XI [92]. Micro Complement Fixation. L. Levine and H. Van Vunakis. Vol. XL [15]. Immunochemical Characteristics of Chromosomal Proteins. F. Chytil. Vol. XL [22]. Use of Antibodies to Nucleosides and Nucleotides in Studies of Nucleic Acids in Cells. B. F. Erlanger, W. J. Klein, Jr., V. G. Dev, R. R. Schreck, and O. J. Miller. Vol. XLIV [48]. Immunoenzymic Techniques for Biomedical Analysis. S. Avrameas. Vol, LVII [6]. Determination of Creatine Kinase Isoenzymes in Human Serum by an Immunological Method Using Purified Firefly Luciferase. A. Lundin.

AUTHOR INDEX

485

Author Index Numbers in parentheses are reference numbers and indicate that an author's work is referred to although the name is not cited in the text. A Aalberse, R. C., 344 Aalund, O., 93 Aassmann, G., 227(25), 239 Abola, E. E., 5 Abraham, G. E., 90, 103, 322 Action, R. T., 7 Adams, A., 388, 406(9) Addison, G. M., 335, 343 Ade, N., 227(17), 239 Adler, K., 227(25), 239 Africa, B., 101 Agate, F. J., 90 Agnoti, F., 230(60), 240 Aharonov, A., 73 Aherne, G. W., 97 Ainsworth, C. F., 5 Aisenberg, A. C., 14 Aito, M.-L., 228(30), 239 Akbar, A. M., 202 Akizuki, M., 228(34), 239 Aladjem, F., 180 Alam, I., 359, 360, 371(24, 25), 372(24) Alder, F. L., 456, 459 AI-Dujaili, E. A. S., 296 Alexander, C. B., 357, 359(21) Alfredsson, G., 230(61), 240 Alkan, S. S., 57 Allen, R. H., 224, 227(16), 239 Allison, A. C., 108 Allison, W. S., 468 Almqvist, S., 271 Amano, T., 12, 179 Amerson, E. W., 207 Amitani, K., 96 Amkraut, A. A., 89 Amzel, L. M., 5 Anantha Samy, T. S., 229(42, 44), 239 Anderer, F. A., 97 Anderson, B., 6, 14(47), 36(47) Anderson, C. W., 95 Anderson, J. K., 235(126), 242

Anderson, M. J., 391,392(43) Anderson, N. G., 300 Andres¢, A. P., 405 Andrieu, J. M., 439 Anfinsen, C. B., 12, 42(91), 43, 44(213), 157, 406 Anken, M., 390, 409(28) Arakatsu, Y., 12, 23(98) Armstrong, J., 329 Arnauld, C. D., 159 Arnheim, N., 468 Arnon, R., 12, 43, 44, 45, 46, 55, 56, 153, 156(10) Ashby, C. D., 225,234(112), 242 Ashe, W. K., 144, 145(11), 146(14), 147(14) Ashwell, G., 12(101), 13, 23(98), 33(101), 34(101), 205 Askonas, B. A., 7, 108 Assan, R., 325 Atanasiu, P., 390 Atassi, M. Z., 6, 41, 42, 45, 46, 47, 50, 51(5, 9), 56 Atkins, R. C., 234(115), 242 Atkinson, L. E., 316 Audran, R., 125 Aug6, C., 29, 36(148), 39(148), 41(148) Augustin, R., 180, 182(39) Aune, K., 472 Aurand, L. W., 232(86), 241 Aurbach, G. D., 280, 289(2) Austen, K. E., 97, 233(103), 241 Austin, M. J., 21 Avey, H. P., 5 Avrameas, S., 123, 131, 132(28), 164, 165, 191, 390, 391, 406, 408(44), 425, 426, 427,431,432(19), 442,447, 457

B

Backhausez, R., 175 Baer, A., 468(34), 469(34), 471(34), 473(34), 474(34), 475(34), 477(34), 478(34), 479

486

AUTHOR INDEX

Bagshawe, K. D., 305, 310(5), 311 Bailey, G. D., 393 Bailey, J. M., 100 Baker, T. S., 101 Baker, W., 156 Baldi, A., 229(46), 239 Ball, F. L., 300 Ballard, F., 232(83), 241 Banck, G., 237(144), 243 Banerjee, S. P., 228(33), 239 Baram, T., 154 Barker, D. L., 468, 478(30) Barlett, A., 388, 406(9), 423, 428(12), 438, 439 Bartos, D., 89, 93(10), 96, 98(10) Bartos, F., 89, 93(10), 96, 98(10) Bassiri, R. M., 102 Bastiani, R. J., 440 Batjer, J. D., 291 Bauman, A., 322, 325(3), 376, 378(2) Bauman, J. B., 61 Baumgold, J., 236(130), 242 Bauminger, S., 153, 154(6), 156(8, 10), 157, 158, 159(6), 204 Bavykiv, S. G., 231(73), 240 Bayse, S., 214, 215,218(5) Bazin, H., 390 Beale, D., 7 Bechhold, H., 166 Beck, P., 337, 352(10) Becker, E. L., 15, 181, 182, 190 Becker, J. W., 5 Becker, M. J., 57 Beckett, G. J., 91 Bedford, D. K., 228(28), 239 B-Efraim, S., 56 Beiser, S. M., 70, 71, 72, 73(20), 74, 88, 90, 91(4), 98(4), 99, 101(4, 5), 102, 154, 155(14), 157(14), 159, 444 Belanger, L., 422 Bell, R. D., 231(78), 241 Bellet, A. J. D., 231(71), 240 Belyovsky, A. V., 231(73), 240 Benacerraf, B., 52, 57, 65, 112, 114, 143, 146(9), 178, 229(53), 240 Bendich, A., 71, 102 Benedict, A. A., 111, 146 Ben-Efraim, S., 57 Benesch, R. E., 102 Beneson, A., 253, 259(14) Benjamini, E., 48 Bennett, J. L., 236(131), 242

Bennett, L. G., I 1, 12(81), 23, 30(81) Bennich, H. B., 377, 382 Bensadoun, A., 232(87), 241 Bentina, V., 206 Berg, D., 23 Berg, H. C., 252 Bernard, C. F., 20, 41(125) Bernstein, D., 229(48, 50), 240 Berson, S., 12, 31, 32(165), 86, 201,202(1), 322(6), 323, 325(2, 3), 328(6), 335, 376, 378(2), 388 Besch, P. K., 388, 406 Bethell, M. R., 467 Beug, H., 227(17), 239 Biberfeld, P., 357, 372(14) Bidwell, D. E., 423,428(12), 438,439 Bieber, C. P., 352, 355(28) Binaghi, R. A., 146 Binoux, M. A., 275,278(5), 292 Birgegard, G., 232(94), 241 Birkenhager, J. C., 231(68), 239 Biro, C., 53 Bizoleon, C. A., 101 Black, P. H., 141 Blake, C. C. F., 43 Blakistone, B. A., 232(86), 241 Blatt, Y., 11, 12(80) Blaustein, J., 34 Bleicher, S. J., 274 Blobstein, S., 100 Bloch, K. J., 143, 146(9) Blomme, W. J., 438, 439, 440(2) Bode, W., 5 Bodey, G. P., 234(114, 116, 118), 242 B6cker, J. F., 391 Boehm-Truitt, M. J., 228(34), 239 Boggs, J. D., 390 Bolton, A. E., 221, 223(1), 224(1), 226(10), 230(58), 238, 240, 344, 357, 359(23) Bonavida, B., 72 Bonner, J., 70 Boorsma, D. M., 132 Bordenave, G., 187 Borek, F., 50, 54(6), 56, 154, 155(14), 157(14), 444 Borek, G., 88, 91(4), 98(4), 101(4, 5) Borel, Y., 75, 78 Borsos, T., 229(40, 41), 233(97, 100), 239, 241, 252, 256, 260, 261(28), 265, 356, 357(7), 358(7), 360(7), 364(7), 370, 372(7, 28), 373, 374(31), 375(31), 390, 408

487

AUTHOR INDEX Borst, A., 394 Boshart, G. L., 92 Bouillon, R., 272 Bowie, L. J., 391,392, 393(47) Boyd, C. M., 388 Boyd, G. W., 113 Boyderi, S. V., 456 Boyle, M. D. P., 229(40, 41), 233(97, 100), 239, 241, 356, 357(7), 358(7), 360(7), 364(7), 370, 372(7, 28), 373, 374(31), 375(31), 390, 408 Bragdon, J. H., 265 Brandt, D. Ch., 356 Brandt, R., 384 Brasfield, D. L., 91, 93(26) Braude, N. A., 79 Braun, D. G., 11, 12(82), 23(82), 24(89) Braun, W., 59, 78, 79(36) Bredehorst, R., 100 Breillatt, J. P., 300 Brenner, P. F., 101 Bretting, H., 30, 34(154, 155) Brezin, C., 190 Brighton, W. D., 378 Brodey, G. P., 93, 96(46) Bronson, P. M., 19 Brooker, G., 392, 451 Brostoff, J., 378 Broughton, A., 93, 96(46), 234(109, 110, 114, 116, 118), 242 Brown, J. B., 45 Brown, L. P., 388, 406 Brown, R. K., 45, 54 Brunfeldt, K., 93 Brunswick, D. J., 97 Bryant-Greenwood, G. D., 225,230(65), 240 Buchanan, T. M., 236(128), 242 Buchanan-Davidson, D. J., 179 Bull, F. G., 233(95), 237(139), 241,243 Bump, S., 467 Bundy, G. L., 93, 98(45) Burger, R. L., 224, 227(16), 239 Burke, J. F., 103 Burman, C. J., 231(68), 240 Burns, J. J., 304 Burrows, G. D., 97 Burtin, P., 48 Burton, D. R., 356 Busby, B., 291 Bush, M. E., 57 Bussard, A., 172, 173(17a), 176 Buster, J. E., 90

Butcher, G. W., 52, 65(22), 137 Butler, G. C., 94 Butler, J. E., 407 Butler, V. P., Jr., 102, 143, 150(6), 159, 203 Buzby, G. C., Jr., 155 Byrnes, D. J., 93

C Cahn, R. D., 467 Callahan, F. M., 95 Callahan, H. L, 58, 202, 205(13), 206(13) Callard, I. P., 228(37), 239 Cambell, D. H., 443 Cammann, K., 444(26), 445 Campbell, A. K., 344 Campbell, D. H., 18 Campbell, P. N., 17 Campbell, R. A., 96 Candler, E. L., 300 Capurso, A., 227(25), 239 Carbonara, A. O., 172(67), 173(67), 190, 191(67) Carlsson, H. E., 423 Carnegie, P. R., 210 Carpenter, G., 217 Cash, J. D., 226(10), 238 Casley, D. J., 225,234(111, 115), 242 Caton, J. W., 300 Catt, K. J., 271,390, 406(15) Catterall, W. A., 238(146), 243 Cautrecasas, P., 406 Cazenave, P.-A., 194, 197, 198(75) Centifano, Y. M., 191 Ceska, M., 378, 390 Chalkley, S. R., 208,305,308, 312(1), 313(1) Chamberlin, W., 81, 82(51) Chan, E., 229(47), 240 Chanock, R. M., 390 Chantler, S. M., 127, 132, 135,206 Chard, T., 207, 208(52), 280, 281,283, 284, 285, 286, 287, 325 Chase, M. W., 4, 6(8), 51, 58(12), 67(12), 88, 112, 169, 192, 338 Chavin, S. I., 19 Chen, B. L., 5 Chen, J. P., 99 Cheng, C.-F., 232(87), 241 Cheng, L. H., 5 Cheng, W. C., 391 Chenoweth, D. E., 233(104, 105), 241

488

AUTHOR INDEX

Chessum, B. S., 390 Chesworth, J. M., 272 Cheung, A. S., 232(87), 241 Chien, Y.-H., 96 Childs, R. A., 39 Chipman, D. M., 12 Chisholm, D. J,, 93 Cho, H. W., 231(81), 241 Chobsieng, P., 153, 154(5), 155(5), 156(5), 159(5) Choi, Y. S., 391 Chong, C. A., 465 Chopro, I. J., 237(137), 242 Chrambach, A., 330 Christine, M., 71 Chu, T. M., 304 Chun, P. W., 286 Chung, S. F., 167 Churchill, W. H., 91, 94(28) Chused, T. S., 228(34), 239 Ciccimarra, F., 233(99), 241 Cinader, B., 49 Cisar, J., 4, 11, 12(19, 77), 21(19), 22, 23(19, 77), 24(19, 77), 25(77), 27(19), 28, 30(19), 34(77) Clark, B. R., 253, 325, 330(20) Clark, D. G., 232(88), 241 Clark, L. G., 58 Clark, M. G., 232(83), 241 Clark, S. J., 89 Claustrat, B., 101 Clotscher, W. F., 291 Clough, J. M., 209 CIriel, J., 189 Cluskey, J. E., 21 Clutton, R. F., 95 Coat, J. P., 72, 100 Cocola, F., 272 Coffey, J. W., 208, 299, 304(3) Coffino, P., 137 Coggin, H. J., 300 Cohen, G. H., 5 Cohen, L. A., 204 Cohen, S. A., 71 Cohen, S. M., 167 Cohn, M., 27 Cohn, Z. A., 214, 252, 253(5) Colburn, W. A., 95 Collins, W. P., 291 Colman, P., 5 Commerford, S. L., 207, 247, 248

Cone, R. E., 214, 215(12), 218(12), 252 Constantoulakis, M., 336 Cook, C. E., 207 Cooke, J. P., 157 Coombs, R. R. A., 377 Coons, A. H., 336 Cooper, L. S., 132 Cooper, N. R., 237(140), 243 Cooreman, W. M., 438, 439, 440(2) Copeland, R. L., 457 Cordoba, F., 101 Cornwell, D. G., 257, 259, 265(24) Costea, N., 336 Cotmore, S. F., 229(55), 240 Cotton, R. G. H., 135, 141(39) Coutts, S. M., 229(51), 233(101), 240, 241 Cowling, G. J., 225,231(74), 240 Crambach, A., 253 Crank, J., 181 Creech, H. J., 96 Cremer, N. E., 4, 6(9), 51, 67(14), 68(14), 443 Cresswell, P., 235(124), 242 Crews, T., 237(138), 242 Crnekovic, V. E,, 230(66), 240 Cross, H. M., 89 Crowle, A. J., 176 Cruickshank, P. A., 92 Crumpton, M. J., 6, 42(43), 48(211) Cuatrecasas, P., 50, 228(32, 33), 239, 314, 325 Cuculis, J. J., 407 Culvenor, J. G., 236(132), 242 Cumber, A. J., 238(145), 243 Cunningham, B. A., 5, 8(67), 9

D Dahlquist, F. W., 32, 209 D'Aiisa, R. M., 72, 99 Dalrymple, G. V., 388 Dameshek, W., 336 Dandliker, W. B., 12 Danks, J., 378 Danner, D. J., 214 Dannies, P. S., 467 Darcy, D. A., 190 Daughaday, W. H., 31,266, 291

David, S., 29, 36(148), 39(148), 41(148), 72, 100

AUTHOR INDEX Davidow, B. J., 93 Davidson, C. S., 17 Davies, A. J. S., 238(145), 243 Davies, C. J., 354 Davies, D. R., 5, 9, 10(72), 19(72) Davies, R. V., 230(59), 240 Davies, S. J., 354 Davis, M. L., 390 Davis, P., 390 Davis, R., 299 Davison, P. F., 467 Dawson, T. R., 228(30), 239 Dean, P. D. G., 84, 90 Degier, J., 260 Delaage, M. A., 91, 98(25) DeLaFarge, F., 149, 150(29) Delaney, A. D., 231(74), 240 Delaney, R., 45 Demartini, P., 143, 150(6) den Hollander, F. C., 98, 271,272(9) Denmark, J. R., 390 Demel, R. A., 260 DeMoor, P., 272 Deodhar, S. D., 97 de Petris, S., 7 de Riggi, M. L., 91, 98(25) Desbuquois, B., 280, 289(2) DeSchryver, C., 228(35), 232(35), 239 Deutsch, H. F., 268 Dev, V. G., 70 Dewdney, J. M., 103 de Weck, A. L., 101 Dickerson, R. E., 6 Dickler, H. B., 143 Diel, F., 230(67), 240 Dienes, L., 108 Dienstag, J. L., 390 Diedan, P. J., 300 Dietrich, F. M., 92 Dinarello, C. A., 227(24), 239 DiNatale, P., 73 Dintzis, H.-M., 238(147), 243 Dintzis, R. Z., 238(147), 243 Dixon, F. J., 206, 210 Dixon, R., 237(138), 242 Dixon, W. R., 91, 98(31) Doebber, T. W., 228(35), 232(35), 239 D61ken, G., 391 Doljanski, F., 252, 259(14) Dolney, A. M., 96 Doniach, D., 17

489

Donohue, D., 299 D'Orazio, P., 444(27), 445 Dorf, M. E., 229(53), 240 Dorner, M. M., 11, 12(77), 23(77), 24(77), 25(77), 34(77) Dorval, G., 357, 358(15), 372(15) Douglas, R. J., 357 Dourmashkin, R. R., 7 Downey, W., 132 Downs, W. G., 51, 64(17, 18) Drake, M. P., 467 Dray, F., 439 Dray, S., 465 Dresser, D. W., 53 Dreesman, G., 146 Drzeniek, Z., 205 Duffus, P. H., 7 Dufour, D., 422 Dulbecco, R., 260 Dunn, P., 63 Dunnette, J., 232(84), 241 Durieux, J., 7, 41(51, 52) Dutton, R. W., 457 Dwek, R. A., 356

E Eastlake, A., 12, 42(91) Eberle, A., 93, 96(53) Ebisu, S., 29 Eby, W. C., 465 Eckelman, W. C., 236(135), 242 Edberg, S. C., 19 Eddy, G. A., 391 Eddleston, A. L. W. F., 228(29), 239 Edelman, G. M., 5, 8(67, 68), 9, 142, 143(1) Eder, H. A., 265 Edmundson, A. B., 5 Edwards, C. R. W., 296 Effenberger, F., 390 Eilat, D. P., 73 Eisen, H. N., 7, 9, 19, 30, 101 Eisenberg, R., 19 Ekins, R. P., 201,202(2), 208, 209(2) Elder, H. A., 93 Elder, J. H., 213 Eiek, S. D., 173, 174(19, 22) Ely, K. R., 5 Emeroth, P., 230(60, 62), 240 Emerson, S. G., 214, 215(12), 218(12)

490

AUTHOR INDEX

Eng, L. F., 352, 355(28) Engel, J., 356 Engelberg, J., 180 Engle, C., 51, 64(16) Engvall, E., 31,344, 388, 406,419, 421,422, 423(3), 428(2), 429(22), 430(20, 21), 433(22), 438, 439(20, 21, 22), 440(6) Epp, O., 5 Erickson, B. W., 233(105), 241 Erlanger, B. F., 70, 71, 72, 73(20), 74, 75(28), 88, 90, 91(4), 95, 98(4), 99, 100, 101(4, 5), 102, 103(62, 64, 65), 154, 155, 157(14), 159, 202, 204, 206(12), 444 Etzler, M. E., 13, 34 Evans, W. H., 236(132), 242 Eveleigh, J. W., 300 Exley, D., 90, 101 F Fabricius, H. A., 357 Fahey, J. L., 191 Fahrenkrug, J., 230(64), 240 Falbriard, J. G., 76 Fang, V. S., 231(81), 241 Farr, L. A., 341, 343(14) Farr, R. S., 69 Farrow, J. T., 89(43), 93 Fasman, G., 77, 92, 96(36), 153 Fasman, G. D., 467 Faure, A., 101 Fausch, M. D., 5 Favre, L., 58 Feairheller, S. H., 160 Fearson, D. T., 233(103), 241 Fehlhammer, H., 5 Feinberg, B. A., 215, 217(14), 218(14) Feinstein, A., 7 Feizi, T., 6, 14(47), 29, 36(47, 148), 39(148), 39, 41 Felber, J. P., 299, 388 Feldman, H. A., 32, 201,209(5) Ferro, A. M., 100 Frsus, L., 227(13), 232(89), 238, 241 Filachione, E. M., 160 Finkelstein, M. S., 61 Fischer, D. S., 51, 64(18) Fischer, J. C., 72, 100 Fischer-Rasmussen, W., 208, 315 Fisher, B. E., 21

Fitzpatrick, F. A., 93, 98(45) Florent, G., 9, 11(75) Fohlman, J., 237(143), 243 Folch, J., 261 Foo, A. Y., 292, 294(11), 295(11) Forghani, B., 391 Forni, L., 142 Forrest, G. C., 272, 292, 391 Forsgren, A., 237(144), 243, 356, 358(6) Forsham, P. H., 288 Foster, H., 354 Fotherby, K., 91, 101 Fox, C. F., 344 Frackelton, A. R., Jr., 422 Franchimont, P., 101 Francki, R. I. B., 80 Frangione, B., 9, 11, 145 Frankel, M. E., 58 Franklin, E. C., 19, 143, 145, 146(9) Franks, W. R., 96 Fraser, A. S., 393 Frazer, G. R., 32, 201,209(6) Freedlender, A. E., 328 Freedman, D. A., 20 Freedman, S. O., 300 Freeman, D. S., 93 Freund, J., 108 Frey, M., 232(85), 241 Freychet, P., 334 Fridkin, M., 153, 154(5), 155(5), 156(5), 159(5) Fried, M., 286 Friedlander, A., 90, 154, 157(15), 158(15) Friesen, H. G., 334 Friman, G., 226(6), 238 Frohman, L. A., 97, 102(82), 161 Frost, P., 108 Fuccillio, D. A., 405 Fuchs, S., 56, 72, 153 Fuchs, Y., 153 Fudenberg, H. H., 229(54), 240, 336, 390 Fuji, H., 464 Fujio, H, 12, 179 Fukuda, M., 39 Fukui, H., 132, 425 Fukushima, D., 90 Fulthorpe, A. J., 172, 173(17) Funding, L., 54 Furmanski, H., 235(121), 242 Furthmayr, H. F., 229(55), 240 Fuxe, K., 230(60, 62), 240

AUTHOR INDEX G

491

Glaudemans, C. P. J., 4, 11, 12(81), 23, 24(87), 30(81) Gadsby, B. W., 155 Glitz, D. G., 70 Gainer, H., 236(130), 242 Glover, J. S., 210, 382 Galen, R. S., 393 Glusman, M., 20 Galfr6, G., 35, 52, 65(22), 137, 140(49), Gochman, N., 391,392, 393(47) 141(49, 51) Goding, J. W., 150, 356, 357, 360(4), 361(4), Gall, W. E., 8(67), 9, 142, 143(1) 457 Gallagher, R. E., 229(47), 240 Goebel, W. F., 98 Gallo, R. C., 229(47), 240 Goebelsmann, U., 101 Gaily, J. A., 9, 11 Goetzl, E. J., 54 Gapp, D. A., 228(37), 239 Gold, P., 300 Garcia, G., 53 Goldberg, M. L., 451,453(32) Goldberger, R. F., 45 Gardner, J., 236(133), 242 Goldensohn, S. S., 20 Gardner, P. S., 390 Garvey, J. S., 51, 67(14), 68(14), 227(21), Goidfarb, D. M., 79 239, 443 Goldie, D. J., 227(18), 239, 391, 392(38), Gassen, H. G., 154 393(38) Gates, R., 214, 218(4, 6) Goldstein, A., 344 Gavin, J. R., 325, 334(18) Goldstein, G., 227(23), 239 Geary, R., 305, 310(2, 3), 314(2) Goldstein, I. J., 4, 12(11), 29, 34, 205, 221, 244(4), 246(4) Gehatia, M., 156 Gehle, W. D., 208, 390, 391(22), 395(48), Golub, E. S., 457 403(48), 405, 409(48) Gomez-Sanchez, C. E., 226(8), 238 Geider, K., 231(69), 240 Gompertz, D., 236(136), 242 Geier, S. S., 235(124), 242 Gonyea, L. M., 232(93), 241 Geis, I., 6 Gonzalez, C., 101 Genazzani, A. R., 272 Goodfriend, T., 325 Genest, D., 85 Goodfriend, T. L., 92, 96(36), 153 Goodman, J. W., 6, 9(40, 41), 12(40, 41), 148 George, J. N., 252,254(11, 12) Gergely, P., 141 Gordon, J. A., 70 Gordon, P., 325, 334(18) Gerhart, J. C., 221,244(2), 245(2) Gottlieb, C., 274, 275 Gerstein, J., 467 Gottlieb, P. D., 8(67), 9 Gerwing, J., 45 Gowland, G., 380 Geurt Van Kessel, W. S. M., 260 Graber, P., 48, 174 Gewurz, H., 233(106), 241 Graf, T., 227(17), 239 Geyer, H., 235(119), 242 Grandien, M., 390 Ghetie, V., 4, 357, 372(14) Granfors, K., 390 Ghose, T,, 104 Gratzes, W. B., 235(127), 242 Giese, J., 315 Gray, G. R., 205 Gilham, P. T., 76 Greaves, J. P., 291 Gill, T. J., 20, 41(125) Green, I., 112 Gillam, I. C., 231(74), 240 Green, N. M., 9 Gilula, N. B., 236(133), 242 Greene, E. J., 114 Ginsburg, V., 48, 205 Greenstein, J. P., 88, 91(3) Girling, R. L., 5 Greenwood, F. C., 210, 300, 327, 343, 357, Gitlin, D., 17 358(22), 382, 406 Giveon, D., 73 Greenwood, H., 272, 391 Givol, D., 12, 24(86) Grettie, D. P., 96 Gjika, H. B., 89(43), 93 Grifliths, F. B., 93 Glass, J., 227(19), 239

492

AUTHOR INDEX

Grigliotti, T. A., 225,231(74), 240 Grisaro, V., 12 Grodsky, G. M., 288 Groner, B., 233(96), 241 Gropper, L., 9 Grossman, L., 467 Grossmiiller, F., 390 Grotjan, H. E., 394 Grover, P. K., 275 Gruenewald, R., 79 Gruezo, F., 23, 33, 34(182), 39 (182) Guesdon, J. L., 131, 132(28), 191, 391, 408(44), 427,432(19) Guigues, M., 84, 85(54) Guilbert, B., 432 Gunther, E., 229(52), 240 Gurd, F. R. N., 154 Gurvich, A. E., 20, 338 Gustafsson, J.-A., 230(60, 62), 240 Gustofson, G. T., 356 Gutierrez-Cernosek, R. M., 205

Hargis, G. K., 159 Harington, C. R., 94, 95 Harisdangkul, V., 30 Harper, J. E., 451 Harris, T. J. R., 231(72), 240 Harrison, E. T., 143, 144(10), 145(10) Hartley, D., 155 Harwig, S. S. L., 224, 227(12), 238 Haryu, A., 439 Havel, R. J., 265 Hawker, C. D., 31 Hawkes, M. L., 207 Hax, W. M. A., 260 Hay, M., 467 Hayashi, K., 390, 391 Hayden, A. R., 182 Hayes, C. E., 4, 12(11), 221,244(4), 246(4) Haynes, W. C., 21 Hehre, E, H., 21 Heidelberger, M., 4, 5(1, 2, 3), 5(1, 2, 3), 13(1, 2, 3, 34-38), 14, 15, 16, 17, 18(1, 2, 3, 36), 19(38), 24, 26(1, 2, 3), 54 Heimer, B., 229(50), 240 H Hellman, N. N., 21 Hellstrom, I., 229(56), 240 Habeeb, A. F. S. A., 103 Hellstrom, K. E., 229(56), 240 Habener, J. F., 204 Helmkamp, R. W., 252,254(13) Haber, E., 31, 92, 97(39), 101, 141, 143, Hengartner, H., 141, 142 150(6) Hennam, J. F., 291 Hagemann, R. F., 299 Henney, C., 191 Haimovich, J., 344, 419 Henry, C., 464 Hainsselin, L., 299 Hepburn, M. P., 208 Haire, M., 135 Herbert, V., 274, 275 Haisjen, J., 440 Herbert, W. J., 112, 114, 464 Hakomori, S.-I., 39 Heremans, J. F., 172(67), 173(67), 190, Halbert, S. P., 390, 409(28) 191(67) Hald, B., 93 Herlyn, D., 140 Hales, C. N., 201,207,208(8), 312, 335,337, Herlyn, M., 140 352(10, 11), 353(11) Herrmann, E. C., Jr., 51, 64(16) Hall, S. J., 7 Herschman, H., 468 Hallgren, R., 226(6), 238 Hersh, L. S., 312, 391 Halliday, J. W., 232(91, 92), 241 Hertzberg, E. L., 236(133), 242 Halloran, M. J., 71, 76(13), 94 Herzenberg, L. A., 52, 65(24), 344 Hamaguchi, Y., 132, 425 Hess, J. P., 151 Hamburger, R. N., 96 Hesse, R. A., 391 Hampton, J., 213 Hewitt, W. L., 234(113, 117), 242 Hanlon, S., 9 Heymer, B., 229(48, 49), 240 Hansen, H. J., 208, 299, 304(3), 305 Higa, O. Z., 322(7), 323 Harboe, M., 7, 41(53) Hill, B. M., 470 Hardman, K. D., 12(100), 13 Hill, H. D., 228(31), 239 Hardy, P. H., Jr., 407 Hilz, H., 100

AUTHOR INDEX Himmelspach, K., 205, 235(119), 242 Hines, L. R., 304 Hinson, C. A., 238(145), 243 Hinton, B. T., 230(59), 240 Him, .M.H., 91, 98(25) Hiroi, M., 101 Hirsch, D., 252 Hirst, J. W., 27 Hlavka, J. S., 92 Ho, N. W. Y., 76 Ho, S. M., 228(37), 239 Hochwald, G. M., 189 Hoffman, D. R., 31 Hofman, A. F., 292 Hokfelt, T., 230(60), 240 Holladay, D. W., 300 Holleman, J. W., 300 Hollenberg, M. D., 228(32), 239, 325 Hollerman, C. E., 34 Hollinger, T. G., 228(38), 239 Holmes, I. H., 390 H0nger, P., 315 Hood, L. E., 49 Hopgood, M. F. H., 232(83), 241 Hopkins, C. R., 230(57), 240 Hopper, J. E., 164 Horesji, V., 30, 31(162, 164) Hori, H., 236(129), 242 Hornbrook, M. H., 378 Horsburgh, T., 236(136), 242 Hotchkiss, J., 316 Hotchkiss, R. D., 98 Howard, A. N., 377 Howard, J. C., 52, 65(22), 137 Howe, S. C., 52, 65(22), 137 Howes, H., 95 Hoyer, G., 235(119), 242 Hubbard, A. L., 214, 252, 253(5) Huber, C. T., 214, 218(6) Huber, R., 5, 233(107), 241 Hughes, G. A., 155 Hughes, W. L., Jr., 210 Hugli, T. E., 233(104, 105), 241 Huisjen, J., 424 Humayun, M. Z., 72, 76(19), 94, 102(56, 57) Hunter, W. M., 31, 120, 210, 221, 223(1), 224(1), 230(58), 240, 300, 322(5), 323, 327,330,343,344, 357, 358(22), 359(23), 382, 406 Hum, B. A. L., 105, 113, 114(1), 206 Hurrell, J. G. R., 48, 85

493

Hurwitz, E., 44, 344, 419 Huser, H., 12, 24(86) Hutchinson, H., 390 Huu, M. C. N., 233(96), 241 Hynes, N. E., 233(96), 241 Hyslop, N. E., 176, 191(27)

lain, P.-R., 91 Imanishi, T., 48 Inomata, K., 236(129), 242 Inoue, H., 236(129), 242 Ishikawa, E., 132, 425 Ishizaka, T., 378 Ismail, A. A. A., 391,392, 393(38) Isselbacher, K. J., 232(90), 241

Jacks, F., 230(59), 240 Jackson, A. O., 80 Jackson, C. M., 226(11), 238 Jacob, T. M., 94, 102(56, 57, 58) Jacobs, L. S., 291 Jacobsen, C., 54 Jacoby, G. A., 92, 97(39) Jaffe, B. M., 89, 91, 92(11) Jahrling, P. B., 391 Jakoby, W. B., 88, 201 Jalanti, R., 191 Jankowski, M. A., 391 Jann, K., 57 Jansen, A. B., 155 Jaross, R. W., 180 Jaton, J.-C., 12, 24(86), 205, 356 Jay, R., 272, 391 Jeanes, A., 21 Jeep, S., 233(96), 241 Jeffcoate, S. L., 153, 154(12), 292 Jencks, W. P., 468(34), 469(34), 471(34), 473(34), 474(34), 475(34), 477(34), 478(30, 34), 479 Jensen, D. M., 228(29), 239 Jensen, K., 356 Jerne, N. K., 464 Johansson, E. D. B., 322 Johansson, S. G. O., 377, 378, 382 Johnson, D. C., 102

494

AUTHOR INDEX

Katchalski, E., 156 Kato, K., 132, 425 Katz, D. H., 50 Kaufman, H. E., 191 Kaufman, L., 390 Kaul, B., 93 Kaushansky, A., 159 Kawanishi, Y., 460 Kawaoi, A., 132, 133(30), 406, 425, 426, 432(17) Kawashima, K., 91, 98(32) Kekwick, R. A., 144 Kelly, K. A., 98, 103(89) Kemp, D., 92 Kendall, F. E., 5, 13(34-37), 15, 16, 17, 18(36) Kennedy, J. H., 425 K Kenny, M. A., 226(4, 5), 238 Kersfeld, R. A., 217 Kabach, R., 235(125), 242 Kabat, E. A., 4, 5(4, 5, 6, 17), 6(4, 5, 6), 7(6), Kessler, D., 467 8, 9(6, 39), 11, 12(6, 12, 13, 19, 39, 77, Khan, F. S., 439 78, 101), 13(4, 5, 6, 38), 14(5, 47, 103), Khar, S. A., 94, 102(57, 58) 15(5), 17(6), 18(4, 5, 6), 19(4,5, 38), 20, Kigushi, T., 96 21(19), 22(19), 23(5, 6, 19, 77, 79, 98), Kim, H. W., 390 24(19, 77, 78), 25(77, 79), 26(4, 5, 6), Kimball, J. W., 12, 24(85) 27(19), 28(19), 29(6, 12, 13), 30(6, 19), King, L. J., 209 31(163), 33(101), 34(77, 101, 105, 154, Kinkade, J. M., 227(20), 239 155, 181, 182), 35(163), 36(6, 13, 47, Kipnis, D. M., 71, 76(14), 93, 98(41), 153, 154(9), 443,453(19) 147), 37, 38, 39(147, 182), 41(6), 54, 68(31), 96, 201, 202(3), 205(3), 206(3), Kirkbride, M. B., 167 Kirkham, K. E., 31, 120 209(3), 267, 443 Kisailus, E. C., 29, 33, 34(182), 39(182) Ka~,aki, J., 432 Kitau, M. J., 280, 325 Kaha, M. R., 233(103), 241 Klarekog, L., 237(143, 144), 243 Kahn, R., 334 Klause, G. G. B., 89 Kaiser, H., 226(7), 238 Klein, G., 141,391 Kaivarainen, A. I., 381 Klein, J. L., 233(102), 241 Kalica, A. R., 390 Klein, W. J., 70 Kalimo, K. O. K., 390 Kleinhammer, G., 205 Kamel, R. S., 236(133), 242 Klinman, N., 19 Kanai, Y., 79 Klotz, I. M., 30, 154 Kapikian, A. Z., 390 Knaub, V., 20 Kaplan, J., 56 Knight, E., 227(22), 239 Kaplan, N. O., 467, 468 Knight, J., 234(118), 242 Kapner, R. B., 20 Knight, L. C., 222, 224(7), 226(3), 227(12), Karlberg, B., 271 238 Karol, M. H., 91, 100(24) Knight, S., 305 Karpov, V. L., 231(73), 240 Knobil, E., 316 Karush, F., 7, 12, 19, 30(60) Kasamatsu, H., 231(70), 235(122), 240, 242 Knoop, F. C., 226(1), 236(1), 237(1), 238 Knowles, B., 137 Kassan, S. S., 228(34), 239 Johnson, M. W., 90 Johnsson, B. G., 344 Johnsson, S. G. O., 336 Johnston, C. I., 234(115), 242 Johnston, M. I., 80 Jondal, M., 141 Jones, J. K. N., 24 Jones, M. E., 467 Jones, S. B., 150 Jonsson, K., 31,419, 428(2), 440 J~rgesen, M., 315 Joslin, F. G., 146 Ju, S., 51, 64(19) Junge, W., 472

AUTHOR INDEX

495

Knowles, J. R., 426 Lamoureux, G., 210 Kobayashi, Y., 96 Lance, E. M., 108 Koch, Y., 153, 154(5), 155(5), 156(5), 159(5) Landon, J., 113,272,280, 288, 289, 290, 325, 390, 408, 439 Kocourek, J., 30, 31(162, 164) K6hler, G., 5, 34(21), 35, 136, 137, 140(45), Landsteiner, K., 26, 86, 90(1), 96(1), 167, 141(45), 206 187(12), 202, 206(11) Landy, M., 52 Koenig, D. F., 43 Lang, S., 232(85), 241 Kohen, F., 153, 154(6), 159(6) Langone, J. J., 204, 206, 207(46), 229(40, Kohler, G., 52, 65(20, 21) 41), 233(97, 100), 239, 241,356, 357(7), Komai, T., 236(135), 242 Koninckx, P., 272 358(7),'359, 360(7), 363, 364, 366, 367, Kopp, H. G., 93, 96(53) 369, 370(27), 371(24, 25, 29), 372(24, 25), 373, 374, 375(31), 390, 408 Koprowski, H., 140 Korn, A. H., 160 Lapresle, C., 7, 41(49, 51, 52), 178, 181 Larsen, J., 208, 315 Korngold, L., 182 Larson, L. J., 235(121), 242 Koros, A. M. C., 464 Lau, K. S., 274, 275 Kricka, L. J., 425 Laurell, C. B., 176, 177, 191(26, 30) Kors, N., 132 Lauer, R. C., 95, 103(62, 64, 65) Koshland, M. E., 9 Lavidor, L., 227(19), 239 Koskimies, S., 141 Lazar, P., 190 Kraus, R., 166 Lazarow, A., 266 Krause, R. M., 229(48, 49, 50), 240 Lazarus, L., 93 Kreuzer, H., 226(7), 238 Leach, S. J., 48, 85,226(9), 238 Krogh, P., 93 LeBeau, L. J., 181 Krisch, K., 472 Lee, C.-J., 235(120), 242 Kruger, F. A., 257, 259, 265(24) Leek, A. E., 280 Krupey, J., 300 Leekeman, G. M., 439, 440(2) Kubota, H., 236(135), 242 Kuchinskaya, N. E., 146 Leeman, S., 153 Kufe, D. W., 229(47), 240 Lees, M., 261 Kuijpers, L. J., 432 Lefkovits, I., 41,464 Kulberg, A. Y., 146 Lefkowitz, R. J., 325 Kumahara, Y., 96 Leger, R. N., 89, 93(10), 98(10) Kunkel, H. G., 4, 7, 41(53), 146 Lehrer, H., 56 Leibach, F., 232(82), 241 Kunz, H. W., 57 Kuppens, P. S., 394 Leinikki, F., 393 Kurosaka, K., 439 Leiva, B., 389, 390(10) Kuzoreta, O. B., 338 Lembach, K. J., 217 Kwok, S. C. M., 225,230(65), 240 Lemieux, R. U., 29, 36(147), 39(147), 40(147) Lemieux, S., 457 L Leng, M., 84, 85(54) Lennette, E. H., 391 Lackner, J. A., 180 Lenusky, R., 299, 304(3) Leon, M., 4, 24(18), 25, 29(18) Lacour, F., 79, 80 Lader, S., 105, 113(1), 114(1) Leonard, E. J., 256 Lerario, A. C., 322(8), 323 Laekeman, G. M., 428 Laki, K., 227(13), 232(89), 238, 241 Lerner, R. A., 213 Leskowitz, S., 103 Lambden, P. R., 357, 359(20) Leslie, R. C. Q., 146 LaMont, J. L., 232(90), 241

496

AUTHOR INDEX

Lesniak, M. A., 325, 334(18) Leung, C. Y., 45, 48 Leute, R. K., 344 Levin, H., 57 Levine, B. B., 103 Levine, L., 45, 70, 71, 72, 79(8, 17), 89(42, 43, 44), 92, 93, 96(36), 153, 205, 359, 360, 369, 371(24, 25, 29), 372(25), 467, 468, 469, 471, 473, 474, 475, 477, 478, 479 Levison, S. A., 12 Levitt, N. H., 391 Levy, A., 91, 98(32) Levy, M., 253 Lewis, J. E., 93 Lewis, J. L., 225 Lewis, P. C., 252, 254(11, 12) Lewis, U. J., 327, 332 Li, C. P., 5 Liao, J., 4, 11, 12(19, 77, 101), 13, 21(19), 22(19), 23(19, 77), 24(19, 77), 25(77), 27(19), 28(19); 30(19), 33(101), 34(77, 101, 155), 36(147), 39(147), 40(147) Liberti, P. A., 58 Lichensteiger, W., 93, 96(53) Lieberman, S., 88, 90, 91(4), 98(4, 31), 101(4, 5), 154, 155(14), 157(14), 444 Liebman, A. J., 91 Liedike, R. J., 291 Liesegang, R. Ed., 166 Lind, I., 360 Lindberg, A. A., 423 Lindberg, B. S., 322 Lindberg, P., 322 Linder, H. R., 90, 153, 154(5, 6), 155(5), 156(5, 8, 10), 157(15), 158, 159(5, 6) Linder, K.-H., 233(98), 241 Ling, N. R., 457 Lipsett, M. B., 252, 330 Lipschitz, D. A., 352, 355(28) Littauer, U. Z., 73 Littlefield, J. W., 136 Liu, C.-T., 456, 459 Live, I., 360 Llenado, R. L., 444, 454(25) Lloyd, K. O., 36, 37 Lodmell, D., 391 Loeckner, C. P., 235(121), 242 Lofstrom, A., 230(60, 62), 240 Longley, C., 235(121), 242 Lonngren, J., 205

Loor, F., 142 Lopez, M., 176, 191(27) Lotan, R., 34 Lowry, O. H., 341,343(14) Ludlam, C. A., 226(10), 238 Lumkin, M. E., 101 Lund, J. O., 315 Lundberg, P. O., 272 Lundblad, A., 27 Lundkvist, U., 378, 385 Luzio, S. D., 337, 352(11), 353(11) Luzzatti, A. L., 141, 142

M McBride, D., 226(2), 238 McBroom, C. R., 205 McConahey, P. J., 206, 210 McCracken, A. W., 390, 391(22) McDermott, K., 108 McDevitt, H. O., 52 MacDonald, A. B., 164, 237(142), 243 McFarlane, A. S., 121,325 McFarlane, I. G., 228(29), 239 McGill, R., 153 McGivern, P. L., 407 McGuigan, J. E., 89, 92(11) McGuire, J., 153 Mclntosh, K., 390 Mclntyre, K. R., 4, 24(18), 25(18), 29(18) Mackey, G., 336, 390 McKeering, L. V., 232(91, 92), 241 McKelvey, E. M., 191 McLean, D. M., 390 McMurtry, J. P., 225,230(65), 240 McPherson, T. A., 210 Madden, D. L., 388 Madsen, L. H., 123 Maegraith, B. G., 167 M~ikel~i, O., 44, 48 Mage, M. G., 143, 144(10), 145(10), 201 Mage, R., 144, 145(11), 357, 359(21) Maguire, K. P., 97 Mahan, D. E., 457 Mahar, S., 145, 146(14), 147(14) Mahley, R. W., 227(26), 239 Maiolini, R., 432 Mair, G. A., 43 Majerus, P. W., 226(11), 238 Makel, O., 141

AUTHOR INDEX Makin, H. L. J., 208, 292, 294(11), 295(11) Maling, B., 81, 82(51) Mancini, G., 172(67), 173(67), 190, 191(67) Mandell, B. F., 228(35), 232(35), 239 Mandy, W. J., 142 Mannick, M., 132 Mansa, B., 360 Marchalonis, J. J., 252, 357, 406 Marchesi, V. T., 229(55), 240 Margoliash, E., 162, 163(6), 215, 217(14), 218(14) Marks, V., 97, 209 Markwell, M. A. K., 344 Maron, E., 12, 43, 44(213), 46, 465 Marsh, W. L., 6, 14(47), 36(47) Marshall, J. C., 253 Martin, M. J., 280, 288, 289, 290 Martinsson, K., 322 Marttila, R. J., 390 Masseyeff, R., 432 Matikainen, M.-T., 272, 390 Matsumoto, G., 236(130), 242 Matsushima, T., 79 Matsuuchi, L., 35 Mattar, E., 322(8), 323 Maurer, P. H., 17, 50, 53, 54, 56, 58, 59(42), 202, 205(13), 206(13) Mayer, M. M., 4, 5(4, 5), 6(4, 5), 13(4, 5), 14(5), 15(5), 17(5), 18(5), 19(5), 20, 22(5), 26(4, 5), 252,443 Meakin, J. C., 305, 310(3) Mecklenburg, R., 252, 330 Mehlman, C. S., 224, 227(16), 239 Meikle, A. W., 93 Melamed, M. D., 146 Melchers, F., 5, 34(22), 35(22), 52, 65(23), 67, 136 Meltzer, H. Y., 231(81), 241 Melvin, E. H., 21 Mendecine, J., 232(82), 241 Mendels, J., 97 Meredith, R. D., 74, 75(28), 100 Merler, E., 233(99), 241 Merrett, J., 207, 208(50), 379, 385 Merrett, T. G., 207, 208(50), 379, 385, 467 Mersel, M., 253,259(14) Metzgar, R. S., 235(126), 242 Metzger, H., 19 Metzger, J. F., 391 Meurman, O. H., 390

497

Meyer, H. G., 407 Meyerhoff, M. E., 444(27), 445, 454(27) Michaelli, D., 227(15), 238 Michaelson, T. E., 148 Michel, M., 196, 197 Michelson, A. M., 80 Michelson, W., 79 Midgley, A. R., Jr., 89, 90(13), 208, 271 Mihara, S., 93, 98(47) Miles, L. E. M., 312, 335, 352, 355(28) Miletich, J. P., 226(11), 238 Miller, E. J., 429, 430(21), 439(21) Miller, H. V., 391 Miller, M. J., 228(35), 232(35), 239 Miller, O. J., 70, 99 Miller, O. N., 274, 299 Millian, S. J., 93 Milstein, C., 5, 35, 52, 65(20, 21, 22), 135, 136, 137, 140(45, 49), 141(39, 45, 49, 51), 206 Minden, P., 69 Minnis, M., 378 Miranda, O. R., 393 Mirzabekov, A. D., 231(73), 240 Mishele, D. R., Jr., 101 Mishell, R. K., 457 Mitchison, N. A., 62 Miwa, M., 79 Miyachi, Y., 253, 330 Modesto, R. R., 425 Moffat, A. C., 209 Moffat, J. G., 93, 98(47) Molinaro, G. A., 465 Moller, N. P. H., 54 Moore, E. H., 305 Moore, S., 226(10), 238 Morell, A. G., 228(36), 239 Morgan, C. R., 266 Morgan, H. G., 228(28), 239 Morgan, W. T. J., 36 Moritsugu, Y., 390 Morris, B., 312 Morris, C. J. O. R., 292 Morris, P., 292 Morrison, M., 206, 207(45), 214, 215(9), 216(16), 217(14), 218(4, 5, 6, 14), 222, 252 Morrison, S. L., 35 Morrod, P. J., 232(88), 241 Moss, A. J., 388 Mota, G., 4

498

AUTHOR INDEX

Mozes, E., 229(52), 240 Miiller-Eberhard, H. J., 146 Mukherjee, A., 231(78), 241 Munck, O., 315 Munoz, J., 15, 51, 64(15), 181 Murakami, W. T., 467 Murayama, A., 425,426 Murphy, B. E. P., 292, 315 Murphy, G. F., 234(115), 242 Murphy, L. A., 29 Murphy, M. J., 253 Murray, J. P., 20 Mynors, L. S., 377

N Nagy, C. F., 299, 304(3) Nahon-Merlin, E., 79, 80 Nair, R. M. G., 229(54), 240 Nakamura, S., 30, 31(161) Nakamura, Y., 237(137), 242 Nakane, P. K., 132, 133(30, 33), 406, 425, 426, 432(17) Nakanishi, K., 95, 103(62, 64, 65) Nambera, T., 439 Nathenson, S., 137 Natvig, J. B., 146, 148 Naus, A. J,, 394 Navia, M. A., 9, 10(72), 19(72) Needleman, B., 97 Neff, J. C., 181 Neimann, H., 36 Nelson, J. C., 93,225 Neporn, J. T., 229(56), 240 Neff, P., 272 Neter, E., 456 Nett, T. M., 202 Neumann, H., 45 Neville, D. M., 334 Newerly, K., 322, 325(3), 376, 378(2) Newton, J. R., 291 Newton, W. T., 89, 91, 92(11) Nezlin, R. S., 20, 381 Niall, H. D., 271,390 Nichoils, A. C., 160 Nicholson, A., 228(29), 239 Nicolson, G., 34 Niedermeyer, W. F., 7 Nielson, M. D., 315

Nieschlag, E., 113 Nilsson, K., 357 Ning, R., 91, 98(31) Ninomiya, I., 96 Nishina, T., 90 Nisonoff, A., 9, 51, 64(19), 142, 162, 163(6), 164 Niswender, G. D., 89, 90(13), 158, 202,271, 291 Nitecki, D. E., 57 Noma, Y., 179 Nomoto, A., 231(72), 240 Nonno, L., 468 Nordin, A. A., 464 Norman, T. R., 97 North, A. C. T., 43 Notkins, A. L., 144, 145, 146(14), 147(14), 390, 391 Novik, N., 44, 344, 419 Nowowiejski, I., 299 Numazawa, M., 439 Nunez, M. T., 227(19), 239 Nye, L., 272, 391

O Oakley, C. L., 172, 173(17) O'Brien, J., 305 Odell, W. D., 31, 103, 208, 275, 278(5), 292 Oette, K., 227(25), 239 Ogihara, T., 96 Ohanian, S. H., 252,255,256, 260, 261(28), 265 Ohlragge, J. B., 228(39), 239 Ohno, T., 229(47), 240 Okabayashi, T., 93, 98(47) Okuhara, E., 79 Oliver, G. C., Jr., 91, 93(26) Olsen, G. D., 89, 93(10), 98(10) Orasz, J. M., 248 Orloff, K. G., 227(15), 238 Orr, A. H., 311 Orth, D. N., 32, 201,206(4), 209(4) Osato, R. L., 91 Osheroff, N., 215, 217(14), 218(14) Osler, A. G., 470 Ostedand, C. K., 7, 41(53) Ouchterlony, O., 48, 172, 173(18), 174(18, 20), 178(20), 182, 186(20), 187

AUTHOR INDEX Oudin, J., 48, 166, 167(1), 168(1), 169, 170(16), 172(1, 53), 173(16, 16a), 174(32), 177(16), 178(1), 179(1, 16), 181, 182, 183(32), 184(53), 185, 186, 187(53), 188, 196, 197, 198 Outschoorn, I. M., 23 Ovary, Z., 103, 143, 146(9) Ovlisen, B., 315 Owen, P., 235(125), 242 Oxford, J. S., 229(45), 239

499

Pepper, D. S., 226(10), 238 Pepys, J., 376 Percy, J. S., 390 Pereira, M. E. A., 4, 12(13), 13, 29(13), 30, 33, 34(105, 155, 182), 36(13), 37, 39(182) Perel, E., 153, 154, 156(10), 157(15), 158(15) Perlmann, P., 31, 344, 388, 406, 419, 421, 423(3), 428(2), 440 Pernis, B., 41, 142 Perry, A., 405 Perry, M. B., 24 Pesce, A. J., 425 P Peskar, B. M., 97 Peters, J. H., 54 Padlan, E. A., 5 Peters, W. P., 229(47), 240 Page, L. B., 92, 97(39) , Petersen, E. E., 391 Painter, A., 231(80), 241 Peterson, E. A., 144 Paldino, R. L., 180 Peterson, M. A., 288 Palezuk, N. C., 59, 78, 79(36) Peterson, P. A., 237(143, 144), 243 Pang, C. N., 102 Petrie, G. F., 167 Pangburn, M. K., 234(108), 241 Petryniak, J., 29 Pant, H. S., 236(130), 242 Phethean, J., 235(127), 242 Papastathopoulos, D. S., 442, 454(17) Phillips, D. C., 12, 43 Papermaster, D. S., 57 Phillips, R. C., 440 Pappenheimer, A. M., Jr., 17, 18, 24 Phillips, S. G., 30 Paques, E. P., 233(106), 241 Phizackerly, R. P., 5 Parikh, I., 50 Hall, E. M., 97 Parker, B. M., 91, 93(26) Picketing, L. K., 93, 96(46), 234(114, 118), 242 Parker, C. W., 9, 30, 31, 32(169), 69, 71, 76(13), 91, 93(26), 94, 231(75, 76, 77), Pickett, R. A., 213 240, 241 Pierce, G. B., 425 Parker, M. L., 266 Pieroni, R. R., 322(7, 8), 323 Parkhouse, R. M. E., 7 Pinchuck, P., 53 PfisillS., S., 393 Pinder, J. C., 235(127), 242 Pastan, I., 325 Pine, L., 390 Pasternak, T. H., 76 Pinto, H., 322(7, 8), 323 Pattee, C. J., 315 Pitt, C. G., 207 Patterson, R. G., 377 Platt, A. S., 305 Patterson, W. R., 400 Platteau, B., 391 Paul, C., 9, 11(75) Plattner, R. D., 21 Paul, W. E., 57, 112, 178 Plescia, O. J., 59, 78, 79(36) Pauling, L., 18 Plotz, P., 19 Paulsen, K., 31 Podit, I., 11, 12(80) Pauwels, R., 391 Poirer, M. C., 100 Peale, J., 390 Poland, R. E., 209, 322, 326(1), 328(1), Pearce, W. A., 236(128), 242 329(1), 333(1) Pearson, T., 137, 140, 141(51) Politz, S. M., 70 Pearl, W. S., 113 Poljak, R. J., 5 Pecht, I., 12 Pollard, A., 305, 307(6), 310(6) Penn, G. M., 146 Ponpipon, M. M., 98, 103(89)

500

AUTHOR INDEX

Ponterius, G., 384 Poonian, M. S., 84 Porath, J., 271 Poretz, R. A., 34 Porter, R. R., 9, 143, 145(7), 147(7) Post, J., 322, 325(2) Potter, M., 4, 5(16), 12(19), 19, 21(19), 22(19), 23(19), 24(19), 25(16), 27(19), 28(19), 30(19), 34(22), 35(22), 52, 65(23), 67, 136, 143, 145(8) Potterf, R. D., 252, 254(12) Potts, J. T., Jr., 204, 205(19) Potuzak, H., 84 Poulik, M. D., 174 Poulsen, K., 93 Pounce, B., 229(54), 240 Poweii, L. W., 232(91, 92), 241 Powell, M. E., 39 Prager, E. M., 468 Prato, C. M., 312, 391 Pratt, J. J., 106 Pratt, K. L., 456, 464(3) Preer, J. R,, Jr., 182, 190(50) Prensky, W., 250 Pressman, D., 18 Price, M. G., 392 Pricer, W., 325 Priess, H., 233(107), 241 Pruzansky, J. J., 377 Prucell, R. H., 390 Putnam, F. W., 9, 11 Putney, F., 9 Q Quabbe, H. J., 230(67), 240 Quick, N. A., 305 Quiocho, F. A., 160

R

Race, R. R., 36 Radding, C. M., 265 Raff, M. C., 7 Rainbow, S. J., 337, 352(11), 353 Rainen, L., 74, 75(27), 78(27), 81, 100 Ram, J. S., 425 Ranadive, N. S., 102

Randall, R. J., 341,343(14) Rankin, J. C., 21 Rapp, H. J., 256, 265 Rappaport, I., 34 Rask, L., 237(143), 243 Raso, V., 81,229(42, 43), 239 Ray, R., 238(146), 243 Raybould, T. J. G., 127 Raynaud, M., 7, 17, 41(50) Raynor, B. D., 143, 150(6) Read, S., 229(49), 240 Reba, R. C., 236(135), 242 Rebar, R. W., 271 Rebers, P. A., 24 Rechnitz, G. A., 442, 444(27), 445(24), 454(17, 24, 25, 27) Reddy, S., 232(82), 241 Reed, C, F., 252, 254(13) Reed, W. P., 24 Reeke, G. N., Jr., 5 Refetoff, S., 322 Rehfield, J. F., 230(63), 240 Reichlin, H., 97, 102(82) Reichlin, M., 54, 55, 161, 162, 163, 164, 204, 467, 468 Reid, D. M., 229(51), 233(101), 240, 241 Reilly, P., 214, 215(12), 218(12) Reiner, L., 167 Reisfeld, R. A., 272, 390, 392 Reiss, E., 390 Reiter, H., 227(14), 238 Rekosh, D. M. K., 231(71), 240 Relyfeld, E. H., 7, 17, 41(50) Renaud, F., 439 Renfer, L., 277(24), 239 Renshaw, A., 208, 305,308, 312(1), 313(1) Repke, D. B., 93, 98(47) Reynoso, G., 304 Riceberg, L. J., 204 Rich, M. A., 235(121), 242 Richards, F. M., 160, 426 Richardson, A. K., 256 Richmond, M. H., 206 Richter, W., 23 Riehm, J. P., 76 Riesen, W. F., 12, 24(86) Riley, M., 81, 82(51) Rist, C. E., 21 Rittenberg, M. B., 89, 456, 464(3) Ritzi, E., 229(46), 239

AUTHOR INDEX Rivera, P., 101 Riviere, J.-F., 101 Riya, I., 172, 173(16a) Robbins, J. B., 113,201,235(120), 242 Roberts, J. R., 272, 391 Roberts, M., 377 Roberts, R., 231(75, 76, 77, 80), 240, 241 Robinson, A. J., 231(71), 240 Robinson, E. S., 17 Robinson, R., 156 Rockey, J. H., 19, 145, 146(16) Rodbard, D., 32, 201,209(5, 6) Roddy, P. W., 72 Rodkey, L. S., 11, 12(82), 23(82), 24(89), 123 Rodman, J. S., 228(35), 232(35, 85), 239, 241 Roelcke, D., 36 Rogers, A. W., 232(83), 241 Roitt, I. M., 17 Rommerts, F. F. G., 291 Ropers, H. H., 228(27), 239 Rosario, T. G., 226(4, 5), 238 Rose, B., 179 Rosebrough, N. J., 341,343(14) Rosen, A., 141 Rosen, F. S., 233(99), 241 Rosenberg, B. J., 73 Rosenberg, R. N., 231(78), 241 Rosenblatt, M., 95 Rosendaal, M., 237(139), 243 Rosenthal, J. D., 390 Ross, G. T., 113 Ross, W. C. J., 238(145), 243 Rosselin, G., 325 Rota, T. R., 237(142), 243 Roth, J., 209, 325, 334(18) Rotheberg, P. G., 231(72), 240 Rothschild, M. A., 322, 325(3), 376, 378(2) Routenberg, .I.A., 390 Rowe, D. S., 191 Rowley, G. L., 424, 440 Roxin, L. E., 226(6), 238 Rubenstein, A. H., 204 Rubenstein, K. E., 419, 420, 424(4), 425(7), 440 Rubin, A. A., 467 Rubin, R. T., 209, 253, 322, 325, 326(1), 328(1), 329(1), 330(20), 333(1) Ruckei, E. R., 23 Rude, E., 229(52), 240 Rudikoff, S., 5

501

Rudkin, G., 70 Ruoslahti, E., 421,422,429(22), 430(20, 21), 433(22), 439(20, 21, 22) Ruoss, C. F., 280 Rupley, J. A., 12 Russell, A. S., 390 Russell, W. C., 231(71), 240 Rutishauser, U., 8(67), 9 Ryall, M. E. T., 344 Rydon, H. N., 160 Rzeszotarski, W. J., 236(135), 242

Sachs, D. H., 12, 42(91) Sacks, D. L., 237(142), 243 Sakato, N., 12 Salmi, A., 272, 390 Salmon, S. E., 338, 390 Salomon, R., 73 Samanen, C. H., 205 Sandberg, A. L., 79, 82(39) Sanderson, C. J., 127, 305, 308(2, 3), 314(2) Sandor, G., 191 Sanger, F., 103 Sanger, R., 36 Santer, V., 252 Sarkar, M., 12(101), 13, 33(101), 34(101) Sarma, V. R., 9, 43 Sarsfieid, J. K. G., 380 Sartorelli, A. C., 51, 64(18) Sato, S , 51, 64(19) Sauerzopf, E. R., 208 Saul, F., 5 Saunders, G. S., 412 Sawyer, J. C., 312, 391 Saxena, B. B., 439 Scatchard, G., 316 Schachman, H. K., 9 Schaffalitzky DeMuckadell, O. B., 230(64), 240 Schaffer, F. L., 312, 391 Schalch, W., 11, 12(82), 23(82), 24(89) Scharff, M. D., 137 Scharpe, S. L., 438, 439, 440(2) Schechter, A. N., 12, 42(91), 73, 231(79), 241 Schechter, B., 48, 56 Schechter, I., 48, 56

502

AUTHOR INDEX

Segars, F. M., 227(20), 239 Schechter, Y., 71, 92, 100(33) Segre, G. V., 204, 205(19) Schedewise, H., 228(31), 239 Sehon, A. H., 98, 102, 103(89), 179 Scheinberg, J. H., 228(36), 239 Sela, M., 4, 6(7), 12, 26(7), 41(7), 42(7), 43, Schenkein, I., 253 44(213), 45, 46, 48, 50, 55, 56, 57, 71, 72, Schepers, G., 11, 12(80) 92, 100(33), 101, 153, 156, 157, 204, Scheraga, H. A., 76 229(52), 240, 344, 419 Schick, A. F., 425 Selby, F. W., 327 Schiffer, M., 5 Senitzer, D., 99 Schiffman, G., 13, 14(103) Senti, F. R., 21 Schild, G. C., 229(45), 239 Sereno, M. M., 390 Schlabach, A. J., 84 Schlager, S. I., 207, 252, 253, 254, 258, Setchell, B. P., 230(59), 240 259(15), 260, 261(15, 28), 262, 263, 264, Sever, J. L., 388 Sevier, E. D., 272, 390, 392 265(15) Seymour, F. R., 21 Schlamowitz, M., 211 Sharon, J., 35 Schleifer, K. L., 229(48, 49, 50), 240 Sharon, N., 12, 13, 34(105) Schlesinger, J., 12, 24(86) Shaw, W., 388 Schlesinger, P. H., 228(35), 232(35), 239 Sheehan, J. C., 92, 151 Schlossman, S. F., 57 Shelton, E. M., 7 Schlumberger, H. D., 97 Shenk, T. E., 70 Schmidt, D. H., 143, 150(6) Shepers, G., 235(119), 242 Schmidt, N. J., 391 Shepherd, J., 228(28), 239 Schneerson, R., 201 Sherwood, O. D., 230(66), 240 Schneider, B., 330 Shick, V. V., 231(73), 240 Schneider, E., 230(67), 240 Shimada, K., 425,426 Schneider, R. J., 224, 227(16), 239 Shimizu, A., 9, 11(75) Schneider, R. S., 345,419, 424(4), 440 Shinoda, T., 9, 11(75) Schnure, J. J., 97, 161 Shiozawa, C., 46 Schoenheit, E. W., 108 Shiu, R. P. C., 334 Schonbaum, G. R., 214 Short, D. J., 112 Schreck, R. R., 70 Shulman, M., 35 Schreiber, R., 237(140), 243 Sia, R. H., 167 Schreiber, S. S., 322, 325(2) Sidebotham, R. L., 21 Schreier, M. H., 141 Siegel, B. A., 236(135), 242 Schuerch, C., 23 Sigel, M. B., 322, 326(1), 328(1), 329(1), Schulman, M. J., 137 Schuurs, A. H. W. M., 271,272(9), 420,432, 333(1) Silver, C., 275 438, 439, 440(1), 441(1), 447, 451 Silverton, E. W., 9, 10, 19(72) Schutz, G., 233(96), 241 Simms, E. S., 19 Schwartz, R., 336 Simpson, J. S. A., 344, 354 Schwyzer, R., 93, 96(53) Singer, S. J., 425 Scoggins, B. A., 97 Singh, R. N. P., 332 Scott, E., 228(28), 239 Sinha, Y. N., 327 Scott, T. A., 21 Sippel, A. F., 233(96), 241 Seaman, E., 71, 72, 79(8, 17) Siskind, G. W., 63, 65, 114 Searle, J. E., 90, 153, 154(12), 292 Sears, D. A., 252, 254(11, 12, 13) Sisler, E. C., 232(86), 241 Secherand, D. S., 135, 141(39) Sjodahl, J., 356 Seed, J. L., 236(131), 242 Sjoquist, J., 356, 357, 358(6), 372(14) Seeger, R. C., 357 Skelley, D. S., 388, 406 Skett, P., 230(60, 61, 62), 240 Segal, D. M., 5

AUTHOR INDEX Slater, R. J., 4, 146 Sloan-Stanley, G. H., 261 Slodki, M. E., 21 Smith, D. F., 205 Smith, E. E., 34 Smith, H., 103, 155 Smith, J. A., 48, 85, 226(9), 238 Smith, J. W., 91 Smith, K. O., 208, 390, 391(22), 395(48), 400, 403(48), 405,409(48) Smith, M., 7, 25 Smith, T. W., 99, 143, 150(6) Smithies, O., 146 Smootz, E., 227(25), 239 Smyth, D. S., 142 Snyder, J. J., 304 Snyder, S. H., 228(33), 239 Sobel, A. T., 237(140), 243 Sobel, B. E., 231(75, 76, 77), 240, 241 Sober, H. A., 57, 144 Sobeslavsky, O., 390 Soergal, M. E., 312,391 Sokal, J. E., 97, 102(82), 161 Sold, G., 226(7), 238 Soloman, P. H., 95, 103(64, 65) Solomon, D. H., 237(137), 242 Soloway, A. H., 103 Sonksen, P., 322 Sonoda, S., 211 Spaar, U., 226(7), 238 Spedden, S. E., 141 Spence, L., 51, 64(17) Spector, S., 91, 96, 98(32) Spiegelberg, H., 150 Spiegelman, S., 229(46, 47), 239, 240 Spiers, J. A., 180, 182(39) Spierto, F. W., 388 Spragg, J., 97 Spratt, J. L., 150 Staehelin, T., 237(141), 243 Stahl, P., 228(35), 232(35, 85), 239, 241 Stahlenheim, G., 356 Stanczyk, F. Z., 101 Stanislavski, M., 191 Stanworth, D. R., 126 Staub, A. M., 12 Stavitsky, A. B., 41, 45(207), 46(207) Steabben, D., 167 Steengard, J., 54 Stefani, D. V., 146, 381 Steinberg, A. D., 73,228(34), 239

503

Steinberg, D., 265 Steinberg, I. Z., 45 Steinberger, E., 394 Sternberger, L. A., 407 Steinbuch, N., 125 Steiner, A. L., 76, 93, 98(41), 153, 154(9), 205, 209(39), 443,453(19) Steiner, D. F., 204 Steinitz, M., 141 Steller, R., 27 Steplewski, Z., 140 Stevens, P., 234(113, 117), 242 Stokert, R. I., 228(36), 239 Stolbach, L., 71 Stollar, B. D., 70, 71, 72, 74, 75(27), 78(27), 79, 80, 81, 82(39), 84, 100, 202, 205(14), 206(14), 467 Stollar, V., 70, 84 Stoltz, F., 190 Stone, M. J., 19, 226(8), 231(78), 238, 241 Straessle, R., 210 Stragand, J. J., 299 Straus, R., 330 Streefkerk, J. G., 132 Strong, J. E., 93, 96(46), 234(110, 114, 116, 118), 242 Stumph, W. E., 70 Stupp, Y., 57 Subbarro, B., 357, 359(21) Subrahmanyam, D., 56, 59(42) Sugimura, T., 79 Sussdorf, D. H., 4, 6(9), 51, 67(14), 68(14), 443 Sutherland, E. W., 76 Suzuki, H., 21 Suzuki, T., 460 Svehag, S.-E.,7 Swanson, P., 407 Sweet, R. W., 229(47), 240 Swerdloff, R. S., 288 Sylvestre, C., 422 Szafran, H., 88 Szaro, R. P., 422 T Tager, H. S., 204 Takasingh, E. S., 51, 64(17) Takenaka, T., 236(129), 242 Takeo, K., 11, 12(78), 24(78), 30, 31(161, 163), 35(163)

504

AUTHOR INDEX

Talmage, D. W., 391 Tanabe, T., 12(101), 13, 33(101), 34(101) Tanaka, S., 460 Tanenbaum, S. W., 71, 91, 99, 100(24), 102 Tanswell, P., 227(14), 238 Tapley, D. F., 91, 95(28) Tasaki, I., 236(130), 242 Tashjian, A. H., 468 Taylor, G. H., 305, 310(2), 314(2) Taylor, R. B., 7 Tawde, S. S., 425 Teale, J. D., 209 Tedder, R. S., 233(95), 241 Teicher, E., 44 Teller, W. H., 182, 195 Templeton, C. L., 357 Tenenhouse, A., 159 Tener, G. B., 225,231(74), 240 Tenoso, H. I., 393 Terasaki, W. L., 392 Ternynck, T., 123, 131, 132(28), 165, 425, 431,447 Terry, W. D., 9 Tew, J. G., 114 Thom, E., 102 Thomas, M. J., 227(18), 239 Thompson, K., 45 Thompson, W., 13, 14(103) Thorbecke, G. J., 189 ThoreU, J. I., 344 Thorpe, P. E., 238(145), 243 Tich~i, M., 30, 31(162, 164) Timple, R., 227(14), 238 Tinelli, R., 12 Ting, R., 231(78), 241 Todd, P. E., 48 Topping, M. D., 378 Torres de ToledoeSouza, I., 322(7,8), 323 Tower, B. B., 209, 253, 322, 325, 326, 328, 329, 330(20), 333 Trachsel, H., 237(141), 243 Trafford, D. J. H., 208,292, 294, 295(11) Treffers, H. P., 18 Tregear, G. W., 204, 205(19), 271,349, 390, 406(15) Trembath, J., 69 Trouet, A., 104 Tsai, C.-M., 48, 205 Tsu, T., 176, 191(27) Tsuchiya, H. M., 21

Tsui, P. T., 98, 103(89) Tsuji, A., 90 Tsvetskova, V. S., 146 Tulchinsky, D., 103 Tumanova, A. E., 338 Tung, A. S., 51, 64(19) Tutwiler, G. F., 332 Twigg, M. B., 229(53), 240 Tyler, F. H. J., 93 Tyler, J. P. P., 291

U Uhr, J. W., 61, 63,214, 253 Ullman, E. F., 344, 345,419, 424(4), 440 Unanue, E. R., 108 Ungar-Waron, H., 71, 92, 100(33), 101,204, 2O5 Uotila, M., 433 Uriel, J., 425 Urosevic, Z., 191 Utermann, G., 228(27), 239 Utiger, R., 71, 76(14), 93, 98(41), 153, 154(9), 443,453(19) Utsumi, S., 142

V Vail, W. J., 357 Vaitukaitis, J., 113 Valdiguie, P., 149, 150(29) Valentine, R. C., 9 Vallotton, M. B., 58 Vance, V. K., 97, 161 Vander Laan, W. P., 209, 322, 326(1), 327, 328(1), 329(1), 332, 333(1) Vander Mallie, R. J., 227(21), 239 van der Straeten, M., 391 Vandevoorde, J. P., 208, 299, 304, 305 Van Eyck, H. G., 150 Van Leeuwen, G., 182 Van Oss, C. J., 19 Van Stenteghem, A. C., 231(79), 241 Van Vunakis, H., 45, 56, 71, 72, 79(8, 17), 89(42, 43, 44), 93, 153,204, 467, 468 Van Weeman, B. K., 439, 440(1), 441(1), 447, 451 Vaughan, J. R., Jr., 91

AUTHOR INDEX Velick, S. F., 9, 30 Venge, P., 226(6), 238 Verger, C., 80 Verwey, E. F., 356 Veyrjeres, A., 29, 36(148), 39(148), 41(148) Vicari, G., 6, 14(47), 36(47), 38 Viljanen, M. K., 390 Visser, T. J., 231(68), 240 Vitello, E. S., 214 Vitius, P., 93, 96(53) Vogelstein, B., 238(147), 243 Vogt, M., 260 Voller, A., 388, 390, 406(9), 423, 428(12), 438, 439 von Fellenberg, R., 467 Von Zur Muhlen, A., 325

W Waaikes, T. P., 71 Wagner, R., 154 Wajchenberg, B. L., 322(7, 8), 323 Wakizaka, A., 79 Waldron, C. B., 305,307(6), 310(6) Walinder, J., 272 Walker, C. S., 89 Walker, J. G., 63 Wallace, R. A., 228(38), 239 Wallace, S. S., 72, 73(20) Wang, R., 272 Ward, P. R., 292, 294(11), 295(11) Ward, S. M., 4 Warlield, D. T., 390 Warner, N. L., 5, 34(22), 35(22), 52, 65(23), 67, 136 Warren, R. J., 91, I01 Wasserman, E., 89(42), 93,467 Wasserman, L. R., 274, 275 Watanabe, F., 96 Watanabe, H., 391 Watanabe, K., 36 Waterman, M. R., 226(8), 238 Watkins, S., 232(83), 241 Watkins, W. M., 36 Watt, P. J., 357, 359(20) Waxdal, M. J., 8(67), 9 Webb, D. R., 299 Webster, R. G., 214, 252 Weed, J. A., 93

505

Weetall, H. H., 98, 391 Weigert, M. G., 27 Weigle, W. O., 59, 457 Weiner, L. M., 95 Weinheimer, P. F., 7 Weinryb, I., 233(101), 241 Weinstein, A., 154, 158 Weinstein, I. B., 100 Weir, D. M., 4, 6(10), 51, 58(13), 67(13) Weisgraber, K. H., 227(26), 239 Weissbach, A., 84 Weissman, I. L., 49 Welch, M. J., 222, 224(7), 226(3), 227(12), 238 Welsh, K. I., 357, 358(15), 372(15) Weliky, N., 98 Weltman, J. K., 422 Werner, I., 272 Werner, R. S., 322(7), 323 West, P. M., 391,392(38), 393(38) Westholm, F. A., 5 Weston, P. D., 132, 164, 165 Westphal, O., 57 Wetmur, J. G., 248 Wetter, O., 233(98), 241 Wettedow, L. H., 17 Wheeler, A. W., 103 White, R., 228(35), 232(35), 239 Whitehouse, M. W., 107 Wide, L., 232(94), 241, 271, 272, 336, 377, 378 Wie, S. I., 146 Wiegert, M. G., 11, 23(79), 25(79) Wienker, T. F., 228(27), 239 Wiesei, F.-A., 230(60, 61), 240 Wigzell, H., 357, 358(15), 372(15) Wilchek, M., 88, 153, 154(5), 155(5), 156(5), 159(5), 201,204 Wilde, C. D., 35, 137 Wilder, R. L., 357, 359(2) Wilding, P., 425 Wilham, C. A., 21 Wilhelmi, A. E., 468 Wilkinson, J. M., 42, 48(211) Willan, K. J., 356 Willerson, J. T., 226(8), 231(78), 238, 241 Willette, R. G., 207 Williams, C. A., 4, 6(8), 51, 58(12), 67(12), 88, 169, 174, 189, 192, 195,338 Williams, D. L., 207

506

AUTHOR INDEX

Williams, G. A., 159 Williams, M. R., 226(2), 238 Williams, P. L., 209 Williams, R., 228(29), 239 Wilson, A. C., 468 Wilson, D. V., 127 Wilson, M. B., 132, 133(33) Wimmer, K., 231(72), 240 Winitz, M., 88, 91(3) Wisdom, G. B., 438, 439 Wissler, F. C., 9 Woernley, D. L., 9 Wolberg, G., 459 Wolberg, W. H., 235(123), 242 Wold, F., 204 Wolff, S. M., 227(24), 239 Wolfrum, D.-I, 226(7), 238 Wollack, J., 100 Wolters, G., 432 Wong, D. C., 390 Wood, E., 29, 36(148), 39(148), 41(148) Wood, F. T., 221,244(2, 3), 245(2, 3) Wood, W. B., 49 Woodhead, J. S., 201,207, 208(8), 337, 344, 349(11), 353(11), 354 Woodnott, D. P., 112 Woods, G. F., 98 Woods, V. L., 357, 359(21) Worsley, I. G., 329 Wotiz, H. H., 89 Wright, B., 35, 137, 140(49), 141(49) Wright, C., 356 Wright, J. K., 11, 12(82), 23(82), 24(89), 233(107), 241,356 Wu, A., 11, 23(79), 25(79) Wu, H., 5 Wu, J. R., 70 Wu, M., 231(70), 235(122), 240, 242 Wu, T. T., 8, 9 Wyatt, R. G., 390

Y Yahr, M. D., 20 Yallow, R. S., 12, 31(168), 86, 201,202(1) Yalow, R. S., 322(6), 323, 325(2, 3), 328(6), 330, 335, 343,376, 378(2), 388 Yamamoto, K:-I., 233(106), 241 Yamamoto, T., 425,426 Yamashita, K., 228(30), 239 Yaron, A., 57 Yaverbaum, S., 312, 391 Yman, L, 384 Yoshioki, T., 236(129, 130), 242 Young, J. D., 48, 54, 93 Young, L. S., 234(113, 117), 242 Young, N. M., 4, 24(18), 25(18), 29(18) Young, R. L., 91, 98(31) Yue, D. K., 337, 349(11), 353(11) Yuen, C. C., 357, 359(21) Yuill, M. E., 94, 95 Yuspa, S. H., 100

Z Zabriskie, J. B., 229(49), 240 Zagyansky, Y. A., 381 Zamchuk, L. A., 79 Zappacosta, S., 142 Zeiss, C. R., 377 Zeitlin, A., 90, 154, 157(15), 158(15) Zeltzer, P. M., 357 Ziegler, A., 140, 141(51) Ziegler, D. W., 390 Zimmerman, J. E., 95 Ziola, B. R., 272, 390 Zopf, D. A., 48, 205 Zor, U., 153,154(5), 155(5), 156(5, 8), 159(5) Zurawski, V. R., 141 Zweig, M. H., 231(79), 241

SUBJECT INDEX

507

Subject Index

A Acetic anhydride tritiation procedure, 359 Acetylcholinesterase subunit, radioiodination, 232 Acetyl salicylic acid, immunogen preparation, 94-95 ACTH, s e e Adrenocorticotropic hormone Actin, radioiodination, 212 Acyl carrier protein, radioiodination, 228 Adenosine 3',5'-cyclic phosphate enzyme immunoassay, 442-454 immunogen preparation, 93, 98, 443 Adenosine-5'-diphosphate-ribose, immunogen preparation, 100 Adjuvant bacterial, 60 B o r d e t e l l a p e r t u s s i s vaccine, 157 choice of injection route, 112-113 Freund complete, 60 preparation, 109-110 composition, 108 effect on immune response, 57 in booster injections, 114-115 incomplete, 60 preparation, 108-109 immune response, 60, 107-108 in immunization, 59-60 mechanism of action, 107-108 types, 59-60 ADP-ribose, s e e Adenosine-5'-diphosphate-ribose Adrenocorticotropic hormone, immunogen preparation, 97, 161-162 Adriamycin, immunogen preparation, 96 Adsorption, of nonspecific antiserum, 122124 Affinity electrophoresis, for association constant measurement, 30-31 AKR virus, radioiodination, 212 Albumin, s e e a l s o Ovalbumin as carrier in immunization, 59 efficiency, 82 behavior in gel diffusion, 178-179

functional groups for conjugation, 87-88 immobilization, 165 immunogenicity, 86-87 induced tolerance, 62 methylated, in immunogen preparation, 79, 82 radioiodination, 212,226, 245-246 Alcohol, immunogen preparation, 97-100 Aldolase, radioiodination, 212 Aldosterone, immunogen preparation, 101 Aldosterone- 18-21-diacetate, immunogen preparation, 101 Alkaline phosphatase activity assay, 433 conjugate preparation for immunoassays, 432-433 Allergen, immobilization procedure, 380 Allergy, screening test, 376-387 Alpha-fetoprotein assay enzyme immunoassay, 433-438 immunoradiometric assay, 351- 352 radioimmunoassay, 212 radioiodination, 212, 300-301 Amikacin, radioiodination, 234 Amino acid polymers, as immunogens, 5051 Aminocellulose, for immunoadsorption, 338 -339 Aminophenyl derivative, use in immunogen preparation, 98, 154-155 Ammonium sulfate, use in radioimmunoassay, s e e Chemical precipitation separation method Ammonium sulfate test, s e e Farr test cAMP, s e e Adenosine 3',5'-cyclic phosphate Anaphylactic shock, during immunization, 64 Angiotensin, immunogen preparation, 92, 96 Antibiotic, radioiodination, 225 Antibody as receptor in immunoassay, 201-202 assay

508

SUBJECT INDEX

enzyme immunoassay, 201-202 Farr test, 69 gel diffusion, 68, 78 immunoelectrophoresis, 82-83 precipitin reaction, 19-20, 68, 78 red cell agglutination, 68 calcium requirement, 58 cell bound, assay, 372-375 coating of plastic tubes, 350-351 definition, 3 from ascites fluid, 64 monoclonal, 141 heterogeneity, 7 immobilization procedure, 427-429 labeling antiserum selection, 130 conjugate evaluation, 134-135 fluorescein method, 130-131 peroxidase method, 131-135 glutaraldehyde procedure, 132-133 periodate oxidation procedure, 133134, 426 with enzymes, 131-135,431,432 monoclonal, s e e Monocional antibody nonprecipitating, 18-19 detection, 192-194 origin, 3 production, 104-143, s e e also Hybridoma; Immunization; Monoclonal antibody purification, 83-84, 122-127,430-431, s e e a l s o specific method radioiodinated, storage, 347-348 radioiodination, 232-233,382 radiolabeling, 343-348 specific, purification, 84-85, 128-130, 337-343 storage stability, 120-121 valence, 7 Antibody specificity factors affecting, 157-159 principle, 72-73 to drugs, 203-204 to haptens, 202-205 to macromolecules, 205 Antigen, s e e also specific antigen assay, s e e specific immunoassay cell surface, rosette test, 466 competition, 64 definition, 3, 167 immobilization procedure, 427-429 on erythrocytes, 456-457

immunization dose, 64 immunogenicity, factors affecting, 53-58 multivalence, origin, 6 particulate, immune response, 61, 115 soluble, immune response, 61 tumor cell, assay for bound antibody, 372-375 type specific, from C h l a m i d i a t r a c h o m a t i s , radioiodination, 237 Antigen-antibody interaction, 3-49 analysis, 166-198 Antigenic determinant binding to antibody, 11-12 cation effect, 58 conformational, 54-55 definition, 3,167 exposure, by solvent, 55-56 factors defining, 42-44 hidden, 7 identification and localization, 41-49 sequential, 54-55 structural studies, 21-30 Apoprotein, radioiodination, 227 1-fl-o-Arabinofuranosylcytosine, immunogen preparation, 93, 98 Ascites fuid, as antibody source, 64, 141 Aspirin, s e e Acetylsalicylic acid Association constant 30-31, s e e also Equilibrium constant; Rate of dissociation measurement by affinity chromatography, 30-31 by equilibrium dialysis, 30 by fluorescence quenching, 30 Autoantibody, in cold agglutinin disease, 35-40 Avidin, radioiodination, 228 2,2'-Azino-di(3-ethyl-benzthiazoline sulfonic acid-6) ammonium salt, 431-432 Azo coupling, in immunogen preparation, 86-87, 155

B B lymphocyte, for antibody production, 141-142 Bacteria as adjuvant, 60 as immunogen, 115, 117-118 Bacteriophage, immune response, 61 Biotin, radioiodination, 236 Bisdiazobenzidine erythrocyte coating procedure, 459-460

SUBJECT INDEX Bleeding techniques, 64 Bleomycin, radioiodination, 236 Blocking factor, polypeptide, radioiodination, 229 Blood group substance, 35-40 substance I, 36-40 Bolton- Hunter radioiodination, 221- 247, s e e also Bolton-Hunter reagent labeling reagent availability, 223 principle, 221-222 procedure, 223-225 reagents for related methods, 221-222 results, 225 specificity, 222-223 Bolton-Hunter reagent, 221-223, s e e also BoRon-Hunter radioiodination availability, 223 preparation, 223-224 structure, 222 Booster injection, s e e Immunization B o r d e t e l l a p e r t u s s i s vaccine, as adjuvant, 157 Bradykinin, immunogen preparation, 96, 97

C Calcium, requirement by antibody, 58 Caprylic acid, for immunoglobulin separation, 125-126 Carbodiimide, s e e also 1-ethyl-3(3-dimethylaminopropyl) carbodiimide chemical action, 92 coupling procedure, 91-94, 156 for immunogen preparation, 76-77, 97100, 151-159 of hapten, 91-95 of nucleotides, 76-77 principle, 153-154 in ester preparation, 152-153 in peptide synthesis, 151-152 water soluble, 92-94 hapten coupling procedure, 76-77, 156 in peptide synthesis, 152 Carbonyldiimidazole, for hapten coupling, 95 Carboxyl derivative, for immunogen preparation, 100, 101 Carboxylesterase, serologic properties, 467 -479 O-(Carboxymethyl) hydroxylamine, for immunogen preparation, 101, 154-155

509

Carcinoembryonic antigen radioimmunoassay, 299-305 radioiodination, 228, 300-301 Carcinogen, immunogen preparation, 96 Carcinogen-deoxyribonucleic acid adduct, detection, 100 Cartier, s e e also Immunogen preparation; specific carrier, available linkage groups, 90-91 choice, 88-89, 106 epitope density, 89 hapten conjugation, 85-104 effect of linkage site, 90, 106, 157-159 use of epsilon amino groups, 87-88 in immunization, 53 Catalase, radioiodination, 212 Cations, effect on immune response, 5758 CEA, s e e Carcinoembryonic antigen Cell surface antigen, rosette test, 466 Charcoal separation method, 274-279 dextran coating procedure, 276-277 dose-response curve, 275-278 dose selection, 279 procedure, 276-278 limitations, 275 procedure, 279 time-response curve, 279 Chemical precipitation separation method, 280-291 advantages, 283-285, 291 choice of agent, 288 disadvantages, 290 principle, 286-288 procedure, 289 use in double-antibody method, 288-290 Chloramine-T, radioiodination, 210-213, s e e also Radioiodination advantages, 210 procedure, 210-211 sources of error, 211-213 Chloramphenicol, immunogen preparation, 96 Chlorocarbonate derivative, for immunogen preparation, 98, 154 Cholecystokinin, radioiodination, 230 Cholera toxin subunit, radioiodination, 237 Cholesterol, immunogen preparation, 100 Cholic acid, immunogen preparation, 91 Chromogen, for horseradish peruxidase assay, 431-432 Chymotrypsinogen, radioiodination, 212

510

SUBJECT INDEX

Clonazepam, radioiodination, 237 Clonazepam-3-hemisuccinate, immunogen preparation, 91 Cloning, s e e a l s o Hybridoma; Monoclonal antibody limiting dilution method, 140 soft agar method, 139-140 Coagulation factor Xa, radioiodination, 226 Cobra venom factor, radioiodination, 212 Cocaine metabolites, immunogen preparation, 93 Cold agglutinin disease, 35-40 Colony-stimulating factor, radioiodination, 237 Collagen, 429-430 CoUagenase, radioiodination, 212 Competitive binding assay, 31-35, s e e a l s o Immunoassay calculation of results, 32 effect of ligand valency, 33-34 principle, 31-32 separation of free and bound ligand, 3233 working range, 33-34 Complement effect on cell membranes, 256-258 effect on precipitation zones, 177-178 effect on radioimmunoassay, 267 Complement components, radioiodination, 213,232-234 Complement fixation test, 467-479 applications, 467-468 detection of conformational change in macromolecules, 467-479 determination of equilibrium constant, 472-473 determination of rate of dissociation, 473-479 Concanavalin A, radioiodination, 212, 229 Conjugation, of haptens, s e e Immunogen preparation from haptens Corticosterone, immunogen preparation, 101 Cortisol, immunogen preparation, 101 Cortisol-21-amine, immunogen preparation, 96 Coupling reagent, s e e Immunogen preparation; specific reagent Creatinine kinase, radioiodination, 231 Creatinine phosphokinase, radioiodination, 231

Cross-reactivity, of antibody, s e e Antibody specificity a-Crystallin, radioiodination, 212 CSF, s e e Colony stimulating factor Cyanogen bromide activation of cellulose particles, 379 of paper disks, 379 Cyclic adenosine monophosphate, s e e Adenosine 3',5'-cyclic phosphate Cyclic nucleotide, immunogen preparation, 71 l-Cyclohexyl-3-(2 morpholinyl-(4)-ethyl) carbodiimide, 152 Cytochrome c, radioiodination, 212 Cytochrome c polymer immunogen preparation, 162-164 immunogenicity, 163 Cytotoxic drugs, s e e specific drug Cytotoxicity, complement-dependent, effect on membranes, 255-258 D Deoxyribonucleic acid immunogen preparation, 102 modified antibody specificity, 79 immunogen preparation, 79 radioiodination, 248-251 reaction with antibody, 79-81 Deoxyribonucleic acid-binding protein, radioiodination, 231 Deoxyribonucleoside, immunogen preparation, 71 Deoxyribonucleotide, immunogen preparation, 71 Dextran antibody, 21-25 for structural studies, 21-26 immunogenicity, 57 precipitin reaction with antibody, 21-25 Dialdehyde derivative, for immunogen preparation, 99-100 O-Dianisidine, 423 Diazocellulose, for immunoadsorption, 339 -340 Diazotized aniline radioiodination, 246-247 procedure, 246 for membranes, 253-255 reagent availability, 253-254

SUBJECT INDEX reagent structure, 222 results, 246-247 Digitoxigenin, immunogen preparation, 93 Digoxin, immunogen preparation, 99-100 Dinitrophenylation, for immunogen evaluation, 103, 157 Diphtheria toxin, radioiodination, 238 DNA~ s e e Deoxyribonucleic acid Domain, of immunoglobulin G, 232 Dopamine B-hydroxylase, radioiodination, 232 Double-antibody separation method, 266274 advantages, 266 in enzyme immunoassay, 440-441 post-precipitation method principle, 266-267 procedure, 268-270 requirements, 267-268 second antibody titration, 270 sources of error, 267 pre-precipitation method disadvantages, 270-271 principle, 270 with nonimmune globulin advantages, 273 precipitate preparation, 273-274 principle, 273 procedure, 274 use of chemical precipitation, 288-290 Drugs, s e e a l s o specific drug antibody specificity, 203-204 cytotoxic, immunogen preparation, 104 synthetic, analgesic and narcotic, immunogen preparation, 93

E Ecdysone, immunogen preparation, 95 Ecdysterone, immunogen preparation, 98 Electrode-based enzyme immunoassay, 439-455 advantages, 447 equipment, 442 procedure conjugate preparation, 442-444 enzyme activity detection, 444-447 optimal conditions, 449-450 reagents, 442

5 11

reproducibility, 454 results, 448-454 Electrophoresis, see Immunoelectrophoresis Electrosyneresis, 176-177, s e e a l s o Immunoelectrophoresis ELISA, s e e Enzyme-linked immunosorbent assay EMIT, s e e Enzyme multiplied immunoassay technique Endotoxin, radioiodination, 212 Enzyme conjugate preparation, 131-135,425426, 431,432-434, 442-444 immobilization procedure, 164-165 immobilized, immunogen preparation, 164-165 immunoassay, s e e specific assay labeling, 131-135, 425-426, 431,432434, 442-444 radioiodination, 231-232 studies with antibody, 49 Enzyme-linked immunosorbent assay, 419 -439, s e e a l s o Enzyme multiplied immunoassay technique advantages, 409 applications, 429-430 automation equipment, 392-394 competitive assay disadvantages, 423 principle, 420-421 enzyme conjugate characteristics, 426-427 preparation, 425-426 enzyme selection, 423-424 noncompetitive assay advantages, 423 principle, 422-423 principle, 419 procedure antigen quantitation, 436-438 antibody purification, 430-431 antigen and antibody immobilization, 427 -429 conjugate preparation, 131-135,431, 432-434 conjugate testing, 434 enzyme activity detection, 431-433 sandwich method, 422-423 sensitivity comparative, 409

5 12

SUBJECT INDEX

factors affecting, 438 separation procedure, magnetic transfer method, 388-416 solid phase advantages, 389-391 materials, 390-391,427-429 Enzyme multiplied immunoassay technique, 419-439, s e e also Enzymelinked immunosorbent assay competitive assay, principle, 420 enzyme conjugate characteristics, 426-427 preparation, 425-426 enzyme selection, 424-425 principle, 419 Epitope definition, 167 density, 89 DL-10, ll-Epoxyfarnesoic acid, immunogen preparation, 95 Equilibrium constant determination by complement fixation, 472-473 effect of salt concentration, 478-499 effect of temperature, 479 Equilibrium dialysis, for determination of association constant, 30 Equivalence zone, of precipitin reaction, 15 Erythrocyte agglutination, 68 agglutination test, 455-466 coating with antigen, 456-457, 459-460 with nucleosides, 75-76 rosette test, 466 tanning procedure, 458-459 E s c h e r i c h i a coli, as immunogen, 117-118 Ester hydroxysuccinimide derivative, for immunogen preparation, 156 synthesis, 152-153 Estradiol, immunogen preparation, 98 17-fl-Estradiol, immunogen preparation, 98 Estrogen, synthetic, immunogen preparation, 91 Estrone, immunogen preparation, 98, 101 l-Ethyl-3-(3 dimethylaminopropyl) carbodiimide, 76-77, 152 Eukaryotic initiation factor, radioiodination, 237

F Fab fragment preparation from immunoglobulin G by proteolysis, 142-150 assay of digest, 145 detection of contaminants, 148 detection of product, 147-148 proteolysis procedure, 144-145 purification, 145-150 removal of contaminating Fc, 148-150 by affinity chromatography, 148-150 by protein A-Sepharose, 150 by miscellaneous methods, 150 structure, 142 uses, 142-143 Farr test, 69 Fc fragment preparation, 146-147 removal from Fab preparation, 148-150 by affinity chromatography, 148-150 by protein A-Sepharose, 150 by miscellaneous methods, 150 Ferritin, radioiodination, 212, 227 Fetuin, radioiodination, 212 Fibrin, radioiodination, 212 Fibrinogen radioiodination, 212, 227 fragment, radioiodination, 227 Fibronectin, 429-430 Flagellin, immunogenicity, 57 Flocculation curve, 17, s e e a l s o Precipitin reaction Fluorescein, for antibody labeling, 130-131 Fluorescence quenching, for association constant measurement, 30 Fluoxymesterone, immunogen preparation, 95 Follicle stimulating hormone, radioiodination, 230 Follitropin, radioiodination, 230 Freund adjuvant, s e e Adjuvant Fructosan antibody specificity, 24-25 precipitin reaction with antibody, 24-25 FSH, s e e Follicle stimulating hormone G /3-Galactosidase, radioiodination, 212 Gastrin

5 13

SUBJECT INDEX immunogen preparation, 93 tetrapeptide, immunogen preparation, 92 Gel centrifugation separation method, 315322 gel column preparation, 316 practicability, 321 procedure, 316-317 reliability, 321-322 results precision, 321 separation efficiency, 317-321 Gel diffusion, s e e a l s o Precipitin reaction in gels analysis of results, 183-192 applications, 194-198 antibody assay, 68, 78 antibody-antigen analysis, 165-175, 183-198 detection of nonprecipitating antibody, 192-194 precipitation zone identification, 183-188 mathematical analysis of position, 179 183 mobility, 179-183 procedure double one-dimensional, 170, 172 two-dimensional, 170, 172, 173-175 quantitative, 170, 171-173, 189-191 simple one-dimensional 169-172, 189-190 two-dimensional, 170, 172-173, 190 -191 Genistein, immunogen preparation, 156 Gentamycin immunogen preparation, 93, 96 radioiodination, 225, 234 GH, s e e Growth hormone Glucagon, immunogen preparation, 97, 162 Glucose oxidase, for lactoperoxidase-catalyzed radioiodination, 330-332 #-Glucuronidasc, radioiodination, 232 L-Glutamic acid polymer, radioiodination, 229 Giutaraldehyde applications antibody purification, 123-124 detection of nonprecipitating antibody, 194 enzyme labeling, 426 -

immunogen preparation, 159-165 of peptides, 161-162 of polymerized proteins, 162-164 of protein-particle conjugates, 164165 peroxidase labeling of antibody, 132133 protein immobilization, 164-165 polymerization, for antibody purification, 123-124 reaction with protein, 160 Glycerol, immunogen preparation, 99-100 Glycol, immunogen preparation, 99-100 Glycophorin A, tryptic peptide, radioiodination, 229 Glycoside immunogen preparation, 99-100 radioiodination, 234-235 Gonadotropin-releasing hormone, immunogen preparation, 155 Gonococcai pili, radioiodination, 236 gp virus, radioiodination, 212 Growth hormone immunoradiometric assay, 352-353 radioiodination, 224-225,230 H Hapten antibody specificity, 202-205 assay for incorporation into immunogen, 103, 156-157 available linkage groups amino, 96-97, 154-155 choice for coupling, 202 carbonyl, 101, 154-155 carboxyl, 91 guanido, 155 hydroxyl, 97-100, 154-155 vicinal hydroxyl, 99-100 azo coupling to protein, 86-87 definition, 3 direct reaction with protein, 102-103 immunogcn preparation, 85-104, s e e a l s o Immunogen preparation from haptens immunogenicity, 85-88 inhibition of precipitin reaction, 26-30 radioiodination, 236-237 HAT medium, for hybridoma production,

5 14

SUBJECT INDEX

65, 136, s e e a l s o Hybridoma; Monoclonal antibody HCG, s e e Human chorionic gonadotropin Helical nucleic acid, s e e Nucleic acid Hemagglutination assay, 455-466, s e e a l s o Hemagglutination inhibition assay; Hemolytic plaque assay; Passive hemolysis assay equipment, 457 principle, 456-457 procedure antigen coating of cells, 456-457, 459460 controls, 461 plate method, 461 tanning of cells, 458-459 tube method, 460 reagents, 457-458 results, interpretation, 461 reverse method, 461-462 sensitivity, 455 Hemagglutination inhibition assay optimal conditions, 468 principle, 456, 462 procedure antibody titration, 462 plate method, 463 tube method, 462-462 Hemisuccinate derivative, for immunogen preparation, 97-98, 154-155 Hemoglobin, radioiodination, 212,227 Hemolytic plaque assay, 463-465, s e e a l s o Passive hemolysis assay preparation of nucleoside coated cells, 75-76 reverse method, 465-466 Hemophilus influenza, capsular polysaccharide, radioiodination, 235 HGPRTase, s e e Hypoxanthine guanine phosphoribosyltransferase HLA antigen, radioiodination, 237 Homopolynucleotlde, s e e a l s o Polynucleotide aggregated, immunogen preparation, 8182 antibody specificity, 79 as immunogen, 81-82 immunogen preparation, 79 multiple helical forms, preparation, 8182 Hormone antibody specificity, 157-159

radioiodinated screening test, 322-334, s e e a l s o Talc -resin-trichloroacetic acid test storage stability, 327-328, 332 radioiodination, 327-330 storage, 327-330 Horseradish peroxidase, s e e a l s o Peroxidase activity assay, colorimetric, 431-432 conjugate preparation, 431 radioiodination, 212 5-HT, s e e 5-Hydroxytryptamine Human chorionic gonadotropin, in urine, radioimmunoassay, 370-371 Human milk virus-like particle, radioiodination, 235 Hybridoma, s e e a l s o Monoclonal antibody for antibody production, 34-35, 65-66, 135-142 production, 34-35 assay of hybrid products, 139 cell fusion procedure, 138 choice of fusion partners, 136-138 cloning of active hybrid, 139-140 growth of fused cells, 139 immunization of spleen cell donor, 138 spleen cell preparation, 138 Hydroxyapatite separation method, 291298 effect of protein, 295-298 procedure for steroids, 292-295 results, 295, 298 3-Hydroxyclonazepam, immunogen preparation, 98 5-Hydroxytryptamine, 96, 102 Hypoxanthine guanine phosphoribosyltransferase, for hybridoma production, 34-35, 65, 136, s e e a l s o Hybridoma; Monoclonal antibody

IgA, s e e Immunoglobulin A IgD, s e e Immunogiobulin D IgE, s e e Immunoglobulin E IgG, s e e Immunoglobulin G IgM, s e e Immunoglobulin M Immune complex, radioiodination, 212 Immune response, factors affecting, 51-58, 105-115 genetic differences, 52-53, 112 lag phase, 61

SUBJECT INDEX primary, 60-61 secondary, 60-62 to adjuvant, 107-108 to bacteriophage, 61 to particulate antigen, 115 Immunization, 59-60, 104-121 adjuvant preparation, 107-110 animal choice, 51-52, 110-112 number, 111 antibody produced affinity, 63 assay, 66-69, 78, 82-83 antigen dose, 62-63, 113-115 antiserum collection and storage, 120-121 defatting procedure, 121-122 immunoglobulin separation, 124-127 purification, 83-85, 122-124 specific antibody separation, 127-130 bleeding techniques, 64 booster injections, 114-115 carriers, 53 homologous, 111 methods, 58-65, 77-78, 115-120 for hybridoma spleen cell donor, 138 guinea pig, 118-119 rabbit, 116-118, 157 sheep, 119-120 procedure for nucleic acid, 77-78 helical, 82 for particulate antigen, 82 for polynucleotide, 82 for protein, 64-65 route, 63, 112-113 Immunoadsorbent characterization, 341-342 preparation, 337-340 reaction with protein, 340-341 storage, 342 Immunoadsorption for Fab preparations, 148-150 for immunoglobulin E preparation, 381382 for specific antibody separation, 127-130 use in precipitin reaction, 20 Immunoassay, s e e a l s o specific method automation equipment, 392-394 complement fixation test, 467-479 enzyme-linked immunosorbent assay, 419-439

5 15

enzyme multiplied immunoassay technique, 419-439 hemagglutination assay, 455-466 immunoradiometric assay, 334-355 magnetic transfer immunoassay, 388416 radioallergosorbent assay, 376-387 radioimmunoassay, 201-209 radiolabeling procedures 221-265 screening test for radiolabeled hormones, 322-334 separation procedures, 266-322, 388416 charcoal method, 266-274 chemical precipitation method, 280291 double-antibody method, 266-274 gel centrifugation method, 315-322 hydroxyapatite method, 291-298 magnetic transfer method, 388-416 microfiitration method, 305-314 zirconyl phosphate gel method, 299305 solid phase automation, 388-416 enzyme methods, 419-439 immunoradiometric assay, 334-355 radioallergosorbent assay, 376-387 radioimmunoassay, 356-375, 376-387 tracer, radioiodinated protein A, 356375 Immunochemical analysis, s e e specific method Immunoelectroosmosis, s e e Electrosyneresis Immunoelectrophoresis analysis of results, 183-192 applications antibody assay, 82-83 antigen-antibody analysis, 166-169, 175-198 precipitation zone identification, 183-188 mobility, 179-183 procedure antigen electrophoresis, 175-176 crossed-fields method, 177 electrosyneresis, 176-177 prior to gel diffusion, 170, 174-175 quantitative, 191 Immunogen, 105-107 definition, 3

5 16

SUBJECT INDEX

factors affecting response, 50, 105-107 antigen dose, 62-63 cations, 57-58 degradation in vivo, 106 degree of polymerization, 162-164 dose, 113-115 molecular size, 57 molecular weight, 57, 105-106 protein aggregation, 53 protein denaturation, 53-54 route of injection, 112-113 Stoke radius, 54 structural complexity, 42-46, 56-57 tertiary structure, 104 Immunogen preparation, s e e also Immunogen preparation from haptens alcohols, 97-100 carbodiimide method, 91-94, 151-159 cyclic nucleotides, 71 deoxyribonucleic acid, modified, 79 deoxyribonucleosides, 71 deoxyribonucleotides, 71 guanosine oxidation procedure, 74 haptens, 85-104 homopolynucleotides, 79 aggregated, 21-22, 82 7-Methylguanosine, hydrolysis prevention, 74-75 nucleic acids, 71-72 helical, 81-82 nucleosides, 97-100 nucleotides, 76 succinylated, 76-77 oligonucleotides, 76-77 peptides, 160-162 periodate oxidation procedure, 72, 73 phenols, 97-100 polynucleotides, single stranded, 78-79 polysaccharides, 97-100 proteins, 59 immobilized, 164-165 polymerized, 162-164 purity, 106-107 ribonucleosides, 71, 74-76 ribonucleotides, 71 steroids, 97-100 sugars, 97-100 use of glutaraldehyde, 159-165 Immunogen preparation from haptens assay for hapten incorporation, 77, 103, 156-157

azo coupling, 86-87, 155 carbodiimide method, 91-94, 153-154 carbohydrates, 97-100 cartier choice, 88-89 effect of linkage site, 90, 157-159, 202 -203 choice of functional group, 202 aminophenyl, 98, 154-155 carboxyl, 94-95, 100, 154-155 from hydroxyl group, 97-100, 154155 carboxymethyl oxime, 101, 154-155 chlorocarbonate, 98, 154 dialdehyde, 99-100 hemisuccinate, 97-98, 154-155 conjugate solubility, 88-89 direct reaction with protein, 102-103 epitope density, 89 estimation of free amino groups, 103, 157 hydroxyl methods, miscellaneous, 100 Mannich reaction method, 102 miscellaneous methods, 101-103 mixed anhydride method, 91 periodate oxidation method, 99-100 Schiff base formation method, 101 water insoluble haptens, 93 water soluble haptens, 156 with amino groups, 96-97, 154-155 aliphatic, 96-97 aromatic, 96 with carbonyl groups, 101, 154-155 with carboxyl groups, 91-96 with double bonds, 154 with guanido groups, 155 with hydroxyl groups, 97-100, 154-155 Immunogenicity, of small molecules, 8588 Immunogiobulin,s e e also specific class antidextran myeloma, structure, 21-25 classes, 3 labeling fluorescein method, 130-131 peroxidase method, 131-135 with enzymes, 131-135, 431,432 separation from serum, 124-127 caprylic acid method, 125-126 ion exchange chromatography, 126127 rivanol method, 124-125

SUBJECT INDEX serum concentration, 377 structure, 7-11 radioiodination, 212,215-216 ImmunoglobulinA J chain, 9 myeloma, precipitin reaction, 24-25 nonprecipitating monomers, 18-19 radioiodination, 212 structure, 9, 11 secretory protein, radioiodination, 233 ImmunoglobulinD radioiodination, 212 structure, 11 valence, 7 ImmunoglobulinE assay radioallergosorbent, 385-386 radioimmunoassay, 370-371 purification, 381 radioiodination, 212, 233 valence, 7 ImmunogiobulinG aggregated, radioiodination, 212 appearance in immune response, 60-62 assay immunoradiometric, 349 radioimmunoassay, 360-364, 371 binding to protein A, 356-357 cell-bound, quantitation, 372-375 Fab fragment, preparation, 142-150 fragments, radioiodination, 212, 233 immobilization, 165 myeloma, precipitin reaction, 24-25 papaln treatment, 144-145 radioiodination, 212,232-233,344 separation from serum, 126-127, 370 storage stability, 120 structure, 8-9, 10-11 domains, 142-143 hinge region, 142-143 subclass properties, 143-144 separation, 144 valence, 7 ImmunoglobulinM appearance in immune response, 60-62 assay, 370-371 cell-bound, quantitation, 374-375 myeloma, precipitin reaction, 24-25 radioiodination, 212 storage stability, 121

5 17

structure, 9, 11 valence, 7 Immunoradiometric assay, 334-355, s e e a l s o Radioimmunoassay antibody coating of tubes, 350-351 antibody, labeled, characterization, 347 elution, 345-347 storage, 347-348 antibody labeling radioiodination, 343-345 miscellaneous, 344-345 antibody purification immunoadsorbent preparation, 337342 immunoadsorption procedure, 342343 automation, 353-354 direct disadvantages, 355 general, 348-349 principle, 348 procedure, 349 direct, two site advantages, 355 principle, 336, 349 procedure, 351 solid phase antibody preparation, 349351 general principle, 334-337 indirect advantages, 352 principle, 337, 352 procedure, 352-353 indirect, two site advantages, 355 principle, 337 procedure, 353 Indomethacin, radioimmunoassay, 372 Inhibition reaction, use in structural studies, 26-30 Inhibition zone, of precipitin reaction, 15 Insulin antibody production, 110, 117-118 radioiodination, 212, 301 radioimmunoassay, 299-305 Interferon, radioiodination, 227 Inulin, precipitin reaction, 24 Iodothyronine, radioiodination, 237 Ion exchange chromatography, for immunoglobulin separation, 126-127

518

SUBJECT INDEX

Isomaltotetraose, radioiodination, 235 Isotope, for radioimmunoassay, 206

J J chain, of immunoglobulin A, 9 Juvenile hormone, see DL-10,1l-Epoxyfarnesoic acid

K

Keyhole limpet hemocyanin as carder, 89 radioiodination, 212 Kinase, radioiodination, 232 Klebsiella pneumoniae subunit, radioiodination, 235 KLH, see Keyhole limpet hemocyanin Kojibiose, 21 in precipitin inhibition structural studies, 27

L Lactoferrin, radioiodination, 227 Lactoperoxidase-catalyzed radioiodination, 214-220, see also Radioiodination advantages, 214-215 factors affecting labeling, 215 for protein localization, 218-220 advantages, 218 disadvantages, 218 procedure, 215-216 procedure for membranes, 219-220, 258-261 recommendations, 219-220 glucose oxidase procedure, 330-332 product isolation, 216-218 Lectin Axinella polypoides, specificity, 30 Bandeiraoa simplifolia, specificity, 2930 definition, 3 radioiodination, 212,228-229, 246-247 Leucovorin, radioimmunoassay, 372 Leuteinizing hormone, radioiodination, 230 Levan, precipitin reaction, 24-25 LH, see Leuteinizing hormone

Liesegang phenomenon, 166-167, 172 Limiting dilution cloning, 140, see also Cloning Lipid cell surface quantitation of release, 261-265 radioiodination, 252-265 plasma membrane, radioiodination, 258261 Lipoprotein cell surface, quantitation of release, 255 -258 radioiodination, 212, 228 Lipoprotein lipase, radioiodination, 232 Liposome, radioiodination, 260-261 Loop peptide, of lysozyme, 43-46 Lutotropin, radioiodination, 230 Lymphocyte, membrane radioiodination, 219-220 Lysergic acid diethylamide, immunogen preparation, 93 Lysozyme antigenic sites, 46-47 effect of structure on antibody specificity, 42-46 structure, 43

M

M protein, radioiodination, 229 o~-Macroglobulin, radioiodination, 212 Macrophage, response to adjuvant, 107108 Magnetic transfer immunoassay, 388-416 advantages, 403-406, 414-416 applications, 410-414 materials, 395-398 procedure for enzyme immunoassays, 398-401 for radioimmunoassays, 401-403 Major histocompatibility complex, effect on immune response, 52-53 Maltose, in precipitin inhibition structural studies, 27-28 Mannich reaction, for hapten immunogen preparation, 102 Medroxyprogesterone acetate, immunogen preparation, 101 a-Melanotropin, immunog©n preparation, 93

SUBJECT INDEX Membrane antigen, rosette test, 466 Membrane components quantitation of release, 256-258 radioiodination, 212, 219-220, 235,252265 diazotized sulfanilic acid method, 253255 peroxidase-catalyzed method, 219-220 applications, 258-259 procedure, 259-261 /3-dl-Methadol, immunogen preparation, 98 DL-Methadol-hemisuccinate, immunogen preparation, 93 Methotrexate radioimmunoassay, 372 radioiodination, 236 Methyl p-hydroxybenzimidate radioiodination procedure, 244-245 reagent structure, 222 results, 245-246 Microfiltration separation method, 305-314 automation, 307-311 filter choice, 314 procedures filter paper, 306-307 CRC thimble, 307-308 radioactive counting, 313-314 sample preparation, 311- 313 ~-Microglobulin, radioiodination, 212 Mixed anhydride coupling method, 91-92 Moloney virus, radioiodination, 212 Monocional antibody, see also Hybridoma from hybridoma, 34-35, 136-140 in cold agglutinin disease, 35-40 production, 65-66, 135-142 assay of hybridoma product, 139 in ascites fluid, 141 in vitro, 140 in vivo, 141 Monosuccinyl ecdysterone, immunogen preparation, 91 Morphine, immunogen preparation, 93 Mucin, desaialylated, radioiodination, 228 Mycobacteria, as adjuvant, 60 Myeloma antibody precipitin reaction, 21-26 radioiodination, 233 antidextran, structure, 21-25

5 19

antifructosan, structure, 24-25 cell lines, for hybridoma production, 136 137 Myoglobin, radioiodination, 226 -

N Neocarzinostatin, radioiodination, 229 Nephritic factor, radioiodination, 237 Nerve growth factor, radioiodination, 228 Netilmycin, radioiodination, 234 Neuraminidase, radioiodination, 232 Nigerose, in precipitin inhibition structural studies, 27-29 a-Nigerosyl-1,3-nigerose, radioiodination, 235 Ninhydrin procedure, for quantitative precipitin reactions, 14-15 Normetanephrine, immunogen preparation, 97 Nortryptyline, immunogen preparation, 97 Nuclear protein, soluble, radioiodination, 228 Nucleic acid, see also Nucleic acid, helical; Nucleoside; Nucleotide antibody, 70-85 assay, 78 purification, 78 as antigen, 70-85 cartier requirement, 70 conjugation to erythrocytes, 75-76 immunogen preparation, 74-77 radioiodination, 231,247-252 precautions, 250 procedure, 250 product storage, 251-252 reaction conditions, 248-251 requirements, 247 Nucleic acid, helical antibody assay, 82-83 purification, 83-85 specificity, 79-81 as antigen, 79-85 immunogen preparation, 81-82 structural studies with antibody, 79-81 Nucleoside antibody specificity, 72-73, 78 conjugation to erythrocytes, 75-76 immunogen preparation, 97-100

520

SUBJECT INDEX

measurement of substitution in immunogen, 77 Nucleotide, s e e a l s o Oligonucleotide antibody specificity, 72-73 immunogen preparation, 76

O Ochratoxin A, immunogen preparation, 93 Oligonucleotide, immunogen preparation, 76-77 Oligosaccharide radioiodination, 234-235 structural studies, 26-30 Orosomucoid agalacto, radioiodination, 228 asiaio, radioiodination, 228 Ouabain, immunogen preparation, 99-100 Ouchterlony diffusion, s e e Gel diffusion Ovalbumin, immune response, s e e a l s o Albumin effect of deamination, 54 effect of denaturation, 54 structural studies, 15-17 Ovarian cyst fluid, blood group determinants, 36-40

P pl5E virus, radioiodination, 212 p30 virus, radioiodination, 212 Papain radioiodination, 212,244 treatment of immunoglobulin G, 144-145 Parathyrin, radioiodination, 228 Parathyroid hormone, antibody production, 117-118 Parathyroid hormone fragment immunogen preparation, 204 radioiodination, 231 Passive hemolysis immunoassay, 463-465, s e e a l s o Hemagglutination assay principle, 463-464 procedure, 464-465 reagents, 464 reverse plaque procedure, 465-466 PEG, s e e Polyethylene glycol Penicillenic acid, immunogen preparation, 101- 102

Penicillin, immunogen preparation, 102-103 Peptide immunogen preparation, 160-162 radioiodination, 226-230 Peptide synthesis, use of carbodiimide, 151-152 Periodate oxidation labeling for antibody, 133-134, 426 for immunogen preparation, 72, 73, 99100 Peroxidase labeling procedure, for antibody, 131-135,431, s e e a l s o Horseradish peroxidase Peroxidase-catalyzed radioiodination, 214220, s e e a l s o Lactoperoxidase-catalyzed radioiodination Phenols, immunogen preparation, 97-100 L-Phenylalanine mustard, immunogen preparation, 103 O-Phenylenediamine, 432 Phosphoenolpyruvate carboxykinase, radioiodination, 232 Phosphorylase A, radioiodination, 212 Phytohemagglutinin, radioiodination, 212 Plasmin, radioiodination, 212 Plasminogen, radioiodination, 212 Platelet factor, radioiodination, 226 Pneumococcal polysaccharide, immunogenicity, 57 Poly (L-lysine) as carrier, 89 radioiodination, 230 Polyacrylamide, dinitrofluorobenzene derivative, radioiodination, 238 Polyethylene glycol for hybridoma production, 65, 138 for radioimmunoassay separation, 286 Polymaleic anhydride particles, 380 Polymerization, of protein, for immunogen preparation, 162-164 Polynucleotide, s e e a l s o Homopolynucleotide; Nucleotide single stranded as haptens, 78-79 immunogen preparation, 78-79 synthetic antibody specificity, 80-81 as immunogens, 81-82 Polypeptide as antigens, 49-70 radioiodination, 229

SUBJECT INDEX Polysaccharide immunogen preparation, 97-100 radioiodination, 234-235 structural studies, 26-30, 35-40 Precipitin reaction, s e e Precipitin reaction in gels; Precipitin reaction, quantitative Precipitin reaction in gels, 166-198, s e e a l s o Gel diffusion; Immunoelectrophoresis analysis of results, 183-192 antigen, number determination, 183 applications, 194-198 detection of nonprecipitating antibody, 194-198 diffusion methods, 169-175 effect of complement, 177-178 effect of reagent concentration, 179-183 electrophoretic methods, 175-177 historical background, 166-167 Liesegang phenomenon, 166-167, 172 partial continuity reactions, 187-188 precipitation zones identification, 183-188 improving visibility, 188-189 independence, 179 mathematical analysis of position, 179-183 mobility, 179-183 multiple, 177-179 principles, 177-183 quantitative, 189-192 sources of error, 177 Precipitin reaction, quantitative, 13-30, s e e a l s o Precipitin reaction in gels applications antibody assay, 19-20, 68, 78 antibody purification, 84 antigen assay, 20-21 structural studies, 21-30 cross-reactions, 26 equations, 19 equivalence zone, 15 inhibition by hapten, 26-30 inhibition zone, 15 nonprecipitating antibody, 18-19 procedure, 13-21 ninhydrin reaction, 14-15 with cold agglutinins, 14 with horse antisera, 14 solubility of precipitate, 13

521

studies with dextrans, 21-25 with fructosans, 24-25 with myeloma antibody, 21-26 with ovalbumin, 15-17 use of immunoadsorbent, 20 working range, 13 zones, 15 Prednisone-2 l-hemisuccinate, immunogen preparation, 93 Primary immune response, 60-62 Pristane, for monoclonal antibody production, 66 Progesterone antibody specificity, 158-159, 202-203 immunogen preparation, 101, 102, 155 synthetic, immunogen preparation, 101 Proinsulin, immunoradiometric assay, 353 Propanolol, immunogen preparation, 98 Prostaglandin, immunogen preparation, 91, 93 Prostaglandin Fa, immunogen preparation, 156 Protamine, radioiodination, 212 Protein, s e e a l s o Protein antigen; specific protein carriers, 86-87, 106 cell-associated radioiodination, 252-265 quantitation of release, 255-258 in gels, radioiodination, 213 radioiodination, 210-220, 224-230 Protein A, s e e a l s o Protein A tracer in radioimmunoassay affinity column, for Fc binding, 150 characterization, 356-357 radioiodination, 212,229, 358-359 tritiation, 359-360 Protein A tracer in radioimmunoassay, 356-375, s e e also Protein A advantages, 372 applications, 370-375 analysis of cell-bound antibody, 372375 immunoglobulin G, 373-374 immunoglobulin M, 374-375 miscellaneous, 370-375 to antigens and haptens principle, 364 procedure, 364-368 results, 370-371

522

SUBJECT INDEX

to fluid-phase immmunoglobulin G, principle, 360 procedure, 360-364 results, 362-364 ligand immobilization procedure, 364365 principle for antigens and haptens, 364 for immunoglobulin G, 360 procedure for antigens and haptens, 364-368 for immunoglobulin G, 360-364 radiolabeling procedure, 357-360 iodination, 358-359 tritiation, 359-360 results for antigens and haptens, 370-371 for immunoglobulin G, 362-364 sources of error, 367-370 test sample pre-absorption, 370 Protein antigens, 49-70, s e e a l s o specific antigen conjugation with haptens, 86-87, 102103 determinants, 54-55 factors affecting immunogenicity aggregation, 59 cations, 57-58 denaturation, 53-54 determinant accessibility, 55-56 molecular size, 57 molecular weight, 57 quaternary structure, 55 structural complexity, 56-57 immobilization procedure, 164-165 immobilized, immunogen preparation, 164-165 immunization procedure, 58-64 immunogen preparation, 59 polymers, immunogen preparation, 162-164 Protein-nucleic acid complex, radioiodination, 231 Prothrombin, radioiodination, 212 Pseudouridine, immunogen preparation, 100 Purogen, human leukocytic, radioiodination, 227 Pyridoxal, immunogen preparation, 101 Pyridoxal phosphate, immunogen preparation, 101

Q Quantitative complement fixation test, s e e Complement fixation test Quantitative precipitin curve, 13-30, s e e a l s o Precipitin reaction, quantitative Quantitative precipitin reaction, s e e Precipitin reaction, quantitative

R Radioiodination, s e e a l s o specific ligand antibody, 232-233, 343-348 complement components, 232-234 enzymes, 231-232 glycosides, 234-235 haptens, 236-237 hormones, 230-231,330-332 screening test for product, 322-334 storage stability, 327-330 lipids, cell surface, 258-261 liposomes, 260-261 membrane components, 219-220, 235236,252-265 miscellaneous, 237-238 nucleic acids, 231,247-252. oligosaccharides, 234-235 peptides, 226-230 polysaccharides, 234-235 proteins, 226-230 in gels, 213 protein A, 357-359 protein-nucleic acid complexes, 231 toxins, 237-238 viruses, 212,235-236 Radioiodination procedure, s e e a l s o Radiolabeling procedure; specific method BoRon-Hunter method, 221-225 chloramine-T method, 210-213 diazotized anifine method, 246-247 diazotized iodosulfanilic acid method, 252-255 lactoperoxidase-catalyzed method, 214220, 258-261 using glucose oxidase, 330-332 methyl p-hydroxybenzimidate method, 244-246 miscellaneous, 244

SUBJECT INDEX Radiolabeling, of specific ligands, s e e Radioiodination Radiolabeling procedure, s e e a l s o specific method acetic anhydride tritiation, 359 BoRon-Hunter iodination, 221-247 chloramine-T iodination, 210-213 diazotized aniline iodination, 246-247 diazotized iodosulfanilic acid iodination, 252-255 iodinations, miscellaneous, 244-247 general considerations, 206-207 lactoperoxidase-catalyzed iodination, 214-220, 258-261 using glucose oxidase, 330-332 reductive methylation tritiation, 359-360 Radioallergosorbent test, 376-387 advantages, 376-377 automation, 388-416 indirect method, 384 mixed allergen method, 385-386 mixed particle method, 386-387 principle, 377-378 procedure allergen insolubilization, 380 allergen particle potency assay, 386 anti-immunoglobulin E purification, 381-382 anti-immunoglobulin E radioiodination, 382 immunoglobulin E reference curve, 385-386 using cellulose particles, 383-384 using paper disks, 383 using polymaleic anhydride particles, 383-384 reagents, 379-382 Radioimmunoassay, 31-35,201-209, s e e a l s o specific assay and method automation equipment, 392-394 assay conditions, choice, 208-209 cross-reactions, 203 disadvantages, 409 hormones, labeled, screening test, 322334 immunoradiometric assay, 334-355 labeling of ligand, 206-207, 210-266 principle, 201-202 protein A, labeled, as tracer, 356-375 radioallergosorbent assay, 376-387 results, calculation, 209

523

sensitivity, comparative, 409 separation procedures, 266-334, 388416 charcoal method, 274-279 chemical precipitation method, 280291 double antibody method, 266-274 gel centrifugation method, 315-322 general, 207-208, 280-283,285-286 hydroxyapatite method, 291-298 magnetic transfer method, 388-416 microfiltration method, 305-314 requirements, 283-285 zirconyl phosphate gel method, 291298 solid phase advantages, 389-391 materials, 390-391 radioallergosorbent test, 376-387 precision, comparative, 410-414 protein A tracer method, 356-375 steroids, separation procedures, 291298, 315-322 RAST, s e e Radioallergosorbent test Rate of dissociation, s e e a l s o ~quilibrium constant from complement fixation test, 473-479 effect of pH, 474-477 Rauscher virus, radioiodination, 212 Reductive methylation tritiation procedure, 359-360 Relaxin, radioiodination, 230 Reserpine, immunogen preparation, 91, 98, 102 Reverse hemolytic plaque assay, 465-466, s e e a l s o Hemolytic plaque assay Rhamnose, 23-24 Ribonuclease, for antibody purification, 84 Ribonuclease B, radioiodination, 232 Ribonucleic acid, reaction with antibody, 79-81 Ribonucleic acid, transfer, radioiodination, 231 Ribonucleoside, immunogen preparation, 71, 74-76 Ribonucleotide, immunogen preparation, 71 Rivanol, for immunoglobulin separation, 124-125 Rosette test, for membrane antigens, 466 RNA, s e e Ribonucleic acid

524

SUBJECT INDEX

Schiff base formation, for hapten immunogen preparation, 101 S c h i s t o s o m a m a n s o n i epidermis, radioiodination, 236 Scripp leukemia virus, radioiodination, 212 Scorpion toxin, radioiodination, 238 Secondary immune response, 60-62 Secretin, synthetic, radioiodination, 230 Separation procedures in immunoassay, 266-334, 388-416, s e e a l s o specific method charcoal method, 274-279 chemical precipitation method, 280-291 double antibody method, 266-274 gel centrifugation method, 315-322 general, 280-283,285-286 hydroxyapatite method, 291-298 magnetic transfer method, 388-416 microfiltration method, 305-314 requirements, 283-285 zirconyl phosphate gel method, 291-298 Serotonin, s e e 5-Hydroxytryptamine Serum collection and storage, 120-121 defatting procedure, 121-122 Sisomycin, radioiodination, 234 Soft agar cloning, 139-140, s e e a l s o Cloning Solid phase immunoassay, 334-416, s e e also Immunoassay; Radioimmunoassay; specific assay immunoradiometric assay, 334-355 magnetic transfer immunoassay, 388416 protein A tracer method, 356-375 radioaUergosorbent test, 376-387 Somatostatin, cyclic, radioiodination, 230 Spacer, for steroid immunogens, 90 Specificity, antibody, principles, 72-73 Spermidine, immunogen preparation, 96 Steroids antibody specificity, 157-159, 202-203 immunogen preparation, 97-100 radioimmunoassay, separation procedure, 291-298, 315-322 Succinylated nucleotide, immunogen preparation, 76-77 N-Succinimidyl 3-(4-hydroxyphenyl) proprionate, s e e BoRon-Hunter reagent Sugars, immunogen preparation, 97-100

Surface stimulation synthesis, 45-48 SV40 virus, lysate, radioiodination, 235 Systemic lupus erythematosus, antibody against nucleic acid, 80

T Talc-resin-trichloroacetic acid test, 322334 advantages, 323 applications, 333-334 atypical hormones, 332 procedure, 332-333 general considerations, 324-325 principle, 323 procedure, 323-324 results, 325-326 Tanning procedure, for erythrocytes, 458459 Testosterone, immunogen preparation, 98, 101, 155 Testosterone- 17-hemisuccinate, immunogen preparation, 91 Tetanus toxoid, radioiodination, 212 Tetrahydrocannabinol, immunogen preparation, 98, 103 Thionein, radioiodination, 227 Thrombin, radioiodination, 212 Thromboxane, radioimmunoassay, 372 Thymidine kinase, for hybridoma production, 34-35, 136, s e e a l s o Hybridoma Thymopoitin, radioiodination, 227 Thyroglobulin as carrier, 89 radioiodination, 212 Thyroid stimulating hormone, radioiodination, 230 Thyrotropin-releasing hormone, immunogen preparation, 102 Thyroxine, immunogen preparation, 91, 95 Tobacco mosaic virus, immunogen preparation, 97 Tobramycin immunogen preparation, 93, 96 radioiodination, 234 Tolerance, 62-63 Toxins, radioiodination, 237-238 Tracers, in immunoassay, protein A, 356375 Transcobalamin, radioiodination, 227 Transferrin, radioiodination, 212, 227

SUBJECT INDEX Transglutaminase, radioiodination, 232 Transplantation antigen, liposome-bound, radioiodination, 237 TRH, s e e Thyrotropin-releasing hormone Tritiation, of protein A, 359-360 Tritiation procedures, s e e a l s o specific method acetic anhydride method, 359 reductive methylation method, 359-360 Trypsin, radioiodination, 212 TSH, s e e Thyroid stimulating hormone Tuftsin, radioiodination, 229 Tumor cell antigen, assay of bound antibody, 372-375 U Urease activity assay, 444-447 conjugate preparation for immunoassay method, 442-444 labeling of antigen, 444-447 Uridine, immunogen preparation, 100 Uridine-5'-carboxylic acid, immunogen preparation, 91, 92 Urokinase, radioiodination, 212

V Virus assays, correlation between, 410-414

525

protein, radioiodination, 229 radioiodination, 212, 235-236 Vitellogenin, radioiodination, 228

X Xanthine oxidase, radioiodination, 232

Z Zirconyl phosphate gel separation method, 299-305 gel preparation, 299-300 procedure, 301-304 for alpha-fetoprotein, 302, 303-304 for carcinoembryonic antigen, 301302, 303 for insulin, 302-303,304 requirements, 304-305 results, 304 Zones of precipitin reaction in gels identification, 183-188 independence, 179 mathematical analysis of position, 179-183 mobility, 179-183 multiple, 177-179 of tolerance, 62-63