Epitope Mapping Protocols

Overview Choosing a Method for Epitope Mapping Glenn E. Morris For most practical purposes, an epitope is easy to define...

Author: Glenn E. Morris

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Overview Choosing a Method for Epitope Mapping Glenn E. Morris For most practical purposes, an epitope is easy to define as that part of an antigen involved in its recognition by an antibody (or, in the case of T-cell epitopes, by a T-cell receptor). Although simple chemical molecules, nucleic acids, and carbohydrates can all act as antigens, the term “epltope mapping” is usually applied to protein antigens, and is the process of locating the epitope on the protein surface or m the protein sequence. The simplicity is deceptive, however, and conceptual problems soon make their practical consequences felt. A considerable understanding of the principles of protein structure and protein folding, and some knowledge of the nature of the immune response may quickly become necessaryfor the correct interpretation of experimental epitope mapping results. The term “epitope mapping” has also been used to describe the attempt to determine all the major sites on a protein surface that can elicit an antibody response, at the end of which one might claim to have produced an “epitope map” of the protein antigen (1). This information might be very useful, for example, to someone wishing to produce antiviral vaccines. Implicit in this view of epitopes is that they are fixed and concrete structures on protein surfaces, which are few in number and uniquely capable of stimulating the immune system. Even if this is true for proteins in their native conformation, it is a limitation imposed by protein structure rather than the immune system, since additional immunogenic determinants are readily revealed by protein unfolding. This kind of “epitope map” also confuses the important distinction between antigenicity (the ability to recognize a specific antibody) and immunogenicity (the ability to produce antibodies in a given animal species). Most people would agree that epitopes should be defined by their antigenicity. From. Methods m Molecular Biology, vol 66, Epltope Mapping Protocols Edited by* G E Morris Humana Press Inc , Totowa, NJ

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It is essential to distinguish between conformational (“discontmuous,” “assembled”) epitopes, m which amino acids far apart in the protein sequence are brought together by protem foldmg, and lmear (“continuous,” “sequential”) epitopes, which can often be mimicked by simple peptide sequences. Parts of conformational epitopes can sometimes be mimicked by peptides, and the term “mimotope” has been coined to describe these peptides. On the other hand, the view that most peptide sequencescan produce antibodies that recognize native proteins (2) has been disputed (‘3). Given the nature of protein structures, most epitopes on native proteins are likely to be “assembled” (4) and, consequently, most antibody molecules m polyclonal antisera raised against native protems do not recognize short peptides (‘3). If assembled epitopes are found most frequently on native proteins, sequential epitopes are found more often on denatured or partially unfolded proteins. Unfolding 1sseldom, if ever, complete under condttions conducive to antibody binding, since conditions that unfold antigens (extremes of pH, chaotropic agents, ionic detergents, and so forth) also affect immunoglobulins and antibody-antigen interactions. There is something of a culture gap between crystallographers, who tend to study assembled epitopes exclusively, and people who use monoclonal antibodies (MAbs) as research tools, for whom assembled epitopes can be something of a nuisance if the MAbs do not work on Western blots. Some authors have preferred to emphasize the distinction between epitopes on native protems and those on denatured proteins by using such terms as “cryptotopes” or “unfoldons” for the latter (5). Apart from their content, the titles alone of reviews by Laver et al. (5) and Greenspan (6) are sufficient to illustrate the extent of this problem of definitions. It might be simpler for the purposes of thus practical manual to adopt the operational view that an epitope is defined by an antibody molecule, I.e., if an antibody exists, then whatever it can be shown to recognize m the antigen is the epitope (or part of it). This view has Its own problems, notably the fact that MAbs often crossreact with sequences or structures other than that of the real antigen. If it sidesteps many important issues (or brushes them under the carpet), it does at least recognize the fact that the extent of conformation dependence of antibody binding is not always known when mapping begins. It also implies that the number of epitopes could be as great as the number of antibodies, depending on how often MAbs recognize identical epitopes. After stimulation by antigen, a B-lymphocyte clone will undergo somatic mutation m a germinal center of the spleen to refine antibody diversity further (7). The slightly different antibody molecules produced in this way ~111generally recognize the same region of protein, but with a different affinity or a different tolerance of amino acid substrtutions. These fine specificities can hardly be regarded as defining different epitopes, although it is difficult to decide where

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exactly to draw the line. At what point should the distinction between two overlappmg epitopes cease to exist? Some may find such questions challenging, whereas others may find them merely tedious.

MAbs that bind to proteinson Westernblots (after SDS-PAGE) will tend to be against sequential epitopes, whereas MAbs that recognize antigens in liquid-phase immunoassays or in frozen tissue sections are often directed against assembled epitopes. It must be remembered, however, that few proteins are completely denatured on Western blots, and epitopes identified by Western blotting may have a considerable conformational element. Another point often overlooked is that the reducing agent (mercaptoethanol or dithiothreitol) in SDS-PAGE may, for proteins with disulfide bridges, have a greater effect on protein denaturation than SDS itself; for example, the binding of a number of MAbs against hepatitis B surface antigen was retained after SDS treatment, but abolished by reduction of the disulfide bridges that maintain the structure of this antigen (81. MAbs can therefore be usefully divided into those that recognize native proteins and are suitable for immunoassays, those that recognize partially unfolded proteins and are suitable for Western blotting, and those that recognize both. The antibody that defines an epitope will, of course, be an MAb, the product of a single B-lymphocyte clone, although epitope mapping methods can also be applied to polyclonal antisera, which should be regarded as a mixture of MAbs. Consequently, unlike MAbs, antisera will usually recognize both native and denatured proteins, but different component antibodies may be involved in the two cases;thus, the antibodies in an antiserum that are used to demonstrate its specificity by Western blotting may be different from those that are active in an immunoassay with that antiserum. X-ray crystallography is often regarded as virtually the only method for pre-

cise definition of an epitopeby identification of all the amino acids in contact with the antibody. As Saul and Alzari show in Chapter 2, the contribution of this technique to our understanding of epitopes has been outstanding. Its prime position, however, is not completely unassailable for a number of reasons. First, there does not seem to be complete agreement on how close amino acids in the antibody and antigen must be to constitute a “contact.” Second, some residues in the antigen could theoretically be “in contact” with the antibody without contributing significantly to the binding. van Regemnortel has made the distinction between “structural” epitopes as defined by X-ray crystallography and related techniques and “functional” epitopes defined by amino acid residues that are important for binding and cannot be replaced (3). Third, the method is restricted by the necessity of obtaining good crystals of antibody-antigen com-

plexes, and it has usually been applied to highly conformational epitopes on the surface of soluble proteins. NMR methods in solution (Chapter 3) avoid the need for crystals, but are limited by the size of the antigen that can be studied

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and are usually applied to peptide antigens. Finally, the time and expense involved in X-ray analysis tend to exclude it as a routine, everyday approach to antibody characterization and epitope mappmg. Electron microscopy has also been used successfully for low-resolution epitope mapping, although usually for very large antigens, such as viruses (9). This method rather speaks for itself, so a protocol has not been included. Competition methods can be very useful when a relatively low degree of mapping resolution is adequate. You may want to establish, for example, that two MAbs recognize different, nonoverlapping epitopes for a two-site immunoassay, or to find MAbs against several different epitopes on the same antigen so that results owing to crossreactions with other proteins can be rigorously excluded. The principle behind competition methods is to determine whether two different MAbs can bind to a monovalent antigen at the same time (in which case they must recognize different epitopes) or whether they compete with each other for antigen binding. Molinaro and Eby describe the simplest possible method based on this principle, using Ouchterlony gel-diffusion plates (Chapter 4); single MAbs or mixtures of MAbs that recognize overlapping epitopes are unable to form precipitin lines. At a more sophisticated and more expensive level, biosensors that follow antibody binding m real time can be used to determine directly whether two or more unlabeled MAbs will bind to the same unlabeled antigen. Johne describes the use of the Pharmacia BIAcore for this purpose (Chapter 7). Such methods as ELISA using microtiter plates are the traditional approaches to competition mapping, and involve labeling either antibody or antigen with enzymesor radioactivity. Kuroki (Chapter 5) and Tzartos (Chapter 6) demonstrate the flexibility of this very popular approach. Chemical modification of amino acid side-chains is a method that is perhaps less widely used today than previously (1). In principle, addition of modifying groups specifically to amino acids, such as lysine, should prevent antibody binding to epitopes that contain lysine residues, and such an approach should be particularly useful for conformational epitopes that are otherwise difficult to map with simple techniques (Chapter 8). Unfortunately, such epitopes are also the most sensitive to indirect disruption by chemicals that cause even small conformational changes, and great care is needed to avoid false positives. The protection-from-modification method described by Bosshard (Chapter 9) is more reliable in principle, since the side-chains in the epitope itself are not altered (protected by antibody) and the modifying groups on the unprotected side-chains are not large (e.g., radioactive acetyl groups). Labeling of individual amino acids is compared in the presence and absence of the protecting antibody. Protection from proteolytic digestion, described by Jemmerson (Chapter lo), is similar in principle; for native pro-

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teins, which are often resistant to proteases, it does depend on the epitope containing a protease-sensitive site, but these, like assembled epitopes, are often associated with surface loops. If the antibodyantigen interaction will survive extensive proteolysis with loss of structure, the antigen fragments remaining attached to the antibody can be identified by mass spectrometry (Chapter 13). An alternative, and simpler, approach for epitopes that survive denaturation is partial protease digestion of the antigen alone, followed either by Western blotting for larger fragments or by HPLC (Chapter 12). The fragments that bind antibodies can be identified by N-terminal microsequencing or by mass spectrometry. Overlapping fragments, produced by different proteases, help to narrow down the epitope location, Chemical fragmentation is an alternative to proteolysis and has the advantage that cleavage sites are less frequent (e.g., for Cys, Trp, and Met residues) so that fragments can often be identified from their size alone; for this reason, antigen purity is less important than for proteolytic fragmentation (Chapter 11). Conditions for chemical cleavage, however, are usually strongly denaturing, so the method is not useful for assembled epitopes. Synthetic peptides have revolutionized our understanding of epitopes to the same extent as X-ray crystallography, although ironically the two approaches are virtually mutually exclusive, since peptides are used for sequential epitopes. Rodda et al. describe the PEPSCAN method in which overlapping peptides (e.g., hexamers) covering the complete antigen sequence are synthesized on pins for repeated screening with different MAbs (Chapter 14). Since the synthesis can be done automatically, this popular approach requires very little work by the end user (and, it sometimes seems, very little thought). The related SPOTS technique for multiple peptide synthesis on a solid phase is described by its originator in Chapter 15. An alternative approach to the synthesis of peptides based on the antigen sequence is the use of libraries of completely random peptide sequences. Pinilla et al. describe a method for the synthesis and screening of such a library using their “positional scanning” approach in Chapter 16. The advent of peptide libraries displayed on the surface of phage (Chapters 17 and 18) took this approach a step further by enabling selection of displayed peptides, as opposed to screening. In this case, random oligonucleotides are cloned into an appropriate part of a phage surface protein, and the peptide sequence displayed is identified after selection by sequencing the phage DNA. Selection of random peptides is unique in producing a range of sequences that are related, but not identical, to the antigen sequence; this enables inferences to be made about which amino acids in the epitope are most important for antibody binding. An advantage shared by all peptide methods is that antigen is not required, which may be important for “rare” antigens, which are difficult to purify.

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New possibilities for mapping arise if the antigen can be expressed from recombinant cDNA. These include the mutation of ammo acids in the epitope, and new methods of generating and identifying antigen fragments. Alexander describes a method for altering individual amino acids in a known epitope by oligonucleotide replacement (Chapter 20), and Shibata and Ikeda deal with the introduction of random mutations into part of the antigen by PCR, followed by screening to detect epitope-negative mutants (Chapter 2 1). An elegant method for conformational epitopes, homolog scanning, described by Wang (Chapter 19) requires two forms of the antigen (e.g., from different species) to be expressible from recombinant DNA as native proteins, one of them reactive with the antibody and the other not. Functional chimeric proteins can then be constructed by genetic engineering, and regions responsible for antibody binding identified. Compared with random mutation methods, this approach is less likely to disrupt the native conformation. Two rapid methods for random shortening of the antigens produced from plasmid vectors are described in Chapters 28 and 29. One of them takes advantage of the spontaneous early termination of translation of mRNA, which occurs in in vitro systems,whereas the other involves the random msertlon of stop codons into plasmid DNA using a bacterial transposon. Extensive DNA manipulation is not required m either method, although transposon mutagenesis has the additional advantage that the site of mtroduction of the stop codon can be identified precisely by DNA sequencing. Brummendorf et al. describe an elegant method for generating shortened fragments at both ends using exonuclease III (Chapter 27). This enables production of overlapping fragments, which can be used to determine epitope boundaries more reliably. As a bonus, this chapter describes a novel vector, pDELF, specifically designed for mapping membrane proteins in mammalian cells. Random digestion of cDNA with DNaseI, followed by cloning and expression, is a popular way of generatmg overlapping antigenic fragments. Stanley described a method using bacterial pEX plasmids in an earlier volume (10), so protocols for yeast plasmids (Chapter 22) and bacteriophage h (Chapter 23) are described here. An additional method for phage display of DNaseI fragments (Chapter 24) has the important advantage that antibody-positive clones can be obtained by selection rather than screening. In all cases,the antigen fragment expressed can be identified by DNA sequencing. Another approach is to clone specific, predetermined (rather than random) fragments that have been generated either by using existing restriction enzyme sites in the cDNA or, more flexibly, by using PCR products that have restriction sites in the primers (Chapter 20). This approach is especially useful if you want to know whether an epitope is in a specific domain of the antigen or whether it is encoded by a specific exon in the gene, since other methods may

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give ambiguous answers to these questions. For PCR products, the necessity to clone may be avoided altogether by including a promoter in the forward primer and transcrlbmg/translatmg the PCR product in vitro (Chapter 21). Another major advantage of the PCR approach is that it is not always necessary to have your full-length antigen already cloned. Provided the cDNA sequence is known, reverse transcriptase-PCR (RT-PCR) can be used to clone PCR products directly from mRNA or even total RNA (II). In Chapter 30, Rodda describes a synthetic peptide method for identification of T-cell epitopes, and Chapter 31 is a simple reminder of the value of naturally occurring sequence variants of antigens, such as isoforms or antigens from different animal species, for identification of individual amino acid residues, which may be important for antibody binding. Finally, the last two chapters describe methods for generating the panels of MAbs that are needed for efficient application of epitope mapping techniques. The traditional hybridoma method has the advantage of 20 years of experience and refinement (Chapter 32), whereas phage display antibodies hold out the promise of better control of antibody specificity and improved possibilities for “humanized” antibodies (Chapter 33). The choice of a method for epitope mapping depends on a number of factors, including: 1. The antigen: Is it available at all? In milligram quantities? As a recombinant protein producedfrom cDNA7 2. The antibody: Does it recognizean assembledor a sequentialepitopev 3. How detailed you want the mapping to be: Some methods identify individual ammo acids essentialfor antibody binding whereas others only show whether two epitopesare sufficiently far apart for simultaneousbinding of the two antibodies, with various levels of detail m between. 4. How much money/equipment/time you have available: Some of the methods require expensive equipment that may not be readily available and other methods are heavy on both consumablesand time. Clearly, the most cost-effective method will vary amonglaboratories. If possible, it is advisable to give some thought to mapping problems at the early stage of antibody production. I would recommend producing a panel of different MAbs instead ofjust one or two, because some MAbs will invariably prove easier to map than others. For detailed mapping at the amino-acid level, it is easier to make antibodies that work on Western blots and probably recognize sequential determinants. However, such antibodies may not be suitable for all purposes, particularly immunoassay, for which they may lack the reqmred specificity and high avidity. In my laboratory, we try to make MAbs that recognize both native and denatured protein, but for many antigens, particularly globular proteins, this may prove extremely difficult or impossible.

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Finally, where the methods in this volume require materials, such as plasmids and bacterial strains, that are not available commercially, the chapter authors will, in most cases,be happy to provide them. Recent developments in epitope mapping include a method for displaying peptide libraries directly on the surface of E. coli in the major flagellum component, flagellin (12). Screening and amplification steps may be simpler than in phage display and kits are available commercially (“FliTrx,” InVitrogen, San Diego, CA). A commercial kit for expressing DNaseI fragment libraries in bacteria is also available (“Novatope,” Novagen, Madison, WI). Another recent development displays random peptide libraries on polyribosomes, and the selected mRNA containing the peptide-encoding sequence is amplified by reverse transcriptase-PCR for reselection or sequencing (13). Peptides have also been chemically synthesized in very large numbers on microarrays for detection of antibody binding by fluorescein-labeled second antibody and immunofluorescence microscopy (14).

References 1. Atassi, M. Z. (1984) Antigenic structure of proteins. Eur J. Bzochem. 145, l-20.

2. Berzoksky,J. A. (1985) Intrinsic and extrinsic factors in protein antlgenic structure. Science 219,932-940. 3. van Regenmortel, M. H. V. (1989) Structural and functional approaches to the study of protein antigenicity. Immunol Today l&266-272. 4. Barlow, D. J., Edwards, M. S., and Thornton, J. M. (1986) Continuous and dlscontinuous protein antigenic determinants. Nature 322,747-748. 5. Laver, W. G., Air, G. M., Webster, R. G., and Smith-Gill, S. J. (1990) Epitopes on protein antigens: misconceptions and realities. Cell 61, 553-556.

6. Greenspan,N. S. (1992) Epitopes,paratopesand other topes: do immunologists know what they are talking about? Bull. Inst. Pasteur 90,267-279. 7. Clark, E. A. and Ledbetter, J. A. (1994) How B-cells and T-cells talk to each other. Nature 367,425-428. 8. Thanh, L. T., Man, N. T., Mat, B., Tran, P. N., Ha, N. T. V., and Morris, G. E. (1991) Structural relationships between hepatitis B surface antigen in human plasma and dimers of recombinant vaccine: a monoclonal antibody study. Vvus Res. 21, 141-154. 9. Dore, I., Weiss,E., Altshuh, D., andvan Regemnortel,M. H. V. (1988) Visualization by electron microscopy of the location of tobacco mosaic vuus epitopes reacting with monoclonal antibodies in enzyme immunoassay. Virology 162, 279-289.

10. Stanley,IL K. (1988)Epitope mapping using pEX. Methods Mol. Biol. 4,351-361. 11. Thanh, L. T., Man, N. T., Hori, S., Sewry, C. A., Dubowitz, V., and Morris, G. E.

(1995) Characterizationof geneticdeletionsin Becker Muscular Dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin. Am. J. Med. Genet. 58, 177-186.

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12. Lu, Z., Murray, K. S., van Cleave, V., LaVallie, E. R., Stahl, M. L., and McCoy, J. M. (1995) Expression of thioredoxin random peptide libraries on the Escherz’chiu coli cell surface as functional fusions to flagellin. Bio/Technology 13,366-372. 13. Mattheakis, J. C., Bhatt, R. R., and Dower, W. J. (1994) An in vitro display system for identifying ligands from very large peptide libraries. Proc. Nat/. Acad. Sci USA 91,9022-9026. 14. Holmes, C. P., Adams, C. L., Kochersperger, L. M., Mortensen, R. B., and Aldwin, L. A. (1995) The use of light-directed combinatorial peptide synthesis in epitope mapping. Biopolymers 37, 199-2 11.

Crystallographic Studies of Antigen-Antibody Interactions Frederick A. Saul and Pedro M. Alzari 1. Introduction X-ray crystallography provides a powerful tool for the study of antigenantibody interactions. Information provided by crystallographtc studies of antigen-antibody complexes includes the topological description of intermolecular contacts and the nature of interactions between amino acid residues. In caseswhere structures of both liganded and unliganded forms of an antigen or antibody fragment are known, crystallographic studies have led to a more dynamic view of binding, often showing conformational changes (induced fit) in the antibody or antigen and displacement of water molecules at the binding interface. Although crystallographic methods are not currently amenable to widespread use in epitope mapping studies owing to intrinsic complexity and the requirement of suitable crystals, crystallographic studies have revealed common features of B-cell epitopes and antigen-antibody mteractions that have greatly increased our understanding of the molecular basis of antigenic recognition. The structural view of epitopes provided by crystallographic studies is complementary in many ways to the functional view obtained by conventional epitope mapping techniques.

2. Three-Dimensional Structures of Antigen-Antibody Complexes 2.1. Lysozyme-Antibody Complexes The crystal structures of lysozyme-antibody complexes that have been determined by X-ray crystallography include hen egg lysozyme (HEL) in complex with murine antilysozyme antibodies D 1.3 (I), HyHEL-5 (2), HyHEL- 10 From

Methods in Molecular Biology, vol 66: Epitope Mapprng Protocols Edlted by G E Morris Humana Press Inc. Totowa, NJ

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(3), and D44.1 (4); pheasant lysozyme (PHL) bound to Fv Dl 1.15 (5); and guinea-fowl lysozyme (GEL) m complex with Fab F9.13.7 (6). These structures reveal a number of common features of antibody-antigen interactions: 1. The total contact surface area excluded from the solvent following association in lysozymeantibody complexes ranges from 1300 AZ in D 1.3-HEL and D 11.15-

PHL to about 1500A2 in the other complexes; 2. The binding of antibody to lysozyme is mediated by van der Waals contacts, intermolecular hydrogen bonds, and to a lesser extent, salt bridges; 3. Amino acid residues from both the light and heavy chains of the annbodies make contact with the antigen, but most contacts come from the CDRs of the heavy cham (in particular, the H3 CDR); and 4. Small conformational changes often take place m the antibodies and in lysozyme on complex formation. The crystallographic

epitopes defined by the six antibody-lysozyme

com-

plexes can be grouped into four distinct, largely nonoverlapping regions covering more than 60% of the total molecular surface of the antigen, thus suggesting

that the entire surface of the innnunogen

is potentially

antigenic.

The epitopes are discontinuous, each formed by two or more discrete segments in the polypeptide

chain. Antibody

D1.3 binds an antigemc

determi-

nant formed by lysozyme residues 18-27 and 117-l 25 (far from the active site cleft) centered at lysozyme residue Gln121. Antibodies HyHEL-5 and D44.1 each bmd an epitope formed by three polypeptide segments (lysozyme residues 41-53,67-70, and 81-84), on the opposite side of the molecule with respect to the epitope recognized by D1.3. Three salt bridges are conserved in the HyHEL-5-HEL and D44.1-HEL interfaces (between Vn residues Glu35 and Glu50 and lysozyme residues Arg45 and Arg68), but a different pattern of intermolecular hydrogen bonds and van der Waals contacts occurs. Antibodies HyHEL-10 and F9.13.7 interact with exposed residues of an a-helix (residues 89-97) and three surrounding loops (residues 20-21, 73-77, 98102) adjacent to the lysozyme active site cleft (Fig. 1). Although HyHEL-10 and F9.13.7 display quite different combining sites and a dissimilar pattern Fig. 1. (opposite page) The three-dimensional structure of the Fab HyHEL10-lysozyme complex (3) provides an example of antibody recognition of protein antigens. (A) Interacting contact surfaces of HyHEL-10 and HEL lysozyme. The antibody-combining site (left) and the lysozyme epitope (right) form contiguous complementary surfaces. (B) Schematic diagram of antigenantibody interactions. Most antibody CDR loops (in dark gray) contribute one or more residues to binding. The contacting lysozyme residues (in light gray) form a discontinuous

epitope. Hydrogen

bonds (represented by dashed lines)

and a salt-bridge (thick line) mediate antigen-antibody interactions.

Crystallographic Studies A

Figure 1. (Caption on oppositepage)

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of crossreactivity with avian lysozymes, these clonally unrelated antibodtes bind the same 12 residues of lysozyme. The crystallographic epitope recognized by antibody Dl 1.15 includes sequence segments 2 l-23, 103-l 06, and 112-l 19. The Hl and H2 loops of Dl 1.15 are closely related to the corresponding loops of F9.13.7. However, these two CDR loops contribute significantly (about 50% of the corresponding contact surfaces) to binding nonoverlapping regions of lysozyme, thus demonstrating that the same structural elements can confer binding specificity in quite different stereochemical environments. The Fab and Fv fragments of antibody D 1.3 have been analyzed in various structural contexts: Fab D1.3 complexed to HEL variants (7), Fv D1.3 m both unliganded and antigen-complexed forms (8,9), and the idiotopeanti-idiotope complexes of Fab DI .3-Fab E225 (10) and Fv D1.3-FV E5.2 (12). The complexes of Fab and Fv D 1.3 with HEL show essentially the same interatomic contacts, and no major structural rearrangements in the antibody or in lysozyme. In particular, the main-chain conformation of the D1.3 CDRs is largely conserved m the various antiget+antibody or idiotope-anti-idiotope crystal structures. Natural variants of avian lysozymes that differ from HEL at a few amino acid positrons have been used to map the epitopes recognized by anti-HEL monoclonal antibodies (MAb), and binding assays allowed partial definition of the antigenic determinants (12-Z 4). In general, the predictions based on these binding assays were reasonably correct, although the limited availability of natural lysozyme variants allowed the identification of only a few of the 15-20 residues belonging to each epitope. For example, recognition by antibody D1.3 is sensitive to mutations at lysozyme position 121. Only avian lysozymes that have a glutamine residue (but not arginine or histidine) at this position bind to D1.3 with high affinity, probably because the side-chain of Gln12 1 sits in a pocket within the antibody combining site that does not allow accommodation of a bulkier side-chain. In a similar manner, other amino acid residues were identified as part of the epitopes recognized by the various antilysozyme antibodies. However, serological mappmg failed in some casesto detect important epitope residues, either because of the limited number of available evolutionary variants of lysozyme (a difficulty that can be circumvented by site-directed mutagenesis) or because a particular substitution produced unexpected binding effects. For example, residue 101, at the periphery of the epitope recognized by HyHEL-10 (Fig. l), is aspartate in HEL and glycine in turkey lysozyme. Since there was less than a twofold difference in competition binding experiments, it was (incorrectly) considered unlikely that residue 101 was involved in the HyHEL- 10 epitope. However, with the development of a yeast expression system for lysozyme, all 20 amino acids were substituted at posi-

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tion 10 1, and a good correlation was found between decreased binding and increased side-chain volume (IS). 2.2. Complexes of Antibodies with Influenza Virus Glycoproteins Influenza virus neuraminidase and hemagglutinin are two membrane glycoproteins embedded in the viral envelope that are recognized by infectivityneutralizing antibodies; mutations in these proteins are the basis of antigenic variation of the vnus. The crystal structure of MAb NC41Qin complex with influenza virus N9 neuraminidase was determined at 2.5-A resolution (16). The Fab fragment and neuraminidase each interact over a surface area of roughly 900 A2, a more extended area than the contact surfaces observed in 1ysozymtiFab complexes. The structural epitope is discontinuous and comprises five distinct chain segments. A total of 19 amino acid residues on neuraminidase make direct contact with 17 residues from five of the SIXhypervariable loops of NC41. Three intermolecular salt bridges and 12 hydrogen bonds are observed at the binding interface. A second antmeuraminidase antibody, NCIO, binds to a structural target that largely overlaps the epitope recognized by NC41. The crystal structure of the Fab NClO-neuraminidase complex, determined at 2.5-A resolution (I7), shows a buried surface area of about 700 A2 in both NC10 and neuraminidase (of which about 80% involves residues common to the epitope recognized by antibody NC41). The epitope comprises four segments of polypeptide chain, and binding contacts involve 14 residues of the antibody and 15 residues of the antigen. Antibodies NC10 and NC41 have identical amino acids within the HI CDR, although this similarity cannot be the basis of crossreaction, since the Hl loop makes no contact with the antigen in the NC IO-neuraminidase complex. The structure of the Fab fragment from neutralizing antibody HC 19, in comaplex with the top domain of X-3 1 hemagglutinin (HA), was determined at 3.3-A resolution (Id). The HA top domain (in this case, residues 28-328 of the HAi polypeptide chain) in complex with Fab HC19 substantially retains the structure of the native form. The general features of HC 19-HA interaction are similar to those of other Fab-protein complexes, although a cavity in the antigen-antibody interface, open to the bulk solvent and presumably filled with disordered water molecules, reduces the overall structural complementarity. The total buried surface area is 1250 A2. Comparison with the unliganded structure of Fab HC19 (19) shows major changes in atomic positions at the Fab combining site, particularly in the H3 CDR, where shifts of up to 10 A allow the H3 CDR loop to interact with the HA receptor-binding site. These conformational changes are associated with four of the 10 intermolecular hydrogen bonds observed in the Fab-HA interface.

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Neurammidase and hemagglutinin are both responsible for the antigenic drift that results in recurrent epidemics of influenza. Antigenic variants of the two proteins have been selected under pressure from various antineuraminidase and antihemagglutinin MAbs, and used in binding assays to map the viral epitopes. As expected from the Fab NC41 neuraminidase crystal structure, most of the substitutions at the antibody-antigen interface markedly reduce binding to NC4 1. However, two such neuraminidase variants (Asn329-Asp and Ile368-Arg) show only marginal decrease of binding to NC41, in spite of the introduction of the additional charge or much larger side-chain. The crystal structure of NC41 complexed with these variants (20) revealed that the substrtutions are accommodated at the antibody-antigen interface with only local structural perturbations. The epitope of HA recognized by HC 19 was mapped in a similar way; all of the mutated residues are located on the surface of hemagglutmin at the periphery of the Fab-hemagglutinin interface. Most involve substitution with a bulkier residue, indicating that steric hindrance of complex formation with the antibody represents an important viral escape mechanism. These studies illustrate the importance of the structural context in analyzing the effect of an amino acid substitution on antibody-antigen association. 2.3. Phosphocarrier Protein HPr-Fab Complex The crystal structure of the Fab fragment of antibody Je142in complex wtth the phosphocarrier protein HPr from Escherichia coli (21) allowed comparison of epitope predictions based on extensive mutagenesis experiments with those from X-ray analysis. All antigen-contacting residues of Fab Je142occur in the hypervariable loops, and as in the other Fab-complex structures reported, most of these are contributed by the heavy chain. The antibody-combining site forms a complementary depression that allows binding to the relatively small (85 residues) HPr antigen, Binding interactions involve 20 residues in Fab Je142 and 14 residues in HPr through a surface contact area of 690 A2 in each protein. Studies by site-directed mutagenesis had correctly predicted nine of the 14 HPr epitope residues. Of these, eight were detected using mutants that introduced larger side-chains, and one, a glutamme residue, was identified by the introduction of an isosteric charged side-chain (Glu) that would be buried in the mutant HPr-Fab complex. Four residues that gave less than a lo-fold change in relative binding were incorrectly assigned to the epitope; these occurred in positions adjacent to epitope residues that were likely to be perturbed by the mutations. Among residues of the structural epitope that were not detected by mutagenesis, two involve contacting residues exposed to solvent at the periphery of the binding site, presumably allowing accommodation of substitutions without disrupting complex formation.

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Crystallographic Studies 2.4.ldiotoph4nti-ldiotope

Complexes

The first reported crystal structure of an idiotopeanti-idiotope complex was that of Fab E225 in complex with the antilysozyme Fab D1.3, determined at 2.5 A resolution (10). The E225-D1.3 complex displays many common features of antigen-antibody structures. The anti-idiotypic Fab E225 interacts with D 1.3 through 14 residues from all six CDRs and one framework residue of VL. The corresponding idiotope of D1.3 comprises 13 residues from five CDRs and a VL framework loop. About 800 A2 of the surface area of each Fab is buried in the complex, and interactions include nine hydrogen bonds and one salt bridge. No significant conformational changes were observed in the main chain of D 1.3 in complex with E225 and with HEL (or in the free state), although the side-chain conformation of three CDR residues in D1.3 differed significantly owing to steric requirements for binding by E225. Only seven of 13 D1.3 residues bound by E225 make contact with lysozyme in the D1.3HEL complex, and the nature of their interactions is different. The structure of a second anti-idiotope D 1.3 complex, the Fv fragment of anti-idiotypic antibody E5.2 in complex with Fv D1.3, was determined at 1.9 A resolution (II). The mteractions of E5.2 with D1.3 involve residues from all six CDRs of each antibody, with a solvent-excluded area of 912 A2 in D1.3 and 974 A2 in E5.2. The H3 CDR of E5.2 accounts for 77% of the total number of contacts with D 1.3. In contrast to the E225-D1.3 complex, similar patterns of hydrogen bonding are observed for 13 Dl.3 residues that are in contact with both E5.2 and HEL. These residues account for 75 and 87% of the binding interfaces with E5.2 and HEL, respectively. The Dl.3-combining site is similar in the D1.3-E5.2 and D1.3-HEL complexes, including 11 conserved water molecules at the interface. The crystal structure of the anti-idiotypic Fab 409.5.3, raised against antibody 703.1.4, was determined at 2.9-A resolution both in complex with Fab 703.1.4 (‘22) and in uncomplexed form (23). Antibody 703.1.4 recognizes the E2 peplomer of feline infectious peritonitis virus (FIPV) and, used as immunogen, can elicit production of anti-anti-idiotypic antibodies (Ab3) that are able to neutralize the virus. Fabs 409.5.3 and 703.1.4 interact with a high degree of structural complementarity. The buried surface area of the interacting CDRs in the complex is 860 A2 for Fab 409.5.3 and 890 A2 for Fab 703.1.4. Interactions involve 19 residues of the idiotope and 17 residues of the anti-idiotope, with 118 van der Waals contacts and at least nine hydrogen bonds. The heavy chain of Fab 409.5.3 dominates binding and contributes 63% of the buried surface area of the anti-idiotope. The idiotope of Fab 730.1.4 involves predominately the heavy chain, which contributes 71% to the buried area on the idiotope. Comparison of the structure of Fab 409.5.3 in complexed and uncomplexed

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form shows small rearrangements in CDR loops (induced fit) that permit optimization of complementarity between the interacting molecules. The greatest conformational change between the supertmposed free and complexed structures of Fab 409.5.3 occurs in the H3 CDR loop, which displays a rigid-body shift of up to 3 A in the main polypeptide chain. The crystal structure of the Fab fragment from the anti-idiotypic antibody T91AJ5 was determined at 2.8 A resolution (24) in both unliganded form and in complex with the Fab fragment of antibody YsT9.1, specific for the lipopolysaccharide A antigen of Brucellu abortus. The contacting surfaces of the two Fabs are highly complementary, although different in character. Interactions include extensive van der Waals contacts and 12 hydrogen bonds. All CDRs in the complex are involved in van der Waals contacts, but the Hl and H3 loops of YsT9.1 do not participate dtrectly in hydrogen bonding to T9 1AJ5. All inter-Fab contacts in the complex involve the hypervariable loops. The solvent-excluded area is 730 A2 for YsT9.1 (Abl) and 760 A2 for T91AJ5 (Ab2). The contact surface of the Abl is divided almost equally between the ltght and heavy chains, whereas roughly 60% of the contact surface of the Ab2 involves the heavy chain. Comparison of the two Fabs with the corresponding unliganded structures (2.5) shows that the H2 CDR loop of T9 1AJ5 undergoes a significant (and energetically costly) conformational rearrangement. This refolding is required in the complexed structure to avoid steric conflict with YsT9.1. 2.5. PeptideAntibody Complexes The crystallographic structures of Fab fragments complexed with peptides ranging in size from 8-20 residues have provided detarled information on the nature and extent of interactions that characterize peptide-antibody interactions. These structures include Fab fragments from antipeptide antibodres m complex with peptide antigens (2631), neutralizing antibodies bound to peptides derived from viral epitopes (32-34), and the complex between an antianti-idiotypic Fab (Ab3) and a small peptide hormone (3.5). The binding sttes of antipeptide antibodies frequently form concave pockets or grooves made up of the six hypervariable loops. The surface contact areas are generally smaller than those observed for protein antigeeantibody complexes, ranging in size from 400-900 A2. A majority of peptide-Fab interactions usually involves residues of the antibody heavy chain. The complementarity of fit at the peptideantibody interface and the nature of intermolecular interactions (van der Waals contacts, hydrogen bonds, and salt bridges) are similar to those observed in complexes of antibodies with protein antigens. Peptide antigens usually adopt well-defined conformations when bound to antibodies. The number of peptide residues seen in the antigen-binding site

Crystalicgraphic Studies

19

varies from g-12. Additional residues outside the binding site are often disordered and not visible in electron density maps. All bound peptides show some degree of regular structure, most frequently tight turns that bind wrthin a cleft at the Fab-combining site, although extended conformations have also been observed (29). The conformation of peptides bound to antibodies may differ significantly from that of the peptides either free m solution or in the context of the native protein from which they were derived (26). However, similar conformations of an antibody-bound peptide with its protein cognate have been observed (or proposed) for peptides derived from viral epitopes (27,32,34). A wide range of conformational rearrangements in the antibody-combining site have been observed in caseswhere the structures of both the complexed and uncomplexed forms of an antipeptide antibody are available. These changes vary from local adjustments of one or more hypervariable loops (26,31,32) to significant quaternary rearrangements in heavy/light chain association (33). This plasticity of structure allows adaptability of the antibody-combining site to optimize structural complementarity at the peptideFab interface.

3. Discussion The crystallographic studies of antibody-protein complexes described here indicate the discontinuous nature of structural epitopes: Two to six discrete polypeptide chain segments form a contiguous topological surface that makes contact with the antibody (Fig. 1). The surface area of interaction varies from about 600-900 A*, and involves 14-20 epitope restdues in contact with a roughly similar number of residues of the antibody. The contacting surfaces can be described as sterically complementary, although quantitative estimates based on known crystal structures suggest that antrgen-antibody complexes display poorer shape complementarity than other protein-protein complexes (36). Binding by the antibody takes place essentially or exclusively through the CDRs, The antibody heavy chain, and in particular, the H3 CDR loop, often makes the greatest contribution to binding. The types of intermolecular interactions involved are van der Waals and hydrophobic interactions, hydrogen bonds, and to a lesser extent, salt bridges. The stereochemistry of interaction may be optimized by induced fit rearrangements of main- and side-chain atoms, and the presence of water molecules can further modulate interactions in the binding interface. However, it should be noted that these studies represent a limited sample of possible antibody-antigen interactions and do not include, for example, complexes involving linear or continuous epitopes, such as exposed loops in the structures of immunodominant viral proteins.

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Crossreactivity is frequently observed in antibody-antigen interactions, A common mechanism of antibody crossreaction with homologous antigens occurs when a critical subset of antigen-antibody interactions is conserved in different antigens, but partial modifications can be accommodated elsewhere by a stereochemically permrssive environment at the molecular interface. For example, the antilysozyme antibody D 11.15 binds with similar affinity to most avian lysozymes because the corresponding epitope is largely invariant in different avian species. In some cases,promiscuity of antibody specificity may be accounted for by true molecular mimicry. Indirect evidence of this has been described in the studies of antiangiotensin II antibodies (35). Evidence of functional mimicry is provided by the idiotope-anti-idiotope complexes Fab 409.5.3-Fab 703.1.4 and Fv D1.3-Fv E5.2, since the anti-idiotypic antibodies, used as immunogens, can elicit Ab3 antibodies that recognize the original antigen, Antibody D1.3 makes similar contacts with lysozyme and Fv E5.2, suggesting a structural basis for antigenic mimicry by the anti-idiotypic antibody in this case (II). A different type of crossreactivity arises when unrelated proteins bind the same structural target. Comparison of the crystal structures of antilysozyme antibody D1.3 complexed to both the lysozyme immunogen and the antiidiotypic antibody E225 shows dissimilar binding targets in HEL and in E225, even though both proteins make contacts with overlapping regions of D 1.3. Similarly, antibody NC 10 binds neuraminidase at a site that extensively overlaps that recognized by another antineuraminidase antibody, NC41 (17), and two clonally unrelated antilysozyme antibodies (HyHEL- 10 and F9.13.7) interact with the same 12 residues of lysozyme (6). Heterologous crossreactivity in these complexes is facilitated by flexibility of protein structure to achieve topographic complementarity, and need not be based on chemical similarity or partial sequence homology. The substitution of bulky or otherwise mcompatible side-chains at the antibody-antigen interface is an important mechanism of antigenic variation allowing viral mutants to escape immune recognition. Crystallographic studies have shown that some substitutions in escape mutants occur outside the area of the structural epitope; these presumably cause local structural perturbations to nearby epitope residues, thereby disrupting complex formation antigen, In contrast, functional studies of antigen variants have in some cases failed to detect epitope residues (often near the periphery of the antibody-antigen interface), presumably owing to local rearrangements of side-chains to allow structural accommodation of the substitutions. These observations emphasize differences between the structural epitope determined by crystallography and functional epitopes determined by conventional methods of epitope mapping.

Crystal/ographic Studies

21

References 1. Amit, A. G., Mariuzza, R. A., Phillips, S. E. V., and PolJak,OR. J. (1986) Threedimensional structure of an antigerrantibody complex at 2.8 A resolution. Science 233,747-753. 2. Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, S. J., Finzel, B. C., and Davies, D. R. (1987) Three-drmensional structure of an antibody-antigen complex. Proc. Natl. Acad. Sci USA 84,8075-8079. 3. Padlan, E. A., Silverton, E. W., Sheriff, S., Cohen, G. H., Smith-Gill, S. J., and Davies, D. R. (1989) Structure of an antibody-antigen complex: crystal structure of the HyHEL-10 Fab-lysozyme complex. Proc. Natl. Acad. Scz. USA 86, 5938-5942. 4. Braden, B. C., Souchon, H., Eisele, J.-L., Bentley, G. A., Bhat, T. N., Navaza, J., and Poljak, R. J. (1994) Three-dimensional structures of the free and the antigencomplexed Fab from monoclonal anti-lysozyme antibody D44.1. J Mol. Biol. 243,767-78 1. 5. Chitarra, V., Alzari, P. M., Bentley, G. A., Bhat, T. N., Eisele, J.-L., Houdusse, A., Lescar, J., Souchon, H., and Poljak, R. J. (1993) Three-dimensional structure of a heteroclitic antrgen-antibody cross-reaction complex. Proc. Natl. Acad Sci USA 90,7711-7715. 6. Lescar, J., Pellegrini, M., Souchon, H., Tello, D., Poljak, R. J., Peterson, N., Greene, M., and Alzari, P. M. (1995) Crystal structure of a cross-reaction complex between Fab F9.13.7 and Guinea-fowl lysozyme. J. Biol. Chem. 270,18,06718,076. 7. Fischmann, T. O., Bentley, G. A., Bhat, T. N., Boulot, G., Mariuzza, R. A., Phillips, S. E. V., Tello, D., and Poljak, R. J. (1991) Crystallographic refinement of the three-dimensional structure of the FabDl. 3-Lysozyme complex at 2.5-A resolution. J. Btol Chem 266, 12,915-12,920. 8. Bhat, T. N., Bentley, G. A., Fischmann, T. O., Boulot, G., and Poljak, R. J. (1990) Small rearrangements in structures of Fv and Fab fragments of antibody D1.3 on antigen binding. Nature 347,483-485. 9. Bhat, T. N., Bentley, G. A., Boulot, G., Greene, M. I., Tello, D., Dall’Acqua, W., Souchon, H., Schwartz, F. P., Mariuzza, R. A., and Poljak, R. J. (1994) Bound water molecules and conformational stabilization help mediate an antiger+antibody association. Proc. Natl. Acad. Sci. USA 91, 1089-1093. 10. Bentley, G. A., Boulot, G., Riottot, M. M., and Poljak, R. J. (1990) Three-dimensional structure of an idiotope-anti-idiotope complex. Nature 348,254-257. 11. Fields, B. A., Goldbaum, F. A., Ysem, X., Poljak, R. J., and Mariuzza, R. A. (1995) Molecular basis of antigen recognition by an anti-idiotope. Nature 374, 739-742. 12, Smith-Gill, S. J., Lavoie, T. B., and Mainhart, C. R. (1984) Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozymes. J. Immunol. 133,384-393. 13. Harper, M., Lema, F., Boulot, G., and Poljak, R. J. (1987) Antigen specificity and cross-reactivity of monoclonal antibodies. Mol. Immunol. 24,97-108.

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14. Smith-Gill,

S. J. and Sercarz, E. E., eds. (1989) The Immune Response to StrucThe Lysozyme Model Adenine, Schenectady, NY. Kam-Morgan, L. N. W., Smith-Gill, S. J., Taylor, M. G., Zhang, L., Wilson, A C., and Kirsch, J. F. (1993) High resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis. Proc. Natl. Acad. Sci USA 90,3958-3962. Tulip, W. R., Varghese, J. N., Laver, W. G., Webster, R. G., and Colman, P. M. (1992) Refined crystal structure of the Influenza virus N9 neuraminidase-NC41 Fab complex. J. Mol. Biol. 227, 122-148. Malby, R. L., Tulip, W. R., Harley, V. R., McKimm-Breschkin, J. L., Laver, W G., Webster, R. G., and Colman, P. M. (1994) The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody. Structure 2,733-746. Bizebard, T., Gigant, B., Rigolet, P., Rasmussen, B., Diat, O., Bosecke, P , Wharton, S. A., Skehel, J J., and Knossow, M. (1995) Structure of Influenza vnus haemagglutinin complexed with a neutralizing antibody. Nature 376, 92-94. Bizebard, T., Daniels, R., Kahn, R., Golinelli-Pimpaneau, B., Skehel, J. J , and Knossow, M. (1994) Refined three-dimensional structure of the Fab fragment of a murine IgGl, 3Lantibody. Acta Cryst D50,768-777. Tulip, W. R., Varghese, J. N., Webster, R. G., Laver, W. G., and Colman, P. M. (1992) Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface. J, Mol. Biol. 227, 149-159 Prasad, L., Sharma, S , Vandonselaar, M., Quail, J W , Lee, J S., Waygood, E B., Wilson, K S., Dauter, Z., and Delbaere, L. T. J. (1993) Evaluation of mutagenesis for epitope mapping. J. Blol. Chem. 268, 10,705-l 0,708. Ban, N., Escobar, C., Garcia, R., Hasel, K., Day, J., Greenwood, A., and McPherson, A. (1994) Crystal structure of an idiotope-anti-idiotope Fab complex. turally Defined Proteins:

15

16.

17.

18. 19.

20.

21.

22.

Proc Natl. Acad. Scr USA 91,1604-1608.

23. Ban, N., Escobar, C. Hasel, K., Day, J., Greenwood, A., and McPherson, A. (1995) Structure of an anti-idiotopic Fab against feline peritonitts virus-neutralizing anttbody and a comparison with the complexed Fab. FASEB J. 9,107-l 14. 24. Evans, S. V., Rose, D. R., To, R., Young, N. M., and Bundle, D. R. (1994) Exploring the mimicry of polysaccharide antigens by anti-idiotypic anttbodies. The crystallization, molecular replacement, and refinement to 2.8 A resolution of an idiotope-anti-idiotope Fab complex and of the unliganded anti-idtotope Fab. J Mol. Biol. 241,691-705.

25. Rose, D. R., Przybylska, M., To, R. J., Kayden, C. S., Oomen, R.OP., Vorberg, E., Young, N. M., and Bundle, D. R. (1993) Crystal structure to 2.8 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide. Protein Sci 2,1106-1113. 26. Stantield, R. L., Fieser, T. M., Lerner, R. A., and Wilson, I. A. (1990) Crystal structure of an antibody to a peptide and its complex with peptide antigen at 2.8 A. Science 248,7 12-7 19. 27. Rim, J. M., Schulze-Gahmen, U., and Wilson, I. A. (1992) Structural evidence for induced fit as a mechanism for antibody-antigen recognition. Science 255,959-965.

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Studies

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28. Altschuh, D., Vix, 0 , Rees, B., and Thierry, J.-C. (1992) A conformation of cyclosporin A in aqueous environment revealed by X-ray structure of a cyclosporin-Fab complex. Science 256,92-94. 29. Rini, J. M., Stanfield, R. L., Stura, E. A., Salinas, P. A , Profy, A. T., and Wilson, I A. (1993) Crystal structure of a human immunodeticiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen. Proc. Nat/. Acad Sci USA 90,6325-6329. 30. Shoham, M (1993) Crystal structure of an anticholera toxin peptide complex at 2. 3 A. J. Mol. Biol 232, 1169-1175. 3 1. Churchill, M. E. A., Stura, E. A., Pinilla, C., Appel, J. R., Houghten, R. A., Kono, D. H., Balderas, R. S., Fieser, G. G., Schulze-Gahmen, U., and Wilson, I. A. (1994) Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 2619. Comparison of two different antibodies bound to the same peptide antigen. J. Mol. Biol. 241,534-556. 32. Tormo, J., Blaas, D., Parry, N. R., Rowlands, D., Stuart, D., and Fita, I. (1994) Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2. EMBO J. 13,2247-2256. 33 Ghiara, J B , Stura, E A , Stanfield, R. L., Profy, A. T., and Wilson, I. A. (1994) Crystal structure of the principal neutralization site of HIV- 1. Sczence264,82-85. 34. Wien, M. W., Filman, D. J., Stura, E. A., Guillot, S., Delpeyroux, F., Crainic, R., and Hogle, J. M. (1995) Structure of the complex between the Fab fragment of a neutralizing antibody for type 1 pohovirus and its viral epitope. Nature Struct Biol. 2,232-242. 35. Garcia, K. C., Ronco, P M., Verroust, P. J., Brunger, A. T., and Amzel, L. M. (1992) Three-dimensional structure of an angiotensm II-Fab complex at 3 A: hormone recognition by an anti-idiotypic antibody. Science 257,502-507. 36. Lawrence, M. C., and Colman, P. M. (1993) Shape complementarity at protein/ protein interfaces J A4ol. Biol 234,946-950.

3 Epitope Mapping Antibody-Antigen Complexes by Nuclear Magnetic Resonance Spectroscopy lrina Kustanovich and Anat Zvi 1. Introduction NMR spectroscopy has been used to study antibody complexes with antigenic peptides. A crucial parameter in NMR studies of large biological complexes is the rate of exchange of a ligand between its free and bound states.In caseswhere the peptide off-rate IS fast (faster than 10 s-r) relative to the decay of magnetic excitation (longitudinal relaxation time) and to the inverse of the mixing period (the time during which magnetization transfer takes place) used in NOESY* experiments, transferred NOE (TRNOE) techniques, including 2D TRNOE difference spectroscopy (1) and the TIP-filtered TRNOE method (21, are very suitable to study antibody-antigen interactions and intramolecular interactions within the bound peptide (3). Alternatively, in the case of tight binding and slow exchange, isotope filtering methods can be applied (4). Tsang et al. (5) used this technique to elucidate amide proton interactions in an antibody-bound peptide complex and to characterize the mobility of these protons on binding. Another approach, the “dynamic filtering” method (6) (applicable to complexes with off-rate slower than 1s-l), was taken by Cheetham et al. (7) to define the size of an antigenic determinant and identify its amino acid composition. On binding the Fab, antigen residues that interact with the antibody lose their mobility and their T, and Tr,, relaxation times approach that of the Fab protons. The chemical shifts of these interacting residues differ significantly from their corresponding values found for the free peptide in solution. On the other hand, peptide segments that are located outside the epitope and do not interact with the antibody retain considerable mobility and long relaxation T2 and T, ,,times. Hence, *Seep. 36 for list of abbreviations used in this chapter. From

Methods in Molecular Biology, vol 66’ Epitope Mapping Protocols Edited by: G E Morris Humana Press Inc , Totowa, NJ

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their protons give narrow resonance lines. Moreover, their chemical shifts are identical or very close to those of the free peptide. The differentiation between peptide protons that are immobilized on binding and those that retain a considerable degree of mobility can be achieved by measuring 2D correlated spectroscopy experiments that are inherently sensitive to dynamtc features (i.e., line width) and exhibit only strong signals of mobile protons with long relaxation times. Therefore, signals of the peptide residues composing the antigenic determinant recognized by the antibody would disappear from the spectrum owing to the substantial line broadening. Cheetham et al. (7) applied this method very elegantly by using the conventional COSY experiment in D20 to map out the epitope recognized by an antibody against a lysozyme peptide. Their findings were solely based on the motional characteristics of the side-chains, and the assignment of the amino acids that retained their mobthty on binding was performed by comparison with the chemical shift values of the free peptide. In our studies of the interactions between antigenic peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) and the HIV-neutralizing antibody 0.5p (8), we found that combined application of HOHAHA (9) and ROESY experiments in Hz0 provides several significant advantages relative to that of the DQF-COSY experiment (7). In addition to the drastically improved signal-tonoise ratio, a HOHAHA spectrum measured in water alleviates the overlap problem caused by the degeneracy of the side-chain resonances since the amide proton resonances are much better dispersed in their chemical shifts. Therefore, the information about the dynamic states of the backbone amide protons, which is lost when recording a spectrum in the deuterated solvent owing to the fast proton exchange, is now readily obtained to elucidate ammo acids involved in the binding with an antibody. The DQF-COSY was not sufficient to obtain an unambiguous assignment owing to the presence of the multiple residues of the same amino acid type. Furthermore, the peptide residues located at the peripheries of the binding site undergo a significant change in chemical shift, and therefore cannot be assigned by direct comparison with the corresponding values obtained for the free peptide. The ROESY experiment contains a long spin-lock pulse during which through-space magnetization transfer occurs between the neighboring protons and provides information about the d,.&z,i + 1) connectivities that is essential for the sequential assignment. The power of the proposed approach is illustrated by our recent determination of the antigenic determinant recognized by an anti-gp 120 HIV-neutralizing antibody O.Sp (8). To observe those peptide residues that retain their mobility after binding to the 0.5/!4 we measured the HOHAHA spectrum of the O.SP/RP135 complex in H20, and compared it to the HOHAHA spectra of the free Fab and free peptide. Figure 1A presents the amide region of the HOHAHA

A

.

-

I

2 .

-

E Ek -K5 B . R4,Ra

!?,I-

Fl7<-

e

-K2z -=-J-R6 GE4

3 G24 A

85

84

83 (f-m)

82

81

80

85

84

83

82 (m-d

81

80

85

84

8-3 (mm)

82

81

&I

Fig. 1. HOHAHA spectra of RP135 and 0.56 Fab in 95% H,0/5% 90 showing amide proton interactions with amino acid sidechams, at pH 5.2 and 34°C. (A) HOHAHA spectrum of free 0.5p Fab. (B) Spectrum of free RP135 (C) Spectrum ofthe complex of RP135 with OSP Fab. Reproduced from ref. 8.

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Kustanovich and Zvi

spectrum of the free Fab. No crosspeaks with detectable intensities are observed, indicating that none of the amide protons are flexible enough to give rise to sufficiently narrow lines. The HOHAHA spectrum of the free peptide in solution is shown in Fig. 1B. The resonances of the free peptide were assigned in combination with a ROESY spectrum measured under the same experimental conditions. Since no Fab crosspeaks are observed in the spectrum of the free Fab (Fig. 1A), the spectrum of the Fab/RP135 complex (Fig. 1C) is not expected to contain any contribution of the Fab protons. By comparing the HOHAHA spectrum of the complex with that of the free peptide, only crosspeaks of Lys22, Ile23, and Gly24 of the bound peptide can be assigned unambiguously. The assigned spectrum of the free peptide provides no clues for the assignment of Lys5 and Ile20 in the complex, and the crosspeaks assigned to Thr3, Arg4, and Gly21 in the bound peptide can potentially be those of Thrl9, Arg8, and Gly14, respectively. To overcome the problems associated with the degeneracy of the side-chain resonances, we measured a ROESY spectrum of the OSP Fab/RP135 complex in HzO. Since the duration of the spin-lock pulse used in the experiment was 100 ms and since T,, of the Fab and interacting protons is approx 10 ms (21, the magnetization of these protons was destroyed during the spin-lock pulse, and the observed spectrum exhibits only signals originating from the flexible residues of the bound peptide. Figure 2 shows a superposition of the HOHAHA and ROESY NH-C&H fingerprint region of RP135 bound to 0.5p. The sequential assignment can be easily followed for all the strong crosspeaks observed in Fig. 1C. These resonances are attributed to two segments of the antigenic peptide: Thr3-Lys5 and Ile20-Gly24. Therefore, we conclude that the epitope recognized by the 0.5p antibody consists of a total of 14 residues (Ser6-Thr19). The observation that Lys5 and Be20 retain considerable mobility in the bound peptide, whereas their amide protons undergo significant change in chemical shift on peptide binding, suggests that they are at the boundaries of the determinant recognized by the antibody. To substantiate the epitope mapping results and to elucidate specific interactions of the individual epitope residues with the amino acids composing the binding site of the Fab fragment, we apply 2D NOESY difference spectroscopy utilizing deuterated derivatives of the antigenic peptide (IO). This technique was demonstrated to be helpful in studying a bound conformation of the tripeptide inhibitor on binding to the pepsin enzyme (1I). The method is based on a calculation of the difference spectrum between the NOESY spectrum of Fab-antigen complex prepared with the unlabeled peptide and the NOESY spectrum of the complex with a peptide specifically deuterated at the desired single position along the chain. The resulting spectrum exhibits exclusively NOE connectivities owing to the intraresidue and intrapeptide interactions of the labeled residue, as

Epitope Mapping by NMR

29 37

39

41

-z a CL

43 I 45

, 85

84

83

82

81

80

(PPrn)

Fig. 2. Superposition of the NH-&H region of HOHAHA and ROESY spectra of OSP Fab-RP135 complex in Hz0 showing sequential connectivities. The spectra were measured at pH 5.2 and 34%. Reproduced from ref. 8.

well as interactions between antibody-binding site and peptide residues. The NOESY difference spectra measured for each specifically labeled peptide derivative are used together to define the bound conformation of the peptide epitope and to elucidate specific interactions with the protein. The NOESY difference spectrum of the OSj3 Fab complexed with a peptide containing the mapped epitope (the truncated derivative of the RP135, RKSIRIQRGPGRAFVT) in which Ile4 was deuterated is shown in Fig, 3C (aromatic region) and D (aliphatic region) (10). Figure 3A,B shows, for comparison, the corresponding NOESY spectrum of the 0.5p complex with the peptide (50 kDa) before the subtraction. It suffers from lack of resolution owing to the large number of protons in the molecule and the broadening of their individual resonances that occurs as the molecular weight of the protein increases. The difference spectrum (Fig. 3C,D) exhibits with a remarkable signal-to-noise ratio only NOE crosspeaks of Ile4; all other signals not involving Ile4 interactions are canceled out. All crosspeaks in Fig. 3C except one were assigned to Ile4 interactions with aromatic protons of the Fab. Figure 3D reveals, in addition to intraresidue connectivities, interactions of Ile4 6CH3 with three peptide protons: Ile6 yCH3, Ile6 o.H, and Val15 PH. Two additional crosspeaks (0.40, 4.14, and 1.70,4.14 ppm) are the result of Ile4 interactions with Fab protons or yet unassigned peptide protons,

Kustanovich and Zvi

30 r

0

I 2

3

4 5 % a. 0

09 61 Irl

7

I

Fob

2 Fab 3

4

FablRPl35a

5 I 8.0

I 75

7.0 PPM

Fig. 3. A comparison between regions of the NOESY spectrum of the 0.5p Fab complex with the truncatedpeptide composing the mappedepitope (RP135a) correspondingto residues3 1l-326 of gp 120 of HIV- 1in (A,B) and the difference between this spectrum and the spectrumof the complex in which Ile4 was deuterated(C,D). The locations of Ile4 resonancesare marked on they-axis. The arrows in (A) mark the locationsof Ile4 crosspeaksobservedin the differencespectrum(C). Crosspeaksowing to intraresidue interactions are marked 14, interactions within the bound peptide are labeled with the symbol of the proton interacting with Ile4, i.e., VlSS, and the crosspeaksthat could be owing to either RP135a-Fab interactions or within RP135a are marked as Fab/RP135a.Reproducedfrom ref. 10.

The extensive network of hydrophobic interactions that we observed for the different specifically deuterated residues in the bound peptide indicates that the mapped epitope forms a 12-residueloop (corresponding to residues 3 14-

31

Epitope Mapping by NMR

gp120 protein) with Ile4, Ile6, and Vall 5 at the base and the conserved GPGR sequence at the top (10). The 12-residue loop formed by internal hydrophobic interactions is part of the larger 36-residue V3 loop formed by a disulfide bridge between Cys303 and Cys338 of gp120 (12). Our studies demonstrate the benefits of using anti-gp120 antibody, which recognizes a large epitope to obtain a global conformation of the V3 loop. Since the 0.5p antibody was raised against gp120, it is expected that the conformation of the flexible antigenic peptide bound to the antibody is similar to the conformation of the corresponding part of gp120 when bound to the same antibody. 325 of HIV-liIr

2. Materials 2.1. Peptide Purification 1. Perdeuterated L-amino acids were obtained from MSD Isotopes, Canada with the minimum isotopic purrty above 98%. Addition of the tert-butyloxycarbonyl (t-Boc) protecting group was done by Oz Chemicals (Jerusalem, Israel). Each antigenic peptide derivative contains a specific deuterated amino acid at a single position. 2 Sephadex G-25 column. 3. HPLC-grade water (BDH Laboratory Supplies, England) 4. Acetonitrrle. 5. 0 03% Trifluoroacetic acid (in H20)

2.2. Antlbody

Synthesis,

Purification,

and Cleavage

6 RPM1 1640 medium (see Gibco [Gaithersburg, MD] technical manual), 2 mM glutamine, 1 n&f pyruvate, 3% fetal calf serum. 7. Sepharose 4B column. 8. Protein A-Sepharose column. 9. Washing buffer: 50 mM Tris-HCl, 150 mMNaC1, pH 9.0. 10. Elution buffer: 0.1M sodium citrate, pH 4.5. 11. Cleavage buffer: O.lM sodium phosphate, pH 7.0, with 4 mkf EDTA, 10 mM sodium azide 12. Papain. 13 Cysteme hydrochloride. 14. Sephadex G- 100 column. 15. Sodium azide. 16. Iodoacetamide 17. Double deionized water.

2.3. NMR Samples 18. NMR sample buffers: 10 m&f sodium phosphate-buffered D20 or 90% HZ00 0% D,O, 0.05% sodium azide (pH is specific to each experiment). 19. D,O (99.9%, CIL, Andover, MA).

32

Kustanovich and Zvi

3. Methods 3.1. Antibody

Production,

Cleavage,

and Purification

1. Grow hybndoma cells as monolayers in 700~mL T flasks with 50 mL RMPI 1640 medium, pH 7.3, supplemented with 2 mM glutamine, 1 mM pyruvate, and 3% fetal calf serum, at 37’C and 5% CO, atmosphere. 2. Collect supematant every 3 d, and replace 50 mL of fresh medium in each flask. 3. Centrifuge the supematant, adjust to pH 9.0, and load on a Sepharose 4B column in series with a protein A-Sepharose column equilibrated with a solution containing 50 mA4 Tris-HCl, 150 mMNaC1, pH 9.0. (The Sepharose 4B column is used first to filter cellular debris and materials that bind nonspectfically to the protein A-Sepharose. The protein A column binds the antibody via the Fc at pH >7.0 and releases it at acidic PH.) 4. Elute the antibody from the protem A column with 0. IM sodmm citrate, pH 4.5. 5. Concentrate the antibody solution (containing approx 100 mg antibody) to about 5 mL and dialyze overnight against 0. 1M sodmm phosphate, pH 7.0, containing 4 mM EDTA. 6. To obtain the Fab fragment, incubate the antibody with 1 mg papain and 20 mg cysteine hydrochloride 4 h at 37’C (see Note 1) 7. To stop the reaction, add 40 mg solid iodoacetamide to alkylate the sulfhydryl groups of the heavy chains, and load on a Sephadex G- 100 column in series with a protein A-Sepharose column. Both columns are first equilibrated as indicated in step 3. The yield is 50-60% Fab.

3.2. Peptide Synfhesis 1. Synthesize the antigenic peptide and its specifically labeled derivatives using an Applied Biosystem 430A automated pepttde synthesrzer. 2. Purify crude peptides partially by gel-filtratron chromatography on a Sephadex G-25 column equilibrated with 0.5% acetic acid in water. 3. Carry out reverse-phase HPLC with a gradient of increasing concentration of acetonitrile in water containing 0.03% tnfluoroacetic acid. (Our peptides are eluted at 28% acetonitrile with a purity better than 98% ) 4. Verify the ammo acid composition of the purified peptides by ammo acid analysis and by NMR sequential assignment.

3.3. NM!? Sample Preparation 1. Prepare Fab solutions at concentrations of l-l.5 mM in 10 n&f sodium-phosphate-buffered D,O or 90% H*O/lO% DzO solution, each contauung 0.05% sodium azide (see Notes 2-4). 2. For the measurements in DzO, dialyze solutions against phosphate-buffered deuterium oxide. The stirred dialysis is conducted continuously for 4-5 d at room temperature at a desired pH, and the sample is transferred to the subsequent phosphate-buffered D,O each day

Epitope Mapping by NMR

33

3. For each difference spectrum, prepare two samples with identical concentrations (l-l.4 mM), at pH 7.0 (one sample contains the Fab complexed with the unlabeled peptide, and the other sample is the complex with the peptide specifically deuterated at a single desired position along the chain). Use Fab from the same cleavage batch and the buffer solutions from the same stock (Note 5).

3.4. Epitope Mapping Protocol 1. All experimental and processing parameters used in our laboratory are given in Note 6. 2. Measure HOHAHA (9) spectrum for the free antigenic peptide in HzO. Perform complete assignment of all crosspeaks observed for the free peptide following the general sequential assignment strategy given by Wuthrich (13). This requires measurement of NOESY or ROESY spectrum in Hz0 (Notes 7 and 8). 3. Measure HOHAHA spectrum of the free Fab m Hz0 (Note 9). 4. Measure HOHAHA spectrum of the Fab-antigenic peptide complex in Hz0 (Note 10). 5. Measure ROESY spectrum of the Fab-peptide complex at the same conditions as the HOHAHA spectrum was recorded (Note 11). 6. Compare the HOHAHA spectrum of the complex with that of the free peptide to identify crosspeaks owing to the residues that do not interact with the antibodybinding site, thus retaining their mobility and exhibiting chemical shift values similar to those found for the free peptide. 7. Compare with the HOHAHA spectrum of the free Fab to identify the residual crosspeaks that might appear from the flexible parts of the antibody. 8. Superimpose NH-C&H region of the HOHAHA and ROESY spectra of the Fabpeptide complex to obtam unambiguous sequential assignment of the flexible residues and those located at the boundaries of the binding sate (Note 12).

3.5. NOESY Difference Spectrum 1. Examine the effect of the temperature and the mixing time on the line width and the signal-to-noise ratio, respectively, in the NOESY spectrum of the Fab-peptide complex (Note 13). 2. Measure NOESY spectra for two complexes (one with unlabeled peptide and the other with the peptide specifically deuterated at a single desired position along the chain) one Immediately after another and at identical expenmental conditions (Notes 14 and 15). 3. Prior to calculating a difference spectrum, process each data set as follows: Zero fill in the F1 dimension, multiply in both dimensions by an apodization window function, and then Fourier transform (Notes 16 and 17). 4. Match pairs of rows from the two spectra in order to estimate a difference factor. It should be 1.Oor very close to 2”, where n is a scaling factor between two spectra. An optimized value should provide a flat baseline and allow the minimization of the number of both positive and negative peaks in the difference spectrum. 5. Multiply one data set by a difference factor, and subtract from the other to obtain a final difference spectrum (Note 18).

34

Kustanovich and Zvi

4. Notes 1. Cysteme acts as a papam activator and as a mild reductive agent for interheavy disulfide bonds. 2. Because of the inherent very low sensitivity of the NMR experiments, htghly concentrated samples are required to obtain meaningful results within a reasonable amount of time. Large proteins may have relatively low solubihty m water (whtch ts also strongly dependent on pH) and tend to aggregate, form complexes, and/or precipitate above specific critical concentration. Usually, an addition of a peptide prevents aggregation and increases stgnificantly the protein solubility However, for the proteins with very low solubility, more dilute samples should be prepared, and more scans (longer experimental times) should be collected m order to achieve a sufficient signal-to-noise ratio. Our typical samples contain about l-l .5 miWconcentrations in Fab, and no solubthty problems were encountered. These concentrations correspond to about 40 mg of a purified FabiNMR sample, and in order to prepare 1: 1 complex between antibody and antlgemc peptide, approx 2 mg of the HPLC-purified peptide are required. 3. The choice of the partrcular pH value could be governed by two conflicting factors: To observe NMR signals from the armde protons, the pH should be as low as possible in order to minimize amide proton exchange with water. In some cases, at low pH, the binding affinity of the peptide to the antibody might decrease significantly. Therefore, the pH value should be adjusted carefully for a system under investigation by taking into account both effects. 4. To assure a molar ratio of 1: 1 between the Fab and the peptide, 20% excess of the free peptide is taken, and then the samples are subjected to two subsequent stirred dialyses against O.OlM sodium phosphate buffer with 0.05% sodium azide. 5 Even small deviations in the NMR sample composition can lead to severe baseline distortions in the difference spectrum. 6. All NMR measurements were performed at 500 MHz on a Bruker AM spectrometer using the time-proportional phase incrementation method (14). The carrier frequency was set on the water resonance signal and it was presaturated during the relaxation time. All spectra were calibrated vs tetramethylsilane. The HOHAHA and ROESY experiments of the 90% HZ00 0% D20 sample were carried out at 34’C. In these measurements, 2048 data points were collected m Fz, with a spectral width of 10-12 ppm. The relaxation delay was set to 3 s. The HOHAHA experiments were performed following the procedure of Bax and Davis (9); 256-5 12 tl increments and 32-80 scans for each t, value were recorded with a typical mixing time of 145 ms. In the ROESY measurements, the duration of the spin-lock pulse was set to 100 ms with a power of 3.5 kHz; 72-80 scans were accumulated for each of 5 12-600 increments of tt . NOESY difference spectra were measured at 42”C, pH 7.15 with a 70 ms mixing time. The residual HOD signal was suppressed by gated decoupling; 256 transients were acquired in the tl domain with 80 x 2000 free induction decays recorded for each t, value. The spectral width was set to 7000 Hz.

Epitope Mapping by NMR

7.

8.

9

10.

11.

12.

13.

14. 15.

35

Data processing was performed using Bruker’s UXNMR software. Zero fllling in F, dimenston and a multiplication of the free induction decay by a shifted sine window function were applied in both dimensions prior to Fourier transformation, The spectra were phase-corrected, and an automatic baselme correction was done using a Bruker program that divided the spectrum into two parts in the F, dimension, allowing definition of a window around the water signal that was not baseline-corrected. Sometimes it is impossible to perform an unambiguous assignment of all resonances, m particular, in cases where an amino acid type is present at more than one position along the sequence. In order to overcome the problem of the degeneracy and hence overlap of the resonances, it is useful to change slightly the pH of the sample and/or temperature of the measurement. The mixing time in the HOHAHA pulse sequence should be long enough to allow sufficient magnetization transfer in order to establish correlations between the amide backbone protons and all side-chain protons. For short peptides, with relatively long correlation times, the typical mixing time intervals are about 400 ms. The mixing time in the HOHAHA pulse sequence should be adjusted to ensure a complete cancellation or at least a significant weakening of the signals of the free Fab antibody The HOHAHA spectra of the free Fab and Fab-peptide complex, which are used for the elucidation of the epitope, should be measured at the same experimental conditions, i.e., temperature, pH values, molar concentrations, and mixing times should be identical or as similar as possible. It is advisable to measure the ROESY spectrum of the Fab-peptide complex immediately after the HOHAHA spectrum has been recorded to minimize various artifacts and phase distortions owing to the instabilities (field drift, temperature fluctuations, and so forth) of the spectrometer. To obtain a good overlay of the HOHAHA and ROESY spectra for performing sequential assignment, it is convenient to record and display them at the same digital resolution. To obtain narrower line width, and thus a better signal-to-noise ratio, the measurements were carried out at 42°C rather than 37°C. To examine the effect of the mtxmg time on the signal-to-noise ratio in the NOESY spectrum, we measured spectra of Fab with unlabeled peptide and with a peptide in which Vall5 was deuterated at two different values of mixing time: 40 and 70 ms. S/N in the NOESY spectrum (of the resolved crosspeak of Ile4) and in the NOESY difference spectrum (VallS cross peak) were 30% higher with 70 ms than those observed using 40 ms. The range of the mixing times used excludes the possibility that spin diffusion contributes considerably to the intensities of the observed crosspeaks (14). The same considerations as in Note 10 should be observed. To obviate the use of a phase correction in the F, dimension (ZS) , the sampling delay (do) was set to do = [MO/Z] - [2PWIn] where IN0 is the dwell time in the FL dimension and PW is the length of the 90” pulse.

Kustanovich and Zvi

36

16. We found that a square sine bell window shifted by 60” gives very good results. 17. To yield minimal distortions in the difference spectrum baseline, the phases of the two spectra should be very carefully adjusted. 18. Typically, a small baseline correction is further applied around the HOD resonance.

Acknowledgments We are most grateful to Jacob Anglister, who headed the studies described in this chapter, and to Rina Levy and Yehezkiel Hayek for their important contributions to the work. This work was supported by a grant of the Israel Ministry of Science and Arts and of the Gesellschaft ftir Biotechnologische Forschung GmbH, Braunschweig (GBF), as well as by a grant from the Israel Science Foundation. I. K. is a Levy E&k01 Postdoctoral Fellow.

Abbreviations NOE, nuclear Overhauser effect; 2D, two-dimensional; NOESY, 2D NOE spectroscopy; TRNOE, transferred NOE; Tr,,, relaxation time in the rotating frame; TZ, transverse relaxation time; COSY, correlated spectroscopy; DQF, double-quantum filtered; ROESY, rotating-frame Overhauser enhancement spectroscopy; d&i, i + l), NOE connectivity between the CaH proton of residue i and the NH proton of the residue i + 1; HOHAHA, homonuclear Hartmann Hahn 2D experiments; Fab, antibody fragment made of the Fv and one constant region of both the light and heavy chains; HIV, human immunodeficiency virus.

References 1. Anglister, J., Levy, R., and Scherf, T. (1989) Interactions of antibody aromatic residues with a peptide of cholera toxin observed by two-dimensional transferred nuclear Overhauser effect difference spectroscopy. Biochemistry 28,3360-3365 2. Scherf, T. and Anglister, J. (1993) A T1,-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens. Biophys. J. 64,754-76 1. 3. Anglister, J., Scherf, T., Zilber, B., Levy, R., Zvi, A., Hiller, R., and Feigelson, D. (1993) Two-dimensional NMR investigations of the interactions of antibody with peptide antigen. FASEB J. 7, 1154-l 162. 4. Fesik, S. W. and Zuiderweg, E. R. P. (1988) Heteronuclear three-dimensional NMR spectroscopy. A strategy for the simplification of homonuclear two-dimensional NMR spectra. J. Magn. Reson. 78,588-593. 5. Tsang, P., Rance, M., Fieser, T. M., Ostresh, J. M., Houghten, R. A., Lerner, R.

A., and Wright, P. E. (1992) Conformation and dynamicsof an Fah’-bound peptide by isotope-edited NMR. Biochemistry 31,3862-3871.

6. Weiss,M. A., Eliason, J. L., and States,D. J. (1984) Dynamic filtering by twodimensional ‘H NMR with application to phage h repressor. Proc. Natl. Acad. Sci. USA 81,6019-6023.

Epitope Mapping by NMR

37

7. Cheetham, J. C., Raleigh, D. P., Griest, R. E., Redfield, C., Dobson, C. M., and Rees, A. R. (1991) Antigen mobility in the combining site of an anti-peptide antibody Proc. Natl. Acad. Scl USA 88,7968-7972. 8. Zvi, A., Kustanovich, I., Feigelson, D., Levy, R., Eisenstein, M., Matsushita, S., Richalet-Secordel, P., Regenmortel, M. H. V., and Anglister, J. (1995) NMR mapping of the antigemc determinant recognized by an anti-gpl20, human immunodeficiency virus neutrahzing antibody. Eur. J. Biochem. 229, 178-187. 9. Bax, A. and Davis, D. G. (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65,355-360. 10. Zvi, A., Kustanovich, I., Hayek, Y., Matsushita, S., and Anglister, J. (1995) The principal neutralizing determinant of HIV-l located m V3 of gp120 forms a 12residue loop by internal hydrophobic interactions. FEBS Lett. 368,267-270. 11, Fesik, S. W. and Zuiderweg, E. R. P. (1989) An approach for studying the active site of enzyme/inhibitor complexes using deuterated ligands and 2D NOE difference spectroscopy. J. Am Chem. Sot. 111,5013-5015. 12. Leonard, C. K., Spellman, M. W., Riddle, L., Harris, R. J., Thomas, J. N., and Gregory, T J. (1990) Assignment of intrachain disultide bonds and characterization of potential glycosylation sites of the type- 1 recombinant human immunodeticiency virus envelope glycoprotein (gp120) expressed m Chinese hamster ovary cells. J Blol. Chem 265, 10,373~10,382. 13. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids Wiley, New York. 14. Marion, D. and Wuthrich, K. (1983) Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of ‘H-‘H spin-spin coupling constants in proteins. Blochem. Biophys. Res. Commun. 113,967-974. 15. Bax, A., Tkura, M., Kay, L. E., and Zhu, G. (1991) Removal of F, baseline distortion and optimization of folding in multidimensional NMR spectra. J. Magn. Reson. 91, 174-178.

4 Mapping Epitopes on Antigens by lmmunodiffusion in Gel Giuseppe

A. Molinaro

and William C. Eby

1. Introduction The mapping of B-cell epitopes on antigen molecules is best done with monoclonal antibodies (MAbs). Most methods require physical chemical procedures for purifying, binding, or labeling the antigen or the MAb. These procedures require some technical proficiency, may present methodological problems, and are based on much trial and error, but, worst of all, these manipulations, including the simple adsorption of molecules to surfaces, may induce the unfolding of the protein antigen, and therefore the loss of native epitopes with the exposure of new determinants (“unfoldons”) (1). Thus, when a mapping technique is successfully set up, there is often the nagging question: “Is the antigen still in its native conformation?’ Our mmrunodiffusion method avoids many of these problems since in most instances, both the antigen and the antibody can be used without any manipulation. The method is based on the fact that a single MAb generates soluble immune complexes with an antigen, but two MAbs to different epltopes, when tested in appropriate conditions, generate insoluble complexes that are detected as a precipitin line. Epitope mapping by immunodiffusion has several advantages over other mapping methods. One can use unlabeled antigen and MAb, and even unpurified MAb and unpurified antigen, as long as the antigen is not a family of related molecules, such as the Ig molecules. The data are mostly based on an all-or-none reaction and are visual. Coreactive MAbs form lines, but competing MAbs give no lines. Notably, the data with competing MAbs are unequivocal, even when the MAbs have different affinities. In contrast, in other competitive assayswith labeled reagents, a high-affinity MAb can displace a low-affinity MAb, but not vice versa. The equipment is minimal, already availFrom

Methods m Molecular Brology, vol. 66 Epitope Mappmg Protocols Edrted by G E Morns Humana Press Inc , Totowa, NJ

39

40

Molinaro and Eby

able in many labs, or can be made easily. The materials are inexpensive. The procedure is fast and deceptively simple. Finally, critical titrations are not necessary since precipitin lines are obtained within wide ranges of antigen and MAb concentrations. Like all techniques, immunodiffusion does have a few limitations. The reactants must be diffusible in the gel. Polymeric antigen and IgM MAb are not suitable for mapping because they form latticed immune complexes. The method is not quantitative, does not give binding constants, and works better and faster at rather high antigen and MAb concentrations (>lOO pg/mL). Finally, the precipitin lines can be photographed, but not stained becausethe antige&vIAb complexesredissolve in washing and staining solutions. The understanding of epitope mapping by immunodiffusion m gel begins with an understanding of the conventional Ouchterlony technique (2). Polyclonal anttbodtes readily precipitate antigen because they form latticed immune complexes that segregate in a gel to form precipitin lines in the respective equivalence zone of each antigen. In contrast, an MAb cannot form antigen-MAb lattices, unless there are at least three copies of the relevant epitope on the antigen, such as a polymer or a hapten conjugate, and the MAb has enough affinity to crosslink the antigen (unpublished). If the antigen carries a single copy of an epitope (“monoepitopic”), the ensuing antigen-MAb complexes are trimolecular and soluble. If the antigen, such as the IgG molecules, carries two copies of an epitope (“biepitopic”), the antiger+MAb complexes are linear or mostly circular, but usually still too small to precipitate in standard conditions. Similar soluble circular complexes are formed by a monoepitopic antigen with two MAbs of different specificities. However, we found that human growth hormone (hGH), a monoepitopic antigen, formed precipitin lines when diffused against two noncompeting MAbs in a gel containing polyethylene glycol (PEG) (3). In similar gels, precipitin lines were also formed by IgG and IgA, when diffused against a single MAb (4,5). Mixtures of hGH with one MAb readily formed lines when diffused against a second MAb of different epitopic specificity (3). Similarly, soluble IgA-MAb complexes formed lines when diffused toward a second MAb (5). These observations led us to develop four gel diffusion methods for mapping epitopes on mono- and biepitopic antigen by testing the antigen with two MAbs in PEG-containing gels. In Assay 1, the two MAbs in one well codiffuse toward the antigen in another well. In Assay 2, the two MAbs and the antigen diffuse independently from three wells at the vertices of a triangle. In Assay 3, the soluble complexes of the antigen with an MAb in one well diffuse toward the other MAb in another well. In Assay 4, the soluble complexes of the antigen with an MAb diffuse toward the soluble complexes of the antigen with the other MAb. Note that in Assay 2, the two MAbs and the antigen, instead of forming a line, may form one or two spurs that may look like a star or like the

lmmunodiffusion in Gel

41

Fig. 1. Precipitin figures formedby two MAbs to humanIgG (upperwells) and humanIgG antigen(lower wells) with 3%PEG(A) and2% PEG(B). In (A), the spurs formed earlier than the lines. In (B), the spursformedwithout the lines. Reprinted from ref. (4) with permissionof the publisher.

stylized silhouette of a flying bird (Fig. 1). At the equivalence of the reactants, the precipitate should be at the center of the triangle. Practically, it may be closer to any of the threewells, or evenbetween the MAb wells if the antigenis small and in excess. The interpretation of precipitin lines formed by monoepitopic antigen is straightforward. Since at least two MAbs are neededfor precipitating the antigen, the very appearanceof a precipitin line between the antigen and the MAb wells is proof that the two MAbs are able to bind to two different and spatially separatedepitopes of the antigen molecule. No line will form when the two MAbs react with the sameor overlapping epitopes becauseof steric competition. All four assayswork very well becausethey are basedon an all-or-none reaction. The interpretation of precipitin lines formed by biepitopic antigen requires the use of a seriesof gels becauseindividual MAb can form precipitin lines in PEG-containing gels. Since different MAbs precipitate the sameantigen at different PEG concentrations, the MAbs are tested in gels containing different PEG concentrations. The gel(s), in which two MAbs precipitate the antigen, but the individual MAb does not, provides the most useful information. It is also useful to monitor the gels in which individual MAb form lines, since theselines form later than the lines formed by two coprecipitating MAbs. Note that Assay 3 and Assay 4 may not work well with biepitopic antigen and high affinity MAb, which probably form bigger MAb-antigen complexes that may precipitate as haloes around wells.

2. Materials 1. GELRITERnsolution:Add 0.9 g GELRITE (Kelco [SanDiego, CA] or Sigma [St. Louis,MO], P 8169)to 100mL of distilledwaterin a graduatedmediabottle. Sincethe solvationof GELRITEtakestime, stir overnight,or stir andwarm up

42

2

3. 4.

5.

6. 7.

8. 9. 10. 11. 12.

Molinaro and Eby the mixture until the powder is evenly swelled and dispersed. Then, heat the disperston m a boiling water bath for about 10 min or untrl the powder is fully dissolved. You can also use a pressure cooker, but turn off the heat as soon as the pressure cooker valve whistles. Whtle still very hot, pipet 8-mL aliquots of the solution into 12 16 x loo-mm glass tubes. When the solution has gelled, cap and store the tubes in a refrigerator. It is stable at 4°C for several months. Stock solutions of PEG 8000 (Baker Chemicals, Phillipsburg, NJ): For monoepitopic antigens, make a single 20% PEG, 0.5% MgClz * 6Hz0, 0.5% NaCl, 0.1% NaN, solution. For blepitopic antigens, make four similar solutions with 15, 10,5, and 0% PEG. You can store them at room temperature for several months Boiling water bath or pressure cooker Polystyrene petri dishes, 100 x 10 mm (Complate, Lab-Tek, Miles, IL): There is less evaporation from these slimmer dishes than from the standard 100 x 15-mm dishes. Evaporation should be mmimized because a higher PEG concentration may precipitate other serum protems m addition to unmune complexes, as indicated by haloes around the wells. Well punchers: They are usually made of beveled stamless-steel hollow cylinders. However, microhematocrtt capillary tubes (Blue-Tips, Fisher [Pittsburgh, PA], 02-668-68) can be used as punchers. A popular puncher is a set of six cylinders, mounted in a hexagonal pattern around a central cylinder In our puncher, the well diameters are about 2 mm, and the distance between wells is 3 mm. Vacuum aspirator* In our lab, we use a home-made vacuum nozzle to aspirate the gel plugs (Fig. 2). Antigen: The antigen need not be purified We have used serum when testing IgA. The working concentration range is wide. We have used rather high antigen concentrations (about 1 mg/mL for IgG and about 0.5 mg/mL for hGH) for faster data. A panel of MAbs as crude ascites. 20-ltL Drummond Microcaps micropipets (Drummond, Fisher, l-000-200). Pleated sandwich bags, 6’12 x 5I/2 x 1 in. A magnifying lens (optional). A camera on a stand and an indirect light source (Immuno-Illuminator, Hyland, CA) to record the precipitin lines (optional): Photograph the precipitin lines without staining them since the washings may redissolve the antigen-MAb complexes.

3. Method One can see precipitin lines in 2-3 h by using crude ascites and the higher concentrations of the antigen working range. The excess of the reactants will guarantee consistent and rapid data, without titration in most instances. For antigens that carry a single copy of each epitope, follow this protocol. For biepltoplc antigens, see Note 1. 1. PEG tube: Pipet 2 mL of the 20% PEG stock solution into a 16 x 100~mm tube for making a 4% PEG-containing dish.

lmmunodiffusion in Gel .’

I’

.c-------

43 3Opl ,’

Mlcroplpet I ,---------

GlassTube

Fig. 2. Homemade vacuum nozzle. Take the glass tube that is in each Drummond Microcaps micropipet box Cap the free end with a cap identical to that already on the other end of the tube. Then, carefully thread a 20-pL Drummond Microcaps micropipet through the hole of one cap, along the tube, and through the hole of the other cap. Slide one end of the nozzle assembly into a plastic tubing connected to a vacuum source via a trap. 2. GELRITE dish: Heat the PEG tube and a GELRITE tube in a water bath, and boll for 10 min, or heat the tubes in a pressure cooker until the valve whistles. While still very hot, hold the PEG and GELRITE tubes with gloved hands or with hardware clamps (Stanley, 43-162P), and pour the GELRITE solution into the PEG tube. Then pour back the mixture mto the GELRITE tube, and finally into a Petri dish rapidly since the solution could become lumpy. The solution gels rapidly, and the gel 1s clearer than agar gels. The dish can be kept for a week on the lab bench if tightly wrapped m a sandwich bag. 3. Gel wells: Cut the gel to create patterns of three or six outer wells around a central well. With one hand, hold the dish firmly on a photocopy of Fig. 3. With the other hand, hold a Fisher Blue-Tips capillary tube, and carefully punch holes into the gel following the patterns, or you can use a multicylinder puncher The dish can accommodate 13 patterns, enough for testing eight MAbs. Suck out the cut gel plugs by using a vacuum aspirator such as the vacuum nozzle of Fig. 2. Do not damage the sides of the wells 4. Well filling: Fill the wells by using 2OqL Drummond micropipets partially filled with the reagents. Tap the microplpet on the bottom of the well. Practice a little to avoid overfilling. In each well pattern, fill the central well with the antigen and every other well with premade biclonal MAb ascites mixtures. The biclonal MAb mixtures should include all the possible pair combinations. You can also fill each outer well first with one MAb and then with the second MAb as soon as the first MAb has been completely absorbed. You should also fill some wells with the mdlvidual MAb. 5. Incubation: Keep the dish m a pleated sandwich bag to minimize evaporation, and at room temperature since changes of temperature from room to refrigerator and vice versa affect the precipltin lines. Remember that the antige*MAb complexes are not latticed. You may score the data the next morning, or after 2-3 h, if you have used undiluted reagents. 6. Data analysis: Look for precipitin lines between central and outer wells. The presence of a line indicates that the two MAbs coprecipitate the antigen, that is, they react with two sterically independent epitopes. In contrast, the lack of a line

Molinaro and Eby

Fig. 3. Template of Ouchterlony patterns suitable for epitopic mapping. Photocopy and place it under the GELRITE Petri dish as a guide. Use the center well for the antigen and every other outer well for the MAb. indicates that the pair of MAbs competes for the antigen, that is, they react with two overlapping epitopes or with the same epitope (see Note 2). If you are screening a large panel of MAbs, tabulate the coprecipitating activities of each MAb in a checkerboard pattern. Then, on the basis of their patterns of reactivities, identify prototype MAbs that identify prototype epitopes. Table 1 shows four hypothetical prototype MAbs, and allows you to map the four prototype epitopes relatively (Fig. 4).

4. Notes 1. For biepitopic antigens, use four gel dishes containing 3, 2, 1, and 0% PEG, instead of a single 4% PEG dish. Briefly, pipet 2 mL of the 15, 10,0.5, and 0% PEG solutions into four 16 x 100~mm tubes, heat these four PEG tubes with four GELRITE tubes, and then follow the above protocol. If reacting with different epitopes, two MAbs form a line as a biclonal mixture, but not as individual MAb at the lower PEG concentrattons. At the higher PEG concentrations, an individual MAb may form a line, but the line formed by the biclonal MAbs appears earlier than the line formed by the individual MAb.

lmmunodiffusion in Gel

45

Table 1 Prototype MAb Reactlvities MAbl MAb2 MAb3 MAW

MAbl + +

MAb2 -

MAb3

-

+ +

MAb4 + + -

From a large number of MAbs, one can identify a small number of prototype MAbs on the basis of their patterns of reactivity. The tabulated hypothetical prototype MAbs Identify four epitopes and their relative positions (Fig. 4).

Fig. 4. Hypothetical map of four prototype epitopes identified with the four prototype MAbs of Table 1. Note that by testing only MAb 1 and MAb2, one would miss that Epitopes 1 and 2 are overlapping, but not identical. Similar misses would occur when testmg only MAb2 and MAb3, or only MAb2 and MAb4. In contrast, the use of a number of MAbs allows the drawing of a map of overlapping and independent epitopes. 2. If you get a double line, one ascite may contain much more MAb than the other ascites (4). Ignore the doubling of the line, or to obtain a single line, dilute the ascites that contains excess MAb. If you do not get a line, the concentration or the affinity of the MAb may be too low to give a precipitin line. Retest the MAb by refilling the wells.

Acknowledgment We are grateful to Skip Brown for his enthusiastic

help.

References 1. Laver, W, G., Air, G. M., Webster, R. G., and Smith-Gill, S. J. (1990) Epttopes on protein antigens: misconceptions and realities. Cell 61,553-556. 2. Ouchterlony, 0. and Nilsson, L. A. (1986) Immunodiffusion and mununoelectrophoresis, in Handbook of Experimental Immunology, vol. 1 (Weir, D. M., Herzenberg, L. A., Backwell, C., and Herzenberg, L. A., eds.), Blackwell, Oxford, Sect. 32.1-32.50. 3. Molinaro, G. A., Eby, W. C., Molinaro, C. A., Bartolomew, R. M., and David, G. (1984) Two monoclonal antibodies to two different epitopes of human growth

46

Molinaro and Eby

hormone form a precipitin line when counterdiffused as soluble immune complexes. Mel Immunol 21,771-774. 4. Molinaro, G. A. and Eby, W. C (1984) One antigen may form two precipitm lines and two spurs when tested with two monoclonal antibodies by gel diffusion assays. Mel Immunol. 21,18 l-l 84. 5. Mohnaro, G. A., Bui, J. D., and Eby, W. C. (1992) Native epitopes of human IgA. Int .I, Clin Lab. Res. 21,235-240.

5 A Simple Solid-Phase Competition Assay with Labeled Antigen Masahide Kuroki 1. Introduction The determination of epitope specificities of monoclonal antibodies (MAbs) has usually been performed using the competitive solid-phase assay in which the antigen is immobilized, and a radiolabeled antibody and competing unlabeled antibodies are mixed in solution (Fig. 1A) (1,2). Although this method facilitates separation of free from bound antibody, it possessesthe problem of labeling all antibodies to be tested. Since the number of MAbs to be screened is usually large, this method is time consuming and tedious, and the instability of radiolabels represents a significant drawback. In addition, radioactive hazards have to be taken into account. Recently, nonisotopic tracers, such as biotin (3‘4) and fluorescein isothiocyanate (4), have been introduced for determination of epitope specificities of MAbs, but these methods still have the problem of labeling all antibodies to be tested. This chapter describes a solid-phase mutual competition assay for determination of epitope specificities of MAbs by using 96-well plates coated with MAbs, competitor MAbs, biotinylated antigen, and avidin-peroxidase conjugate (Fig. 1B) (5). A constant amount of biotinylated antigen is incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated antigen bound to the immobilized antibody is then reacted with avidin-peroxidase conjugate, and the activity of the bound peroxidase is determined by the use of o-phenylenediamine and hydrogen peroxidase. The exchange of competing and immobilized antibodies in this assay system does not demand additional labeling procedures (5,6). Thus, the competition method described here alleviates the laborious procedures of labeling all antibodies to be tested and the confusion From

Methods m Molecular Bfology, vol 66 Epltope Mapping Protocols Edlted by G E Morns Humana Press Inc , Totowa, NJ

47

48

Kuroki

Blocked with Block Ace ’

Fig. 1. Diagrammatic representationof a conventional competition immunoassay with labeled antibody (A) and a new type of competition immunoassaywith labeled antigen (B) used in epitope mapping. In the competition assaywith labeled antibody, the antigen is immobilized, and a radiolabeled antibody as well as competing unlabeled antibodies are mixed in solution. Finally, the radiolabeled antibody bound to the immobilized antigen is detected in a y-counter. Recently, biotinylated antibody is often used insteadof radiolabels.On the other hand,in the competition assay with labeled antigen, a constant amount of biotinylated antigen is incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amountsof soluble competitor MAbs. The biotinylated antigenbound to the immobilized antibody are then reactedwith avidin-peroxidaseconjugate and the activity of the bound peroxidaseis determined.

causedby differential labeling among different MAbs, and is convenient for mappinganalysisof many MAbs if the correspondingpurified antigenis available. The typical competition curves of two Group C anti-CEA (carcinoembryonic antigen) MAbs (F4-82 and F82-61) are shown in Fig. 2. In the MAb F4-82 competition assay (Fig. 2A), of the seven MAbs used as competitors, five MAbs, including the homologousMAb, showedmore than 80% inhibition. Two MAbs could not exhibit more than 50% inhibition in this assay.As shown in Fig. 2B,

Competition Assay with Labeled Antigen

2,500

g

E P z

500 Competitor

49

100

20

antlbody

4

0.8

added (ng)

2 E g 100 P 75

50

25

0 2,500

500 Competitor

100 antibody

20

4

0.8

added (ng)

Fig. 2. Mutual competition assays among Group C anti-CEA MAbs by using biotinylated CEA and purified MAb preparations. Two MAbs of Group C, F4-82 (A) and F82-6 1 (B), were dried onto wells of 96-well plates. Purified IgGs from the Group C MAbs were also used as competitors at the indicated quantities. (Adapted, with permission, from Kuroki et al. 151). Competitor MAbs used: 0, F4-82; 0, F6-22; c1, F84-10; n , FIOI-35; A, F33-49; A, F82-68; X, F82-61.

Kuroki

50 Table 1 Mutual Competition Assays Among Group C MAbsfib MAb immobilized Competitor MAb F4-82 F6-22 F84-10 FlOl-35 F33-49 F82-68 F82-6 1 Epitope

F4-82

F6-22

on polystyrene plates

F84-10

FlOl-35

F33-49

F82-68

F82-6 1

+++

+++

+++

+++

+++

+

-

+++

++-I-

+++

+++

+++

+

-

++t

+++

+++

+++

-

+

-

++-I-

+++

+++

+++

-

+

-

++-t -

+++ -

++ -

+

+

-

+++ -

+++

+++

-

-

-

-

-

+++

+++

C-a

C-a

c-c

C-d

C-a

C-a

C-b

-

“The group C anti-CEA MAbs recognized the epitopes on the domain N of the CEA molecule (Kuroki et al. [.5,7/). bThe amount of competitor MAb required to give half-maximal Inhibition of binding of biotmylated CEA was determined from the respective mhibitlon cures cKey* +++, half-maximal mhibitron at 400 ng; ++, 100-500 ng, +, 500-2500 ng, -, no 50% mhlbition was obtained even at the highest amount (2500 ng) of competitor antibody. Adapted with permission, from Kurokt et al (5).

however, in the MAb F82-61 inhibition assay,only two of the seven competitor MAbs used showed over 80% inhibition at the highest input levels, whereas all five other MAbs demonstrated only trivial inhibition. To quantify the inhibitory effect of each MAb, the amounts of competitor MAbs required to mhibit the blotmylated antigen binding by 50% to each MAE>dried on wells are determined from the respective inhibition curves. Table 1 summarizes the results of mutual competition assaysamong seven Group C anti-CEA MAbs. This presentation allows the comparison of the ability of each MAb to inhibit the binding of other MAbs to CEA with the reciprocal competition of each MAb binding to CEA by the other MAbs (6,7). The nonreciprocal crosscompetitions could result from: 1. Recognition by an antibody of several structurally related sites, only some of which may be recognized by other antibodies; 2. Steric hindrance of an epitope by a second antibody bound to a different site; or 3. Conformational change in the antigen molecule by binding of one antibody, which may affect binding of the second antlbody.

2. Materials

2.1. Biotinylation

of Antigen

1. 1-mL small reaction vials with internal cone (Reacti-VlalTM, Co., Rockford, IL). 2. PBS (0.9% NaCl, O.lM sodium phosphate buffer, pH 7.0). 3. Antigen solution: 1 mg/mL in PBS.

Pierce Chemical

Competition Assay with Labeled Antigen

51

4. N-hydroxysuccinimidobiotin (NHSB, mol wt = 341.4) solution: 2.0 mg/mL (5.86 mA4) in N, N-dimethylformamide. Add 4.0 mg of NHSB, with stirring, to 0.5 mL N, N-dimethylformamide in a Reacti-Vial, and dilute the solution up to 2 rnL with deionized distilled water. Prepare freshly just before use (see Note 1).

2.2. Preparation of Antibody-Coated

Plates

5. 96-Well polystyrene plates. 6. BBS: 0.9% NaCl, O.OlM borate buffer, pH 8.0. 7. Blocking solution: Block Ace (Dainihon Chemical Industries, Osaka, Japan), which includes casein and some other proteins from bovine milk (see Note 2). 8. Washing buffer: 0.05% Nonidet P-40 (NP-40) in BBS.

2.3. Mutual Competltion Assay 9. Assay buffer: 1% bovine serum albumin (BSA), 0.1% methylp-hydroxybenzoate MHB, 0.01% propyl p-hydroxybenzoate (PHB) in BBS (see Note 3). 10. Competitor antibody solutions: Make serial fivefold dilutions of the MAb to be tested in the sample buffer. The starting concentration of each MAb is 100 l@nL. 11. Btotinylated antigen solution: 200 ng/mL m the assay buffer (see Section 3.1.). 12. Horseradish peroxidase (HRP)-avidin D solution: 0.25 pg/rnL in the assay buffer. 13. Citrate/phosphate buffer (CPB): 0.05M citrate, O.lM phosphate, pH 5.0. 14. Substrate stock: 4% o-phenylenediamine (OPD) in methanol. Store in aliquots at -70°C. 15. 30% Hydrogen peroxide (H202). 16. Substrate solution: 0.04% OPD, 0.006% HZ02 in CPB. Dilute 150 pL of 4% OPD and 3 pL of 30% HZ02 up to 15 mL with CPB for one plate. This should be prepared fresh. 17. Stopping solution: 8N H2S04.

3. Method

3.7. Blotinylation

of Antigen

1. Add 25 CAL(50 ug; 146 nmol) of freshly prepared NHSB solution to 0.1 mL (100 pg) of antigen solution in a 1-mL Reacti-Vial with rapid stirring (see Note 4). 2. After incubation for 2 h at room temperature, dialyze exhaustively at 4°C against BBS. 3. Determine the protein concentration of the biotin-labeled antigen by reading the OD at 280 nm (if the extinction coefficiency at 280 nm of the antigen is available) or by another method, such as the bicinchoninic acid method (8). Also, see vol. 32 of this series. 4. Store in aliquots at -2O’C (see Note 5).

3.2. Preparation of Antibody-Coated

Plates

1. Dilute the MAbs to be tested in BBS at concentrations of OS-5 pg/mL (see Note 6). 2. Add 50 pL of each MAb solution into each well of 96-well plates and dry down at 37°C overnight (see Note 7).

52

Kuroki

3. Block nonspecific protein absorption by adding 200 pL of the blocking solutron into each well and incubating for 1 h at 37°C 4. Remove the blocking solution, and wash the plates three times with the washing buffer.

3.3. Competition Assay 1. To each well of 96-well plates previously coated wtth a given MAb, add increasing amounts of competitor MAbs in 25 pL of the sample buffer and 5 ng of biotinylated antigen in 25 pL of the same buffer (see Notes 8 and 9), and shake the plate for 20 s on a plate shaker. 2. After a l-h incubation at 37”C, remove the mixed solutions and wash three times with the washing buffer. 3. Add 100 pL of the HRP-avidin solution and mcubate for 1 h at room temperature. 4. Remove the HRP-avidin solution and wash three times with the washing buffer. 5. Add 150 pL of the substrate solution and incubate for 20-30 min at room temperature 6. Terminate the reaction by adding 20 yL of the stopping solution and read the OD of each well at 492 nm in a plate reader. 7. Determine the amount of competitor MAb required to give half-maximal inhibition of binding of biotinylated antigen from the respective mhibition curves (see Fig. 2).

4. Notes 1. Recently, NHSB has been frequently replaced with long-chain homologs, such as sulfosuccmimidyl-6-(biotinamido) hexanoate (9). The addition of a spacer m biotinylating reagents facilitates subsequent interaction with avidin probes. Water-soluble analogs of NHSB and its derivatives, 1.e , the sulfosuccmimide reagents, are also available from Pierce Chemical Co. (9) In certain cases, these may be favorable, especially when working with proteins that are sensitive to organic solvents, such as N, N-dimethylfotmamide. 2. 5% BSA in BBS can be also used for blocking, but Block AceTM is more effective for preventing nonspecific protein binding. 3. Instead of sodium azide, MHB and PHB are used as preservatives that do not affect the color reaction of OPD. The assay buffer containing these preservatives can be stored for 2-3 mo at 4°C. 4. Biotin can be readily conjugated to a variety of molecules, such as antibodies, enzymes, nucleic acids, and so forth. The small size (mol wt = 341.4) of the biotin molecule prevents the biotinylation procedure from modifymg the chemical, physical, or immunological properties of the molecules to which biotin is bound. Moreover, multiple biotinylation of the same molecule can be performed without any adverse effect (5,10). It is sometimes difficult, however, to determine the exact number of biotin molecules per antigen (protein) molecule. We usually use an NHSB/antigen molar ratio of 1000 for preparing biotinylated antigen, because when carcinoembryonic antigen (CEA) was biotinylated, this ratio did not affect the immunoreactivity of CEA and gave the maximum avidin-binding activity of CEA (5,.

Competition Assay with Labeled Antigen

53

5. Most biotinylated antigens are very stable and can be stored at 4OC for at least 2 yr. 6. The concentrations of MAbs used for coating the plates should be those at which the biotinylated antigen used gave the absorbencies ranging from 1.0-l .2 (~1.5) in the absence of competitor antibody, resulting in good inhibition. 7. When the plates are coated with antibody and dried overnight, ensure that they are completely dry. Otherwlse, maximum antibody binding will not be obtained, and some binding protein will be lost from the plates during subsequent procedures. 8. The competitor antibody solutions should be added into the antibody-coated plates before adding the biotinylated antigen solutions, also resultmg in good inhibition results, 9. Usually, only l-10 ng of biotinylated antigen is enough for this competition assay.

References 1. Kaufman, B. M. and Goldsby, R. A. (1982) Epitope ratio analysis (ERA): a simple radioimmunological method using monoclonal antibodies for the simultaneous analysis of several antigens. J immunol. Methods 54, l-7. 2. Wagener, C., Yang, Y. H. J., Crawford, F. G., and Shively, J. E. (1983) Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epltope speclficitles of monoclonal antibodies. J Immunol. 130,2308-23 15. 3. Bayer, E. A. and Wilchek, M. (1990) Protein biotinylation. Methods Enzymol. 184, 138-160.

4. Harlow, E. and Lane, D. (1989) Antibodies: A Laboratory Manual, 2nd ed. Cold Spring Laboratory, Cold Spring Harbor, NY. 5. Kuroki, M., Wakisaka, M., Murakami, M., Haruno, M., Arakawa, F., Higuchi, H , and Matsuoka, Y. (1992) Determination of epitope specificities of a large number of monoclonal antibodies by solid-phase mutual inhibition assays using blotinylated antigen. Immunol. Invest. 21,523-538. 6. Kuroki, M., Fernsten, P. D., Wunderlich, D., Colcher, D., Simpson, J. F., Poole, D. J., and Schlom, J. (1990) Serological mapping of the TAG-72 tumor-associated antigen using 19 distinct monoclonal antibodies. Cancer Res 50,4872-4879. 7. Kuroki, M., Arakawa, F., Harnno, M., Murakami, M., Wakisaka, M., Higuchi, H., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1992) Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recogmzed by 57 monoclonal antibodies and categorized into 7 groups in terms of domain structure of the CEA molecule. Hybridoma 11, 391-407. 8. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem 150,76-85. 9. Savage, M. D., Mattson, G., Desai, S., Nielander, G. W., Morgensen, S., and Conklin, E. J. (1994) Avidw-Blotin Chemistry: A Handbook, 2nd ed. Pierce Chemical Co., Rockford, IL. 10. Wilchek, M. and Bayer, E. A. (1990) Introduction to avidin-biotin technology. Methods Enzymol. 184,~13.

Epitope Mapping by Antibody Competition Methodology and Evaluation of the Validity of the Technique Socrates J. Tzartos 1. Introduction Most epitope mapping techniques can be classified into two groups: those that use antigenic fragments (subunits, peptides, and so forth) and the specific antibody-binding regions are directly determined, and those that use intact antigen and the epitope determination is usually indirect. Recently, It has become possible to localize epitopes in fine detail by the use of synthetic peptides of various kinds. Some of the recent synthetic and recombinant peptide techniques allow the simultaneous production of many peptides, and make it possible to identify each contributing amino acid in an epitope (1,2). The question therefore arises whether we still require techniques using intact antigens, which only permit indirect and approximate localization. However, peptide mapping techniques are incomplete, since: 1. The fact that a monoclonal antibody (MAb) binds to a peptide corresponding to a sequential segmentof an antigen does not guaranteethat rt also binds to that same sequence m the intact molecule. The peptide may simply mimic an irrelevant conformational epitope on the antigen (perhaps formed from a number of

distantsegments),or the sequencethat is identical to that of the syntheticpeptide may be hidden or have an altered conformation in the intact molecule. 2. Many MAbs do not bmd to any synthetic peptide, probably because their epltope is conformation-dependent. 3. Even when it is unequivocally established that the peptide corresponds to the actual epitope, its relative location on the intact molecule will remain unknown, unless the crystallographic structure of the molecule has been determined,

From

Methods In Molecular Brology, vol. 66. Epltope Mapping frofocols Edlted by G E Morns

Humana

55

Press Inc , Tolowa,

NJ

56

Tzartos

We have performed extensive competition experiments between many MAbs directed against the acetylcholme receptor (AChR) (3-6,,, as well as between MAbs and the natural antibodies in human antt-AChR sera (71, and have compared the data obtained in this way with those from detailed peptide mapping experiments (8-Z4). Overall, the two approaches confirmed and complemented each other, and showed the antibody-competition approaches to be surprisingly accurate. Figures 1 and 2 compare the results of competition experiments between MAbs directed against sites on the cytoplasmic region of the AChR CL-and psubunits (5) with the subsequently derived fine mapping results from antibody binding to synthetic peptides (derived from the a- and P-subunit sequences) attached to polyethylene rods (9,10) according to the Pepscan technique of Geysen (1,2), as well as with earlier peptide mapping experiments using conventional larger peptides (8,13,14). In interpreting competition results, it should be taken mto account that when two epttopes overlap, or even when the areas covered by the arms of two MAbs overlap, competition should be almost complete and mutually crosscompetitive. Thus, only marked mutual crosscompetition should be taken as unequivocal evidence of overlapping epitopes, since weak or one-way inhibition may simply reflect a decrease in affinity owing to steric or allosteric effects. Therefore, we completely ignored casesof weak inhibition (~25%) and essentially only considered inhibition of >50%, paying particular attention to those antibodies giving inhibition values of >70%. It was shown that only those MAbs that bind to very close, or overlapping, sequential epitopes effectively show strong mutual crossinhibition of binding to the antigen. Interestingly, MAbs whose epitopes are separated by as few as six amino acids (anti-a MAb 8 vs MAb 142 in Fig. 1) did not significantly crosscompete. Similarly, in studies using anti-S-subunit MAbs, antibodies to sequential epitopes separated by only five amino acids showed only partial crosscompetition (12). Furthermore, differences between MAbs directed against overlapping epitopes could also be clearly detected by their differential competition pattern with other MAbs: e.g., anti-a-subunit MAbs 8 and 147, which bind to overlapping epitopes, showed complete mutual crosscompetttion, but only one, MAb 147, competed with MAbs 142 and 3 (Fig. 1). Similarly, the binding of anti+subunit MAb 117 to the AChR was inhibited by all MAbs directed against the overlapping epitope VICE-P, but MAbl17 itself could only weakly inhibit the binding of the same MAbs when used as the first (protecting) MAb (Fig. 2). A similar pattern was also seen for several MAbs to the S-subunit (12). It should, however, be noted that these MAbs were derived from animals immunized with SDS-denatured AChR and were selected using intact AChR. Because of this, we probably selected for MAbs directed against antigenic sites

Epitope Mapping by Antibody Competition

a-subunit

-ENKIFADDIDISDISGKQVTGEVIFQTPLIKNPDVKSAIEGVKV 3c.o 3&Q

57

3110

Fig. 1. Mapping the binding sites of MAbs against the AChR a-subunit by competition between pairs of MAbs for binding to the intact Torpedo AChR and comparison with their identified sequential epitopes by synthetic pepttdes. 1251-a-bungarotoxinlabeled AChR was preincubated with a protecting soluble MAb and the complex was incubated with a Sepharose-bound MAb. The Sepharose-bound radioactivity was measured and the percentage inhibition of binding owing to the protecting, soluble MAb was estimated. Large bars denote effective competition (modified from ref. 5). Horizontal bars at the bottom represent the epitopes of the corresponding MAbs identified using synthetic peptides (9,10,14). The epitope for MAb 173 has not yet been determined. The epitope for MAb 19 was only approximately localized by the use of large conventional synthetic peptides (8). Its exact epitope may form only part of the indicated a346-364 peptide and is probably located at its N-terminal end, as Judged from the competition pattern with the other MAbs. The epitopes of the other MAbs have been accurately determined using Pepscan peptides. MAb 149 is an IgM, the large size of which may explain its broader protecting capacity. whose conformation is virtually the same in the two states. This may be partially the reason for the very good correlation between the results for peptide

mapping and antibody competition. Nevertheless, another large group of antiAChR MAbs, derived from animals immunized with intact AChR, showed complete mutual crosscompetition for binding to the AChR (3,6) permitting us to define the main immunogenic region (MIR) of the AChR; several of these anti-MIR MAbs could be mapped by synthetic peptides, and all were found to bind to the same epitope, residues 67-76 of the a-subunit (8).

58

Tzartos

169 -169117 Protecting J 23 soluble mAi%‘/ -A Segment of .li p-subunft

118

15336 I

6469

Fig. 2. Mapping the binding sites of anti+subunit MAbs on the intact Torpedo AChR by competition experiments (modified from ref. 5) and comparison with peptide mapping experiments. The experimental conditions were as in Fig. 1, The large horizontal bar at the bottom represents the region J3336-469, and the small bars above it mark the locations of the sequential epitopes. The locations of the epitopes for MAbs 169 and 172 have only been approximately identified using proteolytic pepttdes (13), whereas the epitopes for the remaining MAbs have been accurately determmed using Pepscan synthetic peptides (IO). VICE-S, very immunogenic cytoplasmic epitope on P-subunit.

2. Materials 1. PBS: 0.9% NaCl, 10 m44 sodium phosphate, pH 7.2. 2. PBS-Tween: PBS, containing 0.05% Tween-20. 3. PBS-bovine serum albumin (BSA): PBS, containing 1% BSA (see Note 1) and 0.02% sodium azide.

4. O.lM sodium bicarbonatebuffer, pH 9.6. 5. Anti-Ig produced by immunization

of rabbits with the relevant Ig.

3. Methods The first antibody to be incubated with the antigen will be referred to as the “first,” or “protecting,” antibody, although that which is incubated with the formed antigen-first antibody complex will be referred to as the “second” antibody. For best results, care should be taken that the protecting antibody is present in excess over the antigen, whereas the amount of second antibody

59

Epitope Mapping by Antibody Competition

should be just sufficient to saturate the antigen (or about 80% of it). Techniques will be described for competition between antibodies from the same species or between antibodies from two different species. In general, one of the three reactants (the antigen and the two antibodies) is labeled by radioactivity or by conjugation to an enzyme. In some cases,indirect labeling can be used, i.e., labeled molecules are used that bind specifically to only one of the reactants (e.g., a ligand of the antigen, an anti-antibody, protein A, and so forth). Usually the assaysare solid-state. Therefore, one of the nonlabeled reactants is immobilized on plastic wells or on Sepharose beads. Antibody competition mainly involves MAbs, but competition between MAbs and polyclonal sera may also be required to determine the percentage of serum antibodies directed against specific epitopes or regions (7). 3.1. Competition

Between Homologous

Antibodles

3.1.1. labeled Antigen, Immobilized Second Antibody A major advantage of this technique is that a single labeling step (that of the antigen) makes it possible to carry out crosscompetition experiments using antiAChR antibodies of any type. It does, however, require that the second antibody preparations be either purified or antibody-enriched (see Note 2). 3.1.1 .I. DETERMINATION OFTHE APPROPRIATE CONCENTRATION OF LABELED ANTIGEN (SEE NOTE 3) 1. Add 50-yL samples of 20 pg/mL of second antibody in O.lMsodmm bicarbonate buffer, pH 9.6 (or in PBS) to the wells of a 96-well microplate (18-24 wells/ antibody), and incubate for 2 h at room temperature or overnight at 4°C (see Note 4) Among the antibodies tested, at least one “control” nonbinding antlbody should be included in order to determinebackgroundbinding. Alternatively, 20 yg/mL of BSA may be used as a negative control. 2. Wash the wells three times with PBS-Tween. 3. Fill the wells completely with PBS-BSA, and incubate for 30-60 mm at room temperature. 4. Wash twice with PBS-Tween. 5. Place 50 pL of 1251-labeled antigen (20,000 cpm) alone, or mixed with variousfold-excesses (e.g., 0, 1, 2, 4, 8, 16, 32, 64 or 0, 1, 3, 9, 27, 81) of unlabeled antigen in PBS-BSA in each well. 6. After 2 h incubation at 4’C, remove nonbound antigen by four washes with PBS-Tween. 7. Release the bound radioactlvity by adding 100 pL of 1% SDS, place in test tubes, and count on a y-counter. 8. After subtracting the background counts, plot the results (bound radioactivity vs antigen concentration) m order to select the proper antigen concentration for the competition experiments. Normally, the plot should show a plateau of bound radioactivity, which then starts to decrease. The antigen concentration at the point

Tzartos

60

at which the values start to fall is consideredequtmolar to the amount of active antibody on the plate. A value of SO-100%of this concentration is used in the subsequentsteps(seeNote 5). 3.1 .I .2. DETERMINATION OF THE REQUIRED CONCENTRATION

FIRST(SOLUBLE) ANTIBODY In this part, competition is between different samples of the same antibody (immobilized and soluble) using varying concentrations of the soluble antibody. 1. Repeatstepsl-4 of Section3.1.1.1. OF

2. Preincubate the antigen at the predetermined concentration for 2-4 h at 4T wtth

increasing concentrationsof the first antibody in PBSBSA 3. Placethe mixture in the coatedwells (50 pL/well). 4. Repeat steps 6 and 7 of Section 3.1.1.1. 5. Plot the results (bound radioactivity vs concentration or dilution of the soluble antibody used). For the subsequent competition experiments between heterologous antibodies, whenever possible, a lo-fold excess of the soluble antibody (calculated as 20 times the concentratron required to bind 50% of the plateau value

[maximum bound radioactivity]) is chosen(seeNote 6). 3.1 .1.3. FINAL COMPETITION EXPERIMENT BETWEEN DIFFERENT ANTIBODIES 1. Repeat steps l-4 of Section 3.1.1.1. for coating, washes, and blocking.

2. Preincubatethe previously determinedconcentrationsof labeled antigen andprotecting antibodies(including controlswith anonbinding antibody or without antibody) for 2-4 h as above. 3. Add 50 pL of the mixture to eachwashedwell, and incubate for 2 h at 4’C. 4. Repeatsteps6 and 7 of Section3.1.1.1. A variation to this technique, in which the second antibody is immobilized on Sepharose beads rather than on ELISA plates, is described in Note 7. 3.1.2. Labeled Second Antibody, Immobilized Antigen The various antibodies to be tested as “second antibodies” must be relatively pure (50% “pure” is adequate), and either radiolabeled (preferably by 1251)or peroxidase-conjugated (see Note 3). 3.1.2.1.

COATING WITH THE ANTIGEN

1. Add 5OyL samples of 20 pg/mL (or less: l-10 yg/mL) of antigen in 0. Msodium bicarbonate buffer, pH 9.6 (or in PBS) to the wells of a 96-well microplate, and

incubatefor 2 hat room temperatureor overnight at 4°C. If the antigen is a membrane protein, which requires the presence of detergent in the buffer, solubilize it in the appropriate concentration of the required detergent, and then dilute it in a buffer lacking detergent (PBS or sodium bicarbonate) to 20 pg/mL or less for the

coating step(a final concentrationof 0.05%Triton X-100 is acceptable).An equal concentration of BSA may be used as a negative control

2. Repeatsteps2-4 of Section3.1.1.1.

Epitope Mapping by Antibody Competition 3.1.2.2.

61

DETERMINATION OF THE REQUIRED ANTIBODY CONCENTRATIONS

1. Add 5O+L samples, containing 20,000 cpm of labeled antibody (mixed with 0, 3-, lo-, or 30-fold excess of the same unlabeled antibody in PBS-BSA) to each well, and incubate for 2 h at room temperature. 2. Wash the plates four times with PBS-Tween. 3. Measure the bound radioactivity and plot the results (bound radioactivity vs amount of antibody used) as in steps 7 and 8 of Section 3.1. I. 1. Normally, the plot should start with a plateau that suddenly starts to decrease. The antibody concentration at the end of the plateau (when it starts decreasing) is considered equimolar to the active antigen on the plate. 4. Repeat steps 1 and 2 in Section 3.1.2.1. 3.1.2.3.

COMPETITION EXPERIMENT

1. Add 50 pL of unlabeled first antibody (10 times the above determined “equimola? concentration) in PBS-BSA to the antigen-coated wells, and incubate for 3-4 h at room temperature. 2 Add (without removal of the unlabeled antibody) 50 ltL of 1251-labeled second antibody (if necessary, mixed with unlabeled antibody to give the “equimolar” concentration) m PBS-BSA, and incubate a further 2 h at room temperature. 3. Wash the plates and count the radioactivity as in steps 2 and 3 of Section 3. I .2.2. (see Note 6).

3.2. Competition Between Antibodies from Two Different Species Competition between antibodies from different species has an additional advantage. Using anti-Ig sera, which bind selectively to Ig from the species in which the second antibody was raised (e.g., of human origin), the competition test can be performed totally in solution with the only labeled specres being the antigen. For example, we often perform competition experiments between rat MAbs and human sera for binding to the AChR. 1. Label the antigen as m Section 3.1.1, 2. Obtain antiserum specific for Ig from the species m which the second antibody was raised (e.g., human sera: use antihuman Ig). 3. If this antiserum partially crossreacts with the first antibodies, preincubate it for at least 3 h with normal serum from the corresponding first species, e.g., rat (the appropriate amountshave to be determined experimentally,but in my laboratory we mix 1 mL of anti-Ig serum with 50 yL of normal serum) and centrifuge to eliminate any aggregates formed.

4, Add an additional 50 yL of normal serum and incubate for at least 2 h, usually wtthout any further pellet becoming visible. The pretreated antiserum must then be tested to verify that it does not bind the protecting antibodies and to determine the amount neededto precipitate the secondantibodies,as follows.

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5. Incubate 20 p,L of PBS-BSA contaimng 20,000 cpm (10-100 fmol) of labeled antigen with 20 pL of PBS-BSA containing a IO-fold (or greater) molar excess of protecting first antibody or an equimolar amount of second antibody (or 80% of this). To ensure that a significant pellet will be formed in the subsequent steps, the 20 pL of antigen should also contain 0.1 pL of normal serum from the species in which the second antibody was raised to act as a carrier. 6. After 3 h of incubation, add 20-PL dilutions (from undiluted to l/IO) of the pretreated anti-antibody, and incubate for 1 h. 7. Add 1 mL of washing buffer and mix. 8. Centrifuge for 3-5 min and wash the pellets one to two times. 9. Count the radioactivity of the pellets. The results should (1) show that the tubes with the protecting antibody contam only background level of radioactivity, and (2) deterrnme the appropriate dilution of anti-antibody for the subsequent experiments.

3.2.1. Final Experiment 1. Incubate 20 pL of PBS-BSA contaming about 20,000 cpm (10-100 fmol) of labeled antigen plus 0.1 mL of normal serum from the species m which the second antibody was raised, for 3 h with 20 pL of PBS-BSA containing a lo-fold molar excess of “protecting” antibody. 2. Add 20 pL of a sample contammg second antibody equivalent to about 80% of the antigen used, and Incubate for a further 2 h. 3. Add 20 pL of a sample containing the above predetermined dilution of pretreated antGIg, and incubate for 1 h. 4. Add 1 mL of washing buffer, mix, and centrifuge. 5. Wash the pellets one to two times, and count their radioactivity (see Note 8). Alternatively, by using enzyme-conjugated antiserum specific for the Ig of the second species, the competition can be performed as a solid-phase assay without any requirement to label any of the three reactants: antigen, first, and second antibody (see Note 9).

4. Notes 1. Some investigators use 3% BSA; in my laboratory we have not found it significantly better, but we found it significantly more expensive. An efficient economical alternative is the use of 3% powdered milk instead of BSA 2. Ammonium sulfate precipitates of ascites fluids are sufficiently pure, but hybridoma culture supematants contaming serum are not appropriate. However, an easy means of obtaining sufficiently pure antibody preparations is to culture the hybridomas for 24 h in serum-free medium (DMEM); under these conditions, in our experience, the cells contmue to produce antibody at about one-third of their normal rate. This can then be concentrated by ultrafiltration (e.g., by Amicon, Beverly, MA), but not by ammonium sulfate precipitation, since the protein concentration is too low.

Epitope Mapping by Antibody Competition

63

3. The anttgen that is routmely used in my laboratory, the AChR, is easily indirectly labeled by premcubation with 1251-labeled a-bungarotoxin, which binds very strongly in the region of the acetylcholine-binding sites. It is, therefore, a simple process to label AChRs without the need for purifying them. Should such a method not prove suitable, the antigen can be conjugated to peroxidase or labeled directly with 1251,using the chloramine-T or other methods (15). The use of the radiolabeled antigen is described below, but the method can be easily modified for use with peroxidase-conjugated antigen. If the concentration of the unlabeled antigen is known, a reasonable estimate of the molarity of the labeled antigen can be made assuming about 20-30% loss during the labeling and purification procedures. However, it is probable that a large percentage may be inactivated during labelmg. 4. If necessary, much lower concentrations of antibody can be used (diluted just before use), but the incubation times should be increased (e.g., 4 and 6 h for 4 and 1 pg/mL of antibody, respectively). 5. If the amount of bound radioactivity is low and does not exhibit a plateau, this may mean either that the amount of active antibody 1s low or that the specific radioactivity of the antigen is very low. The experiment must be repeated using 5000, 10,000, and 20,000 cpm of antigen without unlabeled antigen. If the plateau is very low, this may mean either that the affinity of the antibody-antigen interaction is very low or that the radioactive antigen has been damaged, possibly during labeling. In this case, if the background is much lower, the experiment may be repeated with the use of much more radioactivity. If the whole plot is a high plateau, the experiment must be repeated using antigen mixtures with higher amounts of unlabeled antigen. Alternatively, the concentration of the coating antibody may be decreased to 0.1-l pg/mL, so that lower amounts of labeled antigen can be used. 6. To ensure further that the first antibody gives sufficient protection even when its affinity for the antigen is much lower than that of the second antibody, it can be tested at varying degrees of excess (e.g., 5- to 50-fold excess); their effect should not be very different; otherwise, even higher excesses may be needed. 7. Second antibody immobilized on Sepharose beads: This method has the advantage that immobilized antibodies can be prepared in large quantities and remain active for more than a year, which obviously improves the reproducibility of results. In addition, there is not the strict necessity for the antibody preparation to be purified before immobilization, since CNBr-activated Sepharose beads have a high protein-binding capacity. Nevertheless, the method does have significant disadvantages, such as the need to use a large number of test tubes (and many tedious washes) instead of a smgle multiwell plate. The work can be reduced by the use of flexible polyvinyl chloride V-bottom multiwell plates (Dynatech Laboratories), which are washed by centrifugation in special rotors and the wells individually cut out to measure the bound radioactivity. If enzyme labeling is used instead of radiolabeled antigen, the colored supernatants must be transferred to ELISA plates for measurement. I would recommend this approach only in conjunction with the use of radioactive antigen.

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a. Antibody immobilization on CNBr-Sepharose beads: This is performed according to the manufacturer’s instructions. However, since the use of large Sepharose beads is inconvenient, in my laboratory we break them up into small pieces by sonication just before conjugation. b. Titration of immobilized antibody: The titers of the immobibzed antibodies are estimated m order to determine the amount of immobilized antibodies and of radioactive antigen to use. Make l/5, l/15, l/50, and l/l50 suspensions of the Sepharose-antibody beads in PBS-BSA, and incubate (with shaking), in Eppendorf tubes, 50 pL of suspension and 50 yL of labeled antigen (20,000 cpm 1251-Iabeled antigen plus increasmg concentrations of unlabeled antigen as above) for 2-4 h at room temperature. Wash two to three times (brief centrifugation) with 1 mL of washing buffer (we use PBS-0.5% Triton X-100, which is suitable for AChR solubilization), and count the remammg radioactivity Plot the data (preferably bound radioactivity vs antigen used), and select an appropriate pair of antibody-antigen concentrations: Choose the mmimum amount of Sepharossantibody that shows a plateau and a corresponding antigen concentration just after the plateau (i.e., eqmmolar concentration). c. Final steps: The methodology used to determine the required concentration of first antibody and for the final competition experiments between different antibodies will be obvious from the above information. 8. Similar differential precipitation of labeled second antibody may be applied to competition experiments between antibodies from single species when only one of the antibodies binds protein A (or protein G). Sepharose-immobilized protein A (or G) can replace the precipitating anti-Ig, whereas 1251-labeled or peroxidaseconjugated protein A (G) can replace the labeled anti-lg. In such cases, however, an antibody may be used only as second or first antibody depending on whether or not it binds protein A (or protein G). 9. Heterologous antibody competition in solid state (ELISA or RIA): Only the prmciple of the techmque will be described here, since the details will be evident from the previously described techniques. Any of the three reactants may be unmobilized m the wells. If the second antibody is to be immobilized, the procedure is the followmg: a. The second antibody is plated on the microwells. b. After washes, a preincubated mixture of predetermmed amounts of the antigen and an excess of first antibody are added and further incubated. c. After washes, peroxidase-conjugated anti-Ig specific for the species of the first antibody (preabsorbed with normal serum from the species of the second antibody) is added, and the mixture is incubated. This is followed by washes, addition of substrate for color development, and absorbance measurement.

Acknowledgments This work was supported by grants to S. J. T. from the Association Francalse contre les Myopathies, the BIOMED-I program of EC (BMHl-CT93-1 loo), and the Human Capital and Mobility program of EC (CHRXCT94-0547).

Epitope Mapping by Antibody Competition

65

References 1 Geysen, H. M., Meloen, H. R., and Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81,3998-4002. 2. Geysen, H. M., Tainer, J. A., Rodda, S. J., Mason, T. J., Alexander, H., Getzoff, E., and Lerner, R. A. (1987) Chemistry of antibody binding to a protein. Science 235,1184-l 190. 3. Tzartos, S. J., Rand, D. E., Einarson, B. E., and Lindstrom, J. M. (198 1) Mapping of surface structures of Electrophorus acetylcholine receptor using monoclonal antibodies. J. Biol. Chem. 256,8635-8645. 4. Tzartos, S. J. and Kordossi, A. (1986) Acetylcholine receptor conformation probed by subunit-specific monoclonal antibodies, in Nicotinic Acetykholine Receptor, NATO ASI series vol. H3 (Maelicke, A., ed.), Springer-Verlag, Heidelberg, pp. 3w7. 5. Kordossi, A. and Tzartos, S. J. (1987) Conformation of cytoplasmic segments of acetylcholine receptor a and b subunits probed by monoclonal antibodies. Sensitivity of the antibody competition approach. EMBO J. 6, 1605-1610. 6. Kordossi, A. A. and Tzartos, S. J. (1989) Monoclonal antibodies against the main immunogenic region of the acetylcholine receptor. Mapping on the intact molecule. J. Neuroimmunol. 23,35-40. 7. Tzartos, S. J., Seybold, M., and Lindstrom, J. (1982) Specificities of antibodies to acetylcholine receptors m sera from myasthenia gravis patients measured by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 79, 188-l 92, 8. Tzartos, S. J., Kokla, A, Walgrave, S., and Conti-Tronconi, B. (1988) Localization of the main mrmunogenic region of human muscle acetylcholine receptor to restdues 67-76 of the a-subunit. Proc. Natl. Acad. SIX USA 85,289~2903, 9. Tzartos, S. J. and Remoundos, M. S. (1992) Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor a-subunit. Dissecting a potentially myasthenogenic epitope. Eur. J Biochem. 207, 915-922. 10. Tzartos, S. J., Valcana, C., Kouvatsou, R., and Kokla, A. (1993) The tyrosine phosphorylation site of the acetylcholine receptor P-subunit is located in a highly immunogenic epitope implicated in channel function. Antibody-probes for /3 subunit phosphorylation and function. EMBOJ. 12,5141-5149. 11. Tzartos, S. J., Tzartos, E., and Tzartos, J. S. (1995) Monoclonal antibodies against the acetylcholine receptor y-subunit as site specific probes for receptor tyrosine phosphorylation. FEBSLett. 363,195-198. 12. Tzartos, S. J., Kouvatsou, R., and Tzartos, E. (1995) Monoclonal antibodies as site-specific probes for the acetylcholine receptor S-subunit tyrosine and serine phosphorylation sites. Eur. J. Biochem. 228,463-472. 13. Ratnam, M., Sargent, P., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., and Lindstrom, J. (1986) Location of antigenic determinants on primary sequences of the subunits of the nicotinic acetylcholine receptor by peptide mapping. Biochemistry 25,262 l-2632.

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14. Ratnam, M., Le Nguyen, D., Rivier, J., Sargent, P., and Lindstrom, J (1986) Transmembrane topography of the nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity protile. Blochemutry. 252633-2643. 15. Harlow, E. and Lane, D. (eds.) (1988) Antibodzes, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Epitope Mapping by Surface Plasmon Resonance in the BlAcore Bet-it Johne 1. Introduction 7.1. The BlAcore lnsfrument The BIAcore (biomolecular interaction analysis) analytical system consists of a detection unit, an autosampler, and a liquid delivery system, controlled by a computer. Karlsson et al. (1) have given a detailed description of the analytical system, the detection principle, and the theoretical background for binding measurements. The system combines a microfluidic unit in contact with a sensor for surface plasmon resonance (SPR) detection. Figure 1 shows the principle of SPR detection. The sensor chip consists of a glass slide mounted in a plastic frame. On one side of the glass, a thin film, approx 50 run, of gold is deposited, and the dextran matrix is attached on top of this film. The sensor chip is inserted into the instrument with the dextran/gold side in contact with the flow cells. When the injected sample is passing through the flow cell, antigen binds to immobilized antibody in the dextran matrix. Light covering a span of angles of incidence falls on the glass side and is reflected mto a 2D array detector where the intensity of the reflected light is measured. SPR occurs at a certain angle and is seen as a minimum in reflected light intensity. When the refractive index close to the gold film is changed, for example, when immobilized antibody binds antigen, the angle at which SPR occurs is changed. This change is proportional to the amount of bound protein, and is expressed as refractive units (RU) on the Y-axis of a sensorgram; 1000 RU corresponds to 1 ng protein/mm2 of the 100-nm thick dextran layer. Reproducibility of the system has been validated by Fagerstam et al. (2).

From: Methods m Molecular Biology, vol. 66: Eprtope Mapping Protocols Edited by* 0. E Morns Humana Press Inc , Totowa, NJ

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68 Reflected intensity Light source

Detection unit

.

Reflected intensity

C Resonance signal [RU]

Time [s]

Fig. 1. The principle of SPR signal detection. Surfaceplasmon resonancedetects changesin refractive index of the surfacelayer of a solution in contactwith the sensor chip. The changein refractiveindex is causedby variation of the masson the sensorchip surfaceowing to interactionsof the biomolecules(A,B). A dip in the intensity of the reflected light occurs at a certain angle, which is referred to as the resonancesignal (RU) (C). The shift in resonancesignal is plotted against time and displayed in a sensor-gram(from ref. 1).

1.2. Antigehhtibody

Interaction

An epitope is a specific site on an antigen molecule defined by the binding characteristics of one monoclonal antibody (MAb) (3,4). Structural and fimc-

tional approachesto the study of protein antigenicity have led to two different

Surface Plasmon Resonance

69

perceptions of the nature of protein epitopes (4). The structural approach concentrates on the spattal arrangement of atoms found in the antigerrantibody complex and shows that at least 15 amino acid residues may be implicated in each epitope. The functional approach, which introduces the additional dimension of time, takes the form of crossreactive binding measurements and leads to the view that a smaller number of residues are implicated in each epitope. Functional binding assaysare operational in character, and it must be considered that different types of epitopes are identified by the use of different probes (4). With the BIAcore, we are taking the functional approach. 1.3. Epitope Mapping Epitope mapping in the BIAcore may be performed to characterize an antigen or a group of specific monoclonal antibodies, or both (5). High affinity antibodies are important in most immunochemical techniques and essential to immunoassay sensitivity (6). However, binding kinetics are affected by binding conditions, immobilization, labeling, or conjugation of the reactants as well as the flexible nature of proteins (6-8), as we have shown in a recent BIAcore study (7) by comparing native and colloidal gold-labeled MAbs. Epitope mapping with conventional EIA or RIA is time-consuming, includmg elaborate labeling methods, and considerable amounts of purified reactants are needed. With the BIAcore, there is no labeling of the reactants, purification is not necessary (cell supernatants or ascites can be used), and small amounts of the reactants are sufficient for epitope mapping. The fully automated system can handle large mapping matrices overnight. The association and dissociation rate of the molecular binding is monitored in real time, and can be analyzed separately. The BIAcore is thus a unique tool to analyze functional aspects of molecular interaction. 1.4. Analysis of Molecular Binding Patterns Characterization of epitope specificity patterns with a panel of MAb gives valuable information for utilizing MAb in clinical, diagnostic, and technical contexts. A functional epitope map is created. In order to probe the surface topology of the antigen down to the level of smaller structural elements, one should perform inhibition studies with peptides or fragments of the antigen. In the present chapter, we focus on epitope mapping for the purpose of characterizing antibodies for use in immunoassays, thus illustrating the BIAcore and its potential in any molecular interaction study. A method for epitope mapping by pairwise binding is presented(Fig. 2), but the BIA core can also be used for multisite binding analysis and for affinity measurements (see Notes 1 and 2). As an example of epitope mapping by multisite binding, we have recently worked with a complex antigen, calprotectin (9), a 34.5-kDa protein consisting

70 A Resonance

Signal [RU]

24000 23000 22000 21

000

20000 120

240

360

460

600

720

840 Time [s]

C

0 M4 MS

Fig. 2. Epitope mapping of MAbs against myoglobin. (A) Sensorgramof a mapping sandwich.The resonancesignal from the secondMAb is indicatedby the vertical arrow. This value is enteredin the matrix for each sandwich.(B) Matrix for pairwise epitope mapping. Circles denotepair function. (C) Epitope map of myoglobin. Heavy circles: full mapping of six MAbs in the matrix above. Light circles: data from an extended mapping matrix shown in the samepaper (6). Overlapping circles denote MAb that cannot bind concurrently (from ref. 6). of three protein chains (one 8- and two 14-kDa chains) with repeated epitopes. In this case, multisite binding is the preferred mapping method. Table 1 shows a multisite binding matrix with one low-affinity and two high-affinity MAbs.

Surface Plasmon Resonance Table 1 Multisite Binding

71

of MAbs to Covalently

Bound

CalprotectirP

Second MAb MAb CPl First First MAb MAb CPl MAb CP2 MAb CP5

1116b 0 1068

MAb CP2

MAb CP5

Second

First

Second

First

Second

249 1314 752

1114 0 1055

-554 0 -29

1101 0 1018

874 1425 464

“MAbs (100 pg/mL) were injected sequentially on covalently bound calprotectm, with a buffer wash between each sample See Fig. 3 for selected sensorgrams. Mab CPl and CP5 are hrghaffinity MAb against two different epitopes. CP2 is a low-affinity MAb with no apparent binding to covalently bound calprotectm. However, competition with CPl epitope binding was demonstrated. All three MAb bind to soluble calprotectin unmobilized by binding to another MAb m a BIAcore two-site binding experiment (data not shown). bThe table shows RU values for each step m each cycle of two sequentially injected MAb.

Calprotectin was covalently bound to the BIAcore chip, and two MAbs were sequentially injected in the pattern shown in the table. We found that CPl and CP5 have separate epitopes, since they bind sequentially in any order, and the total signal is stronger than CPl + CPl or CP5 + CP5 (Fig. 3A, Table 1). Furthermore, the low-affinity MAb CP2 competes with MAb CP 1binding leading to a notable dissociation of CPl when CP2 is injected as the second MAb (Fig. 3B and Table 1). This suggests common or overlapping epitopes. CP5 binding is not affected by a subsequent injection of CP2 (Table 1). Calprotectin is a calcium-binding protein, and we found that EDTA in the BIAcore running buffer destroys the binding of certain MAb to their epitopes possibly owing to changed epitope conformation by removal of Ca*+ (data not shown). This is an example of binding conditions that can completely alter an epitope’s binding properties. The above experiments were run without EDTA. With pure myoglobin (a single-chain protein without repeating epitopes) as antigen (6) and high-affimty antibodies, the stoichiometry of multisite binding is easy to calculate (Table 2). Our results (6) show that myoglobin, a small 17-kDa globular protein, can simultaneously bind four MAbs of approx 150 kDa each. Myoglobin was bound by the first MAb and a mix of three MAbs were injected (Table 2). Since they had four separate epitopes, the RU signal created was three times as strong as when one MAb was injected as a second MAb. Methods for measuring affinity and rate constants in the BIAcore are very well described in the BIAcore manual and in several publications. Reproducible calculations were easily obtained with purified human transferrin and a

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R

15300

U 14800

-4

FIW Second

mAbCP1 mAbCP5

1101 RU 874RU

13800

0

200

400

600

800 Time

1000

1200

1400

1600

1800

1600

1800

181

15300.

I

14800.

9

R u

14300 Second

1114RU -554RU

mAbCP2

1

0

Iv----200

400

600

800

Time

1000 [sl

1200

1400

'

t..

Fig. 3. Multisite binding to immobilized calprotectin (see Table 1). (A) Multisite binding of two MAbs (CPl and CP5) to separate epitopes. The sensorgram shows that CP 1 binds well as first MAb, and that almost the same amount of CP5 is bound simultaneously. Table 1 shows the similar results for CP5 as first and CPl as second. The two different MAb give a larger RU signal than only one of them injected in the same sequential way, thus indicating separate epitopes. (B) Competition

ity MA\, CP2 and high-affinity

between low-affin-

MAb CP 1. The low-affinity MAb CP2 shows no bind-

ing when injected alone (Table 1). However,

when mjected as a second MAb after

CPl binding competrtlon IS seen, it results in the loss of CPl from the chip surface (-554 RU). This is not seen with CP2 after CP5 (Table l), thus indicating interference with the CPl epitope, but not with the CP5 epitope.

73

Surface Plasmon Resonance Table 2 Stoichlometry of Multisite Binding to Myoglobina Sample

First MAb Antigen Second MAb

SecondMAb Responseper myoglobin

Ml Myoglobin M3+M5+M6 M6

Run 1

Run2

745 30 971

742 30

30.0

289 9.6

*The experiment was performed on a chtp with covalently bound RAM in a two-site binding as described in Fig 2A (data from ref 6)

group of high- and medium-affinity mouse MAbs (B. Johne, unpublished results), whereas reliable results were more difficult to obtain with complex antigen preparations. Thus, it is strongly recommended that the antigen is pure and well defined, before affinity measurements are initiated. Affinity ranking of different MAbs is, however, easily performed with most antigens. We have observed large variations in on and off rates (6,7), particularly with MAb 2D2 against human albumin. This MAb showed an extremely rapid dissociation of antigen when immobilized on RAMFc, but no dissociation when binding as an unlabeled second MAb to immobilized albumin (7). In order to dissect the structural elements involved in a functional epitope, inhibition studies can be performed with overlapping peptide sequences along the protein sequence of the whole antigen (see Note 3). Keep in mind that the probes (antibody or other ligand) are different for each epitope. Furthermore, steric inhibition may occur. To obtain a complete understanding of the relations between structure and function, mapping of the same epitopes must be performed with different methods taking structural as well as functional approaches. 2. Materials 1. BIAcore instrument with PC, including software to run the instrument and to evaluate data (Pharmacia Biosensor AB, Uppsala, Sweden). 2. Sensor Chip CM5, with a carboxylated dextran layer suited for covalent binding of proteins: For unmobllization of biotinylated molecules, Sensor Chip SA, coated with streptavidin, can be recommended (see Note 3). 3. Rabbit-antimouse-Fc-y (IWMFc) (Pharmacia Biosensor AB, Uppsala, Sweden): Polyclonal antibody for immobilization of murine MAbs. 4. Immobilization reagents: (EDC) (N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride), N-hydrosuccinimide (NHS), ethanolamine, acetate buffer, pH 5.0 (Pharmacia Biosensor AB).

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5. Running buffer and sample dilution buffer: HEPES-buffered saline, pH 7.3, with 3.4 mA4 EDTA and 0.005% BIAcore surfactant (HBS) (Pharmacia Biosensor AB). 6. Protein antigens: Examples include myoglobin (Scripps Laboratories, San Diego, CA), human serum albumin (Novo Nordisk, Glostrup, Denmark), transferrm (Scripps Laboratories) and calprotectin (Nycomed Pharma AS, Oslo, Norway). 7. A panel of murine MAbs resulting from fusions of spleen cells from mice immunized with the antigen. Negative control antibodies and blocking antibodies in sandwich methods. 8. 100 mM HCl for regeneration of the covalently bound protein surface. 9. Automatic pipets and tips. 10. Plastic and glass tubes for samples and reagents (fitting the BIAcore racks). 3. Method When you purchase the BIAcore, a short training course 1s included. The instrument is well equipped with extensive manuals and guides to methods and applications (see Note 4).

3.1. Preparation

of the Instrument

1. 2. 3. 4. 5. 6. 7.

Switch on the processing unit and the computer. Take out the sensor chip from the refrigerator. Warm the running buffer, and filter and degas it (see Note 5). Create a directory to store your data in. Load the BIAlogue program under Windows. Undock the optical unit, remove the old sensor chip, insert a new chip, and dock it Initiate the system with buffer. Normalize the signal response by injecting 40% glycerol in water. 8. Perform a dipcheck to control the four flow channels available.

3.2. Immobilization Covalent binding of RAMFc to the carboxylated dextran layer may be performed automatically by a prewritten program. The procedure takes approx 1O-20 min. Alternative binding chemistries are described in the manual (3). 1 Activate the carboxyl groups in the hydrogel by injection of NHS/EDC. 2. Inject ligand diluted in acetate buffer (30 pg/mL are recommended for the RAMFc) (see Note 6). 3. Deactivate unreacted NHS-esters on the sensor surface by treatment with 1M ethanolamine.

3.3. Epitope Mapping by Pairwise Binding A typical sandwich in a pairwise mapping Notes 7 and 8).

matrix is shown in Fig. 2A (see

Surface Plasmon Resonance 1. Inject the first MAb over the covalently bound RAMFc, followed by buffer rinse. 2. Inject a blocking MAb with high affinity for the RAMFc and no crossreactivity against the antigen is injected, followed by buffer rinse (see Note 8). 3. Inject antigen and then the second MAb, followed in each case by buffer rinse (see Note 9). 4. Regenerate the RAMFc surface with 100 m&f HCl. 5. Set up the results from the individual sensorgrams in a matrix (Fig. 2B), and translate the data into a functional epitope map (Fig. 2C) (see Notes $7, and 8).

4. Notes 1. Multisite binding may be compared to the classical competition assays, since the epitope information is obtained from competition between different antibodies. An important difference in the BIAcore is that the MAb are unlabeled, and that association and dissociation can be followed in real time. Competition or inhibition with peptide sequences may give further information about the epitope structure. 2. When the antigen is a complex of two or more protein chains, possibly with repeating epitopes, mapping by multisite binding is preferred rather than pairwise mapping. With this method, sequential binding of different MAbs to an antigen is analyzed as described (3). 3. Biotmylated peptides may be immobilized on sensor chip SA with a Streptavidin surface. On this surface, biotinylated peptides or other biotinylated molecules are readily bound, and subsequent binding of specific antibodies or other ligands can be studied. The risk of conformational changes in a small peptide immobilized in this manner is considerable compared to the corresponding epitope on the larger molecule. 4. Use the programming examples included in the BIAlogue guide, and edit and modify from them. The windows-based BIAlogue software is user-friendly, and contains a guide section and several program examples. 5. Degassed buffers and inspection of samples for gas bubbles are important in order to avoid ruined sensorgrams. 6. Immobilized amount is regulated by mjection time and ligand concentration. A suitable amount of RAMFc for epitope mapping lies between 8000 and 15,000 RU (6). 7 Negative sandwich results may be owing to rapid dissociation of a low-affinity ligand, conformational changes in the reacting molecules, or steric inhibition. Inspect the whole sensorgram. 8. Stoichiometry calculation should be used as a control of binding efficiency. For most biomolecules, 1000 RU corresponds to 1 ng/mm2 of the IOO-nm thick dextran hydrogel (3). 9. Optimize your binding conditions with alternative running buffers. 10. Competition studies give added information to a two-site binding study. The binding sequence IS not always irrelevant in multisite binding.

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References 1. Karlsson, R., Michaelsson, A., and Mat&son, L. (1991) Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor-based analytical system. J. Immunol. Methods 145,229-240. 2. Fagerstam, L. G , Frostell-Karlsson, A., Karlsson, R., Persson, B., and Ronnberg, I. (1992) Biospeciflc interaction analysis using surface plasmon resonance detection applied to kinetic, bindingsite and concentration analysis J Chromatogr 597,397-410. 3. Anon, (1991) BIAcoreTM System Manual, Pharmacia Biosensor AB, Stockholm. 4. van Regenmortel, M. H. V. (1989) Structural and functional approaches to the study of protein antigen&y. Immunol. Today 10,266-272. 5. Fagerstam, L. G., Frostell, A., Karlsson, R., Kullman, M., Larson, A., Malmquist, M., and Butt, H. (1990) Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping. J. Mol. Recogn. 3,208-214. 6. Johne, B., Gadnell, M., and Hansen, K. (1993) Epitope mapping and binding kinetics of monoclonal antibodies studied by real time biospecific interaction analysis using surface plasmon resonance. J. Zmmunol. Methods 160, 191-198. 7. Johne, B., Hansen, K., Mark, E., and Holtlund, J. (1995) Colloid gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard nnmunoassay format. J. Immunol. Methods 183, 167-174. 8. Campbell, A. M. (1991) Monoclonal Antibody and Immunosensor Technology, Elsevier, Amsterdam, p. 427. 9. Fagerhol, M. K., Andersson, K. B., Naess-Andresen, C. F., Brandtzaeg, P., and Dale, I. (1990) Calprotectin (the LI leukocyte protem), in Stimulus Response Coupling: The Role of Intercellular Calcium Binding Proteins. (Smith, V. L. and Dedman, J. R., eds.), CRC, Boca Raton, FL, pp. 187-210.

Identifying Residues in Antigenic Determinants by Chemical Modification N. Martin Young and Raymond

P. Oomen

1. Introduction Chemical modification of the side-chains of residues in protein antigens was one of the first methods developed to investigate epitopes. Together with proteolytic fragmentation, it played a major role in the pioneering efforts of Atassi and others to assign antigenic determinants on the surfaces of lysozyme and myoglobin (1,2). The principle of the method is that alteration of the structure of a key residue in an epitope by a chemical modification reagent will greatly change its reactivity with an antibody to that epitope. The steps m the procedure are modification of the protein antigen with the chosen reagent, removal of byproducts after the reaction, and immunoassay of the modified protein for the expression of the epitope. The chemical modification is usually performed in solution, but in a convenient variation described here, the antigen is modified when it is already adsorbed to an ELISA plate (3). Although epitope mapping via the synthesis of overlapping peptides offers greater precision, chemical modification has the advantage that it can be applied to discontinuous as well as continuous epitopes. Moreover, continuous epitopes can be conformationally constrained in the context of the folded protein and may not always adopt a recognizable conformation when removed from that context. Since chemical modification can only rarely identify a particular residue in an antigen, rather than a residue type, as being a key epitope constituent, it is necessarily an adjunct to other tools that can locate the individual residue of the type being modified. The exception is when a particular type of residue occurs infrequently, i.e., only once or perhaps twice in the protein. This is either more common in smaller proteins or involves rare residues, such as cysteine. Examples include an analysis of the roles of histidines in the From: Methods m Molecular Biology, vol. 66: Epltope Mapping Protocols Edited by G E Morns Humana Press Inc , Totowa, NJ

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expression of Gm allotypes on IgG (4) and investigation of a cysteme residue in HLA-B27 (5). Generally, identification of the exact residue will not be achieved. However, the range of chemical reactivities of residues has made it possible to prepare partially or even singly modified derivatives of some antigens (6,7), which thus permits direct identification of key residues. In the ideal case, chemical modification would lead to abrogation of antigen recognition solely by modification of a key residue. However, chemical modification can have indirect effects on the display of an epitope, notably by disrupting the protein’s structure. Some reagents modify hydrophilic sidechains by substituting them with hydrophobic groups, which may affect the protein’s solubility or cause aggregation. Other reagents may cause oligomeric proteins to disaggregate, with loss of epitopes at subunit interfaces or reduction of effective valence for binding. With chemical modification studies in solution, it is possible to make some independent assessmentof the structural integrity of the protein after modification, either by physical methods, such as circular dichroism, or by checking retention of biological properties, such as enzyme activity. Since chemical modification is often used to investigate these properties, functional and antigenic investigations can easily be combined. The above factors are also important to consider when choosing an immunoassay system with which to measure changes in a protein’s antigenic properties. In enzyme-linked or radioimmunoassays, the modification may affect the adsorption of antigen to the plate or tube for direct assays.Sandwich assaysof monomeric proteins require two different determinants, one for capture and one for detection, and additional tests will be necessary to distinguish which epitope has been modified. In addition, the experiments may involve dialysis of many small samples of the protein after modifications with a set of reagents under different conditions, with concomitant recovery problems. These considerations led us to develop a combined EIA and modification approach (3), in which the antigen is first adsorbed to the EIA plate, and then chemically modified in sm. The reagents and byproducts are removed by plate washing, and the appropriate antibodies are added for the assay. Detection is further simplified by using any of a number of commercial secondary antibody conjugates that catalyze measurable color reactions. This approach permits easier analysis of a range of reagents and various condittons, yet it is highly economical on antigen, Reagents for the modification of proteins in solution are well described in general reviews of the field (8-11), as well as parts of several volumes in the Methods in EnzynoZogv series. Although many reagents have been described, certain ones have proven most useful, which are summarized in Table 1, The reagents vary in their specificity for a particular side-chain, and virtually all can give some side products. These are generally rare and can be minimized by

79

Chemical Modification of Epitopes Table 1 Specific Chemical Modification Reagents ReagentResiduetype Aspartate, Carbodiimide glutamate + nucleopbile Lysine, -NH2 Acetic anhydride Succtnicanhydride Tyrosine Iodination, nitration Histidine Diethylpyrocarbonate Arginine p-Hydroxyphenylglyoxal 2,3-Butanedione Cysteine Iodoacetamtde Methionine Methyl iodide Tryptophan 2-Hydroxy-5-nitrobenzylbromide

Stdereactions Tyrosine Serine,threonine

Lysine Histidine

choice of reaction conditions. The side products in some casesare more labile than the desired product, and treatment with hydroxylamine can remove the unwanted modilications. In addition, there is a useful more general reagent, 2,4-dinitro-fluorobenzene, which modifies Lys, His, Tyr, and Cys residues. As Table 1 indicates, specific reagents are available for almost half of the 20 amino acids. Of the remaining resrdues, most are hydrophobic and are less common on protein surfaces. The lack of reagents for the amide and hydroxylic residues is more serious, particularly since glutamine is a key residue in an epitope of lysozyme (12). Possibly the enzyme transglutaminase could be applied here, since it has been used to modify glutamine residues in proteins (13). Chemical modification is also used to obtain protein antigen derivatives for immunoassay purposes, notably by radioiodination of tyrosines or biotinylation of lysines. The products are usually checked to confirm retention of antigenicity, and hence, these experiments are probably the most common, if unintended, examples of residue mapping in epitopes. 2. Materials 1. Reagentsfor the ELISA assaychosenby the investigator (protein antigen, antibody, secondantibody conjugate,and calorimetric substrate). 2. ELISA equipment (multichannel pipeter, microtiter plate shaker,plate washer, andplate reader). 3. Phosphate-bufferedsaline:O.OlMsodiumphosphatebuffer, pH 7.2,O.15MNaCl. 4. T&buffered saline: 0.IM Tris-HCl, pH 8.0 or 8.5,O.15MNaCI. 5. Modification reagents(seeNote 1). a. l-Fluoro-2,4-dinitrobenzene: 1M in dry acetonitrile. b. p-Hydroxyphenylglyoxal: 0.1M in PBS.

80

Young and Oomen c. Diethylpyrocarbonate: 0.4M in anhydrous ethanol. d. Acetic anhydride: 2M in dry acetonitrile. e. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide: 1M in water, adjusted to pH 4.7. f Propylamine: 1Min water, adjusted to pH 4.7 with HCl. g. Hydroxylamine: 0.5M in water, adjusted to pH 7.5 with HCI.

3. Method The precise details of the assay system will be determined by the characteristics of the antigen-antibody

system to be studied, but as indicated

above, for

the approach we describe, the antigen is directly adsorbed to an ELISA plate. Buffers incorporating amines generally cannot be used, and PBS is preferred. The investigator must determine in preliminary experiments the amount of antigen and antibody needed, as well as incubation and wash conditions, and

conditions for color development with the second antibody enzyme conjugate, to yield approx 1 absorbance unit of chromophore. The method was developed with an idiotypic antibody system (3), and utilized an anti-antibody alkaline phosphatase

conjugate

as the disclosure reagent.

1. Antigen coating: Coat the wells of the ELISA plate with the predetermined amount of antigen, and wash three times with PBS (350 pL/wash) No blocking protein can be added at this point, since this would also react with the chemical modifying reagents. (See Note 2). 2. Chemical reaction: Add reagents chosen from the list below to give dilution series m 100 pL aliquots of the appropriate buffer in the wells. Care should be taken to ensure that all reagents are added into the solution in the microtiter plate wells, away from the well walls. The modification reactions should all be run in duphcate or triplicate, with control wells of antigen that receive no chemical treatment. With reagents that are added in orgamc solvent, controls with solvent alone should be included. The reactions are run at room temperature with gentle shaking on a plate mixer. The cited reviews (34) should be consulted for details of the following reactions, including mechanism and side-reactions. a. Fluorodinitrobenzene modification of tyrosine, lysine, histidine, and cysteme residues: Prepare dilutions m anhydrous acetonitrile (5 pL; 2-600 @4) and add to the buffer in the well (95 pL of PBS or Tris-NaCl buffer, pH 8.5) Leave the reaction overnight in the dark. b. p-Hydroxyphenylglyoxal modification of arginine residues: This reagent can be used in buffers around pH 8.0 with 2-h reaction times or PBS overnight. Add aliquots (100 l.tL) of dilutions in the concentration range of 1 p.M to 100 mM in the buffer. c. Diethylpyrocarbonate modification of histidine residues: Caution: this reagent is particularly hazardous. Add dilutions in anhydrous ethanol (5 pL; 10 @QAOO mM) to 10 mMphosphate buffer in the wells (95 pL; pH 6.0), and leave the reaction for 1 h.

Chemical Modification of Epitopes

3. 4.

5. 6.

81

d. Acetic anhydride modification of lysine and N-terminal amino groups: Add dilutions in anhydrous acetonitrile (5 pL; 0.1 mM-2M) to Tris-saline, pH 8.0 (95 uL) in the wells, and leave the reaction for 2 h. e. Carbodiimide-nucleophile modification of carboxylate groups. The basis of this modification is that the carbodiimide activates the carboxyl group for reaction with a nucleophile, preferably an amine. The water-soluble reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide is used, with and without an amine nucleophile and also at pHs where carboxylates preferentially react, e.g., pH 4.7, or at higher pHs where tyrosine can react. For reactions with amines, add freshly-prepared carbodiimide (5 uL; 1M in water, adjusted to pH 4.7) to the various concentrations of nucleophiles in the wells, such as propylamine or ammonium chloride adjusted to pH 4.7 (95 l.tL; 3 u.bGlM) and leave the reactions for 2 h. The reactions without amines are carried out similarly (100 uL; 1.5 pi+1 00 mM carbodiimide) at pH 4.7 or 7.0 in water. f. Additional reagents: As Table 1 indicates, reagents are available for several other types of residue, and reagents such as iodoacetamide for modification of cysteines should be readily adapted to this type of protocol. Rinsing: Following the chemical modification reactions, remove reagents and byproducts by washmg with PBS three times (350 pL/wash). Hydroxylamine treatment: Several side reactions of the carbodiimide, acetic anhydride, and diethylpyrocarbonate reagents involve formation of esters or other labile groups, and these can be reversed by hydroxylamine treatment. Following the above step, treat the protein-coated wells with 100 pL of OSMhydroxylamine (adjusted to pH 7.5) for 1 h, and rewash with PBS as in step 3. Antibody reaction: Add the predetermined amount of antibody in 0.1% BSA in PBS. If required, a separate blocking step can precede the antibody reaction. Color development. Complete the assay by reaction with antibody-enzyme conjugate followed by the chromophoric substrate, under the predetermined conditions (see Note 3).

4. Notes 1. Most of the reagents are reactive with water, and must therefore be prepared in anhydrous organic solvent, such as acetonitrile, and used immediately. Several of them are hazardous, particularly diethylpyrocarbonate, and appropriate precautions should be taken to avoid contacting them or inhaling them. 2. If the antibody-antigen reaction is readily dissociated, it is possible to carry out a confirmatory experiment by using a protective blocking step. Aliquots of the antigen are treated with an excess of antibody prior to the chemical modification, to conceal the residues in the epitope from the reagents. After the chemical reaction, the blocking antibody is removed by exposure to low pH or other means, and the remainder of the ELISA procedure is carried out as above. Comparison of the antibody reactivity of the protected and unprotected antigen samples can then confirm the importance of the residue in the epitope.

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3. Virtually all the reagents when used at high enough concentration will abolish antigenic reactivity for the reasons described above, The sensitivity of the antigen to the reagent, i.e., at what concentration it is effective, therefore has to be the criterion of involvement of the particular residue in the determmant. Additional confirmation can be obtained m two ways. First, a protection experiment as above can be used to see if the epitope is concealed from reaction by antibody. Second, the set of reagents is partly redundant in that some of the residues can be modified by more than one reagent. Thus, if acetic anhydride treatment points to involvement of a lysine residue, fluorodinitrobenzene should also abolish antigenie reactivity. Once a particular type of residue has been established as an epitope constituent, synthetic peptide or mutagenesis approaches can be planned to identify the exact residue involved, unless, of course, the residue is a unique one. In the case of the idiotope investigation m which the above techmque was developed (3), the assignments have been confirmed by X-ray crystallography of the Id-anti-Id Fab-Fab complex (14).

References 1. Atassi, M. Z. (1975) Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigemc structures of proteins. Immunochemistry 12,423-438. 2. Atassi, M. Z. (1978) Precise determination of the entire antigenic structure of lysozyme. Immunochemistry 15,909-936. 3. Gudmundsson, B.-M. E., Young, N. M., and Oomen, R. P. (1993) Characterisation of residues in antibody binding-sites by chemical modificatton of surfaceadsorbed protein combined with enzyme immunoassay. J. Immunol. Methods 158, 215-227. 4. Williams, R. C., Malone, C. C., and Solomon, A. (1993) Conformational dependency of human IgG heavy chain-associated Gm allotypes. Mol. Immunol. 30, 341-351. 5. MacLean, L., Macey, M., Lowdell, M., Badakere, S., Whelan, M., Perrett, D., and Archer, J. (1992) Sulphydryl reactivity of the HLA-B27 epitope: accessibility of the free cysteine studied by flow cytometry. Ann. Rheum. Dis 51,456-460. 6. Yang, C. C., Chang, L. S., Ong, P. L., and Tung, T. H. (1992) Immunochemical properties of Nuju nuju utru (Taiwan cobra) phospholipase A2 using polyclonal and monoclonal antibodies. Toxzcon 30, 15 l-l 59. 7. Lm, S. R., Chang, IS. L., and Chang, C. C. (1993) Chemical modification of amino groups in cardiotoxin III from Taiwan cobra (Nuju nuju utru) venom. Bzochem Mol Biol. Znt. 31, 175-184 8. Means, G. E. and Feaney, R. E. (1971) Chemlcul modification ofproteins. HoldenDay, San Francisco, CA. 9. Glazer, A. N., DeLange, R. J., and Sigman, D. S. (1976) Chemtcal modification of proteins, in Laboratory Techniques in Biochemistry and Molecular Biology, vol. 4 (Work, T. S., and Work, E., eds.), North-Holland Publishing, Amsterdam, pp. 3-205.

Chemical Modification of Epitopes

83

10. Lundblad, R. L. and Noyes, C. M. (1984) Chemical Reagents for Protein Modificatzon, ~01s. 1 and 2. CRC, Boca Raton, FL. 11. Imoto, T. and Yamada, H. (1989) Chemical modification, in: Protein Functzon: A Practical Approach (Creighton, T. E., ed.), IRL, Oxford, pp. 247-277. 12. Amit, A. G., Mariuzza, R. A., Phillips, S. E. V., and Poljak, R. J. (1986) Threedimensional structure of an antibody-antigen complex at 2.8 A. Science 233, 747-753. 13. Yan, S. C. B. and Weld, F. (1984) Neoglycoproteins: in vitro introduction of glycosyl units at glutamines in @casein using transglutaminase. Biochemistry 23, 3759-3765. 14. Evans, S. V., Rose, D. R., To, R., Young, N. M., and Bundle, D. R. (1994) Exploring the mimicry of polysaccharide antigens by anti-idiotypic antibodies: the crystallisation, molecular replacement, and refinement to 2.8 A resolution of an idiotopwurtt-tdiotope Fab complex and of the unliganded anti-idrotope antibody. J. Mol. Biol. 241,691-705.

9 Epitope Mapping by Differential Chemical Modification of Antigens Hans Rudolf Bosshard 1. Introduction Antibodies are directed against three-dimensional features of proteins, and the recognition of an epitope by an antibody is always a fit of structures in three-dimensional space. In the case of antibodies to native proteins, most or perhaps all epitopes are discontinuous (I). Because of the large size of a typical contact epitope m an antigell-antibody crystal, it is unlikely for an antibody to bind exclusively to a contiguous stretch of the polypeptide chain and not also to contact residues apart in sequence, but close in space. Space-filling models of proteins show few linear stretches longer than four to five residues in direct peptide linkage accessible on the molecule’s surface. This complicates epitope mapping. Apart from X-ray diffraction analysis, there is no other single method to map an antigenic determinant on a folded, native protein at atomic resolution. However, the approximate location and outline of a protein epitope may be established by a combination of methods that can be applied to the antigen-antibody complex in solution. One such method is the differential chemical modification of the free and antibody-bound protein. Other methods complementing differential chemical modification are described elsewhere in this volume. This method reveals steric protection of amino acid residues at the interface of a proteitqrotein complex; it was developed originally to map electrontransfer interaction domains (2,3) and was later adapted to the mapping of B-cell epitopes (4). The rationale is to compare the degree of chemical modification of appropriate residues of a protein in the presence and absence of a monoclonal antibody (MAb) and to deduce the location of the epitope from the differential degree of modification of amino acid side-chains. The rate at which From

Methods m Moleculer Biology, vol. 66 Epftope Mapping Protocols Edited by G E Morns Humana Press Inc , Totowa, NJ

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a side-chain functional group reacts with a modifying reagent is a quantitative measure of the degree of protection of that residue. Absolute rates are difftcult to measure, and it is easier to compare the relative rates of chemical modification in the free and antibody-bound antigen. The ratio of the relative rates is called the protection factor R, which is defined such that R > 1 indicates protection of a residue in the antigen-antibody complex, R = 1 means no effect of the antibody on the chemical reactivity, and R < 1 increased chemical reactivity in the presence of the antibody. R can be determined from the degree of modification of a residue if the modification reaction 1s performed under pseudo-first-order reaction conditions (3,5). The differential chemical moditication experiment has three principal steps (Fig. 1). Step 1. Antibody-bound antigen (experiment B) and free antrgen (experiment F) are reacted with a trace amount of a suitable radioactive reagent to obtain tracelabeled derivatives of the antigen. Step 2: The trace-labeled derivatives from step 1 are completely labeled with nonradioactive reagent to obtain derivatives that are heterogeneous with respect to the radioactive label and homogenous with respect to chemical modification. Step 3: The degree of radioactive labeling of individual residues is determined. To this end, the protein is cut into peptrdes, and the pepttdes are chromatographltally separated, purified, and sequencedby stepwiseEdman degradation. The protection factor R is calculated from the amount of radioacttvtty released at each Edman degradation step.

In principle, amino (Lys), thiol (Cys), carboxyl (Asp, Glu), imidazole (His), guanidino (Arg), and hydroxyl (Ser, Thr, Tyr) groups can be modified (6) However, only the specific chemical modification of a unique class of functional groups is suitable for the procedure. Thus, the method is essentially limited to the modificatton of the side-chams of Lys, Cys, and His, of which Lys is the most frequent on a protein surface. So far only differential chemical modification of lysine s-amino groups has been applied to the mapping of epitopes. The reagent of choice is acetic anhydride, which produces Wacetyl-lysine and an acetylated amino terminus. O-ace@ derivatives of Ser, Thr, and Tyr are formed to only a small extent. The following discussion is restricted to the differential chemical modification of Lys.

R is a valid measure of the chemical reactivity of an amino group if R ts proportional to the ratto of the rates of acetylation in the free and the antibodybound antigen (see Note 1 for theory):

[d(AR)ldtlfreelCd(AR)ldtl~und =R

87

Protection from Chemical Modification EXPERIMENT

w

EXPERIMENT

F

STEP 1 React with trace of [3H]acetlc anhydnde Add pm-made “C-acetylated antigen Separate antigen from antibody

STEP 2 Modify extensively with excess radioactIve acetic anhydnde -t

Digest, Determine

B

-t

non-

STEP3 separate and purify peptides Sequence peptldes 3H/“C-ratio of labelled residues

w

Fig. 1. Flowchart of the differential chemical modification experiment described in Section 3.

AR is the Wacetyl group of a modified Lys or the P-acetyl group of the modified N-terminal residue. An easy way to measure R is to use radioactively labeled acetic anhydride and to equate d[AR]&dt and d[AR]bound/dt wrth the amount of radioactivity incorporated into the free antigen and the antibody bound antigen, respectively. However, this is only a valid approach if the overall degree of acetylation is low and the reaction proceeds under pseudo-first order conditions with the concentration of acetic anhydride much higher than

Bosshard

88

that of the reactive amino groups. Using 3H- and *4C-labeled acetic anhydride, the experiment can be conducted in such a way as to make R proportional to the 3H/14C ratio of an NE-acetyl group. In two parallel experiments, free antigen (experiment F) and antibody-bound antigen (experiment B) are reacted with a small amount of [3H]acetic anhydride (step 1). In this step, only a few amino groups are acetylated, preferably
(2)

where RA is the thiohydantoin derivative of NE-acetyllysine. An example of the procedure is described m detail in Section 3. (see Note 2). Care must be taken to adjust the reaction conditions in the two parallel experiments of step 1in order that any altered chemical reactivities truly reflect the side-chain protection in the antigen-antibody complex. One precaution is to use a small amount of reagent in step 1, as already discussed. Furthermore, buffer and temperature conditions must be identical. A difficult problem is caused by the extra amino groups of the antibody present in experiment B. The ratio of reagent to modifiable amino groups is smaller in experiment B if the same amount of acetic anhydride is added to both experiments. The resulting difference in reaction conditions can be roughly corrected by adding more reagent to experiment B. Another way of correction is to add an internal standard antigen (7). This “innocuous” antigen is not recognized by the antibody, but “feels” the different reaction conditions while it is acetylated. A short peptide containing Lys is a good choice. The peptide is added to experiments B and F, separated from the labeled antigen after Step 2, and its 3H/14C ratio is determined. R of the internal standard is 1 if the reaction conditions are

Protection from Chemical Modification

89

Table 1 Differential Protection of Horse Cytochrome c by Antibody E8 (Data from ref. 11)’ residue

3W14C in experiment B F

5 7 8 13 22 25 27 39 53 55 60 72 73 79 86 87 88 99 100

7.4 13.3 12.9 16.4 14.6 14.0 14.9 13.6 8.2 7.9 14.8 5.7 3.4 10.1 12.8 14.3 10.7 7.9 9.0

LYS

6.1 15.9 10.6 12.4 3.0 7.9 17.4 6.4 4.2 7.8 0.6 3.8 3.2 12.2 7.4 9.4 9.8 0.25 14.0

R 1.2 0.8 1.2 1.3 4.9 1.8 0.9 2.1 2.0 1.0 24.0 1.5 1.1 0.8 1.7 1.5 1.1 32.0 0.6

Lr

0.9 0.6 0.9 1.0 3.8 1.4 0.7 1.6 1.5 0.8 18.0 1.2 0.8 0.6 1.3 1.2 0.8 24.0 0.5

aActual radioactivities were at least 5000 cpm for 3H and 1000 cpm for 14C.R calculated according to Eq. (2). The average R for all residues except Lys-22, Lys-60, and Lys-99 is 4, = 1.29 f 0.42. R was divided by KV to obtain $,,, shown in the last column.

equivalent; R f 1 if the reaction conditions differ. R values of the antigen are correctedaccordingly. For example,if R of the internal standardis 2, this indicatesthat the degreeof acetylation is twice as high in experiment F, independent of whetheror not a lysine is protectedby the antibody.Division by 2 of all the 3W’4C ratios in experiment F corrects the nonequivalenceof reaction conditions. Addition of an internal standardis recommendedif the R values of the protectedresiduesare not very high and difficult to distinguish from “noise” (9). Use of an internal standardis less important if the protection factors are high, asin the following exampledescribing the results of the differential protection of cytochrome c by MAb E8 (Table 1). No internal standardwas used in this experiment to correct for differencesof reaction conditions in experimentsB and F. R valueswere first averagedto obtain R,. R,,, was defined as

90

Bosshard

R/R,,. Lys 60 and 99 were st!ongly protected in the antigen-antibody complex. The two residues are 7 A apart in the native molecule. The contribution of Lys-60 and Lys-99 to the epitope of antibody by other mapping procedures (8,9).

ES was later confirmed

2. Materials 1. Buffer 1: O.lMHEPES (Sigma, St Louis, MO), pH 8.0. HEPES is N-2-hydroxyethylpiperazme-l\r-2-ethanesulfonic acid. 2. Buffer 2: 6Murea (ultrapure-quality free of isocyanate, e.g., SigmaUltra or Fluka MicroSelect) in buffer 1. 3. [3H]Acetic anhydride (Amersham #TRA38 1) and (1 -14C)acetic anhydride (Amersham CFA296), 5-20% solutions in toluene. The specific radioactivity of the commercial material is reduced as appropriate by the addition of nonradioactive acetic anhydride. 4. Acetic anhydnde: analytical-grade, e.g., Fluka purissimum. 5. Reacti Vial from Pierce: cone-shaped thick-walled glass vial for 3-5 mL reaction volume, closed by a screw cap with septum and equipped with a small stnring bar. 6. HPLC equipment, amino acid sequencer, p-counter, faclhties for handling radioactive material, device for ultrafiltration (e.g., Amlcon).

3. Methods The followmg method is for the mapping of an epttope on cytochrome c recognized by MAb E8 (20,ll). Experiment B is described in detail. Experiment F is described as far as it differs from experiment B. Steps 1 and 2 of experiment B and F are conducted in parallel at the same time and can be completed

in 2-3 d.

3.1. Trace-Labeling of Free and Bound Antigen with pH]Acetic Anhydride (Step 1) 3.1.1. Experiment B 1. Place 375 ug (30 nmol) cytochrome c and 3750 ug (50 nmol with respect to antibody-binding site) MAb in 1 mL of buffer 1 in a React1 Vial (see Note 3). Incubate at 25’C for at least 10 min to allow for the formation of the immune complex (see Note 4) 2. Add 10 yL of a toluene solution containing 1 umol E3H]acetic anhydride (0.5 mCi, 185 MBq, specific radioactivity 0.5 Ci/mmol, 18.5 MBq/mmol) (see Note 5). Use a Hamilton syringe (or an equivalent glass syringe with stainless-steel plunger) to add the reagent as fast as possible into the vigorously stirred solution. The acetylation reaction is fast and completed after a few minutes. Because most of the acetic anhydride is hydrolyzed by water at pH 8.0, the effective anhydride concentration reacting with protein amino groups is small, and the acetylation reaction becomes pseudo-first-order. Less than 1 Lys/cytochrome c is acetylated

Protection from Chemical Modification

3.

4.

5. 6.

91

under these condmons. A higher degree of trace-labeling is not desirable (see Section 1.1. and Note 1). Add 1 pCi (37 kBq) of *4C-labeled cytochrome c in approx 100 uL of buffer 1. 14C-acetylated cytochrome c (see Note 6), has a specific radioactivity of approx 70 Ci/mol; 1 pCi 14C-acetylated cytochrome c corresponds to approx 15 nmol cytochrome c (see Note 7). Add 300 nmol of nonradioactive cytochrome c in 200-300 pL of buffer 1. This extra cytochrome c is added as a carrier to ease protein and peptide handling m subsequent steps (see Note 8). Separate the 3H-trace-labeled cytochrome c from the antibody and low-mol-wt reaction products by chromatography on a column (0.9 x 50 cm) of Bio-Gel P100 equilibrated and eluted with 7% (by volume) formic acid (see Note 9). Collect, pool, and freeze-dry the cytochrome c-containing fractions identified by their heme absorption band centered at 410 run. Add a few drops of concentrated NaOH to the later fractions to neutralize the volatile radioactive acetic acid produced by hydrolysis of the anhydride, and dispose of them properly. Discard the early fractions that contain the slightly 3H-acetylated antibody.

3.12. Experiment F Proceed exactly as in experiment B, except omit the MAb and use only 0.25 mCi (9.25 MBq) of [3H]acetic anhydride in 5 pL of toluene (see Note 10). 3.2. Complete Acetylation

(Step 2)

1. Dissolve each sample of freeze-dried 3H,14C-acetylated cytochrome c from step 1 in 1 mL of a half-saturated aqueous solution of sodium acetate in water, transfer into Reacti Veals, and cool on ice. 2. Add to the vigorously stirred solution five 5-pL portions of acetic anhydride, and wait about 10 mm between addmons. This amount of acetic anhydride corresponds to an approx 50-fold excess of anhydride over the total amount of lysines in the 3H,14C-acetylated cytochrome c from experiments B and F, respectively (approx 250 nmol cytochrome c with 19 reactive amino groups/molecule). 3. Separate from reaction products, and change to buffer 1 by ultrafiltration or by gel filtration. 4. Divide material mto three to five portions, and keep samples frozen until used in the subsequent analysis.

3.3. Pro teolysls, Peptide Separation, Edman Degradation, and Determination of 3H/‘4C-Ratios (Step 3) This is by far the most time-consuming step of the entire procedure. A few weeks are required for the complete analysis of a small protein of the size of cytochrome c. The analysis of a larger protein antigen will take longer. If the researcher has no previous experience in peptide analysis and amino acid sequencing, collaboration with an expert is strongly recommended.

Bosshard

92 3.3.1. Proteolysis and Peptide Separation

The choice of proteases and of the chromatography to separate peptides very much depends on the size and sequence of the 3H/14C-labeled protein. In the case of cytochrome c, the 3H/L4C-labeled protein from step 2 is divided in three equal batches, which are digested with a-chymotrypsin, thermolysm, and protease from Staphylococcus aweus V8 (cleaves after Glu and Asp), respectively (see Note 11). The method of choice for the separation of peptides is highperformance liquid chromatography on a reversed-phase column, using as eluent a gradient of acetomtrile in 0.1% aqueous trifluoroacettc acid (seeNote 12). 3.3.2. Sequencing and Determination of 3H/‘4C-Ratios Purified peptides are subjected to manual (12) or automated Edman degradation. If automated Edman degradation is used, the reaction protocol has to be changed to allow for the collection of the thiohydantoin derivative. This derivative is counted in a P-counter to obtain the 3H/i4C ratto. A p-counter with an external standard facility to correct for quenching is highly recommended (see Note 8). By sequencing many overlapping peptides, obtained from the digestion with three (or more) different proteases, the same Lys is often found more than once, allowing for multiple determination of the 3H/14Cratio. 4. Notes 1. The chemical reactivity of the a-amino group is governed by its pKa value and nucleophilicity, which in turn are influenced by the microenvironment of the group. The microenvironment encompasses neighboring atoms of the antigen as well asnearbyatomsof the antibody.The chemicalreactivity of ana-amino group is affected by a change of pKa or/and by steric factors. Proximity effects can increase or decrease the pKa. If, in the antigen-antibody complex, a Lys sidechain is in an area of negative potential, the pKa increases, if in an area of positive potential, the pKa decreases. A change to a less polar environment lowers the pKa. Only the deprotonated amino group reacts with acetic anhydrrde. The rate of acetylation is proportional to the concentration of the unprotonated ammo group, (A), which depends on pH according to: @)=WWXA

+

(WI=

~~6%)

(3)

[At] is the total concentration of the amino group, KA is its acid dissoctation

constant,and oA = KA/(KA+ [H+]) denotesthe fraction of the amino group in the deprotonated form (0 2 oA .S 1). The amino group reacts with acetic anhydride

(RX) according to A + RX + AR + X, where AR is Wacetyl (or Na-acetyl), and X is acetate. The rate of acetylation is:

d(AR)/dt = kA(RX)(At)KA/[KA + (H+)] = k@X) aA

(4)

Protection from Chemical Modification

2.

3. 4.

5. 6.

7.

where kA is the pa-independent second-order rate constant of the acetylation reaction. Equation (4) shows that the rate of acetylation decreases if the pKa of the amino group increases (decrease of KA). However, the situation is more complicated because kA also depends on KA. The relationship is given by the Bronsted equation Zogk, = SpKa + 7, where /3 and y are constants. The equation predicts that logk, increases with increasing pKa provided the steric accessibility of the amino group is unchanged. This means that part of the rate change caused by a change of the pKa is counterbalanced by a change of k,. If, however, access of the reagent to the amino group is sterically hindered-as may happen at the antigen-antibody interface-k, decreases. The result is a displacement from the straight line described by the Bronsted equation. It was shown that surfaceaccessible a-amino groups of a free protein behave as predicted by the Bronsted equation (23), whereas buried groups fall below the Bmnsted line (X14). As an alternative, [3H]acetic anhydride can be used for the acetylation of the free antigen and (1 -14C)acetic anhydride for the acetylation of the antibody-bound antigen in step 1. The trace-labeled derivatives from experiments B and F are then mixed together and completely acetylated with excess acetic anhydride under denaturing conditions m step 2. This has as an advantage that in step 3, the 3H/14C ratios are equivalent to the R values: 3W14C = R. However, in order that R = 1 for all residues of equal reactivity in the free and the antibody-bound antigen, the specific radioactivity of [3H]acetic anhydride and ( 14C)acetic anhydride must exactly match. This procedure is conceptually simpler, but has not yet been applied to epitope mapping. It was used in an experiment with an electron-transfer complex (1.5). Other buffers can be used, but they must not contain a reactive amino group; ammonium bicarbonate or Tris is unsuitable. The concentration of antigen and antibody should be at least 100 trmes higher than the dissociation constant (&) of the complex in order that the concentration of the free proteins becomes very small. In the experiment with cytochrome c, concentrations were about 1000 x &. The use of toluene solutions of radioactive acetic anhydride is strongly recommended, since the pure liquid is difficult to handle. r4C-acetylated antigen for use in step 1: Incubate 3.5 mg (280 nmol) cytochrome c in 1 mL of buffer 2 for at least 1 h to unfold the protein completely. Add 250 &!i [14C]acetic anhydride as a toluene solution (9.25 MBq, specific radioactivity 20 Ci/mol, 740 GBq/mol) to the vigorously stirred solution placed in a Reacti Vial. Separate from low-mol-wt reaction products by gel chromatography or ultrafiltration into buffer 1 (see Section 3.2.). The material obtained in this way contains 8-14 [ 14C]acetyl groups/cytochrome c and can be stored frozen for several months. The total 14C radioactivity added in the form of 14C-acetylated cytochrome c should be smaller than the total 3H radioactivity incorporated in the trace-labeling reaction because the determination of 3W’4C ratios is more accurate if 3W14C >I. It is recommended to perform a preliminary acetylation experiment to estimate the amount of 3H radioactivity incorporated during trace labeling.

94

Bosshard

8. The amount of added carrier antigen depends somewhat on the expected overall loss of material during the purification and analysis procedures of step 3. One should note that the radioactive antigen is diluted out by the addition of carrier antigen, and the determination of the 3H/14C ratio may become inaccurate if too much carrier is added. For the analysis of 3W14C ratios, at least several hundred cpm of each isotope should be available for counting at every sequencing step. 9. Other gel-chromatography materials may be adequate depending on the size of the antigen. The use of ion-exchange chromatography IS not recommended since the acetylated antigen has a different isoelectric point than the nonacetylated antigen. 10. As discussed in Section 1.1.) the amount of acetic anhydride must be reduced in experiment F because there are less amino groups to react with acetic anhydride. However, it is virtually impossible to match exactly the ratio of anhydride to amino groups m both experiments. In our experience, halving the concentration in experiment F has been a good compromise. By addition of an internal standard, the nonequivalence of reaction conditions can be corrected more accurately (see Section 1.1. and ref. 9). 11. Because Lys is acetylated, cleavage with trypsin will produce large fragments ending at Arg. 12. The choice of the reversed-phase cohnnn material and the shape of the acetonitrile gradient depend on the nature of the peptldes to be separated. Condttions for peptide separation may be optimized in a preliminary experiment. Good separation of peptides is achieved on a Nucleosil lOO-5Cts column (Marchery-Nagel, Dliren, Germany) using a binary, linear gradient composed of buffer A (0.067% [by vol] trifluoroacetic acid [Fluka, Microselect]) and buffer B (60% [by vol] acetomtrile [Fluka purtssimum] in buffer A).

References 1. Barlow, D. J., Edwards, M. S., and Thornton, J. M. (1986) Continuous and discontinuous protein antigenic determinants. Nature 322,747,748. 2. Rieder, R. and Bosshard, H. R. (1980) Comparrson of the bindmg sites on cytochrome c for cytochrome c oxtdase, cytochrome c reductase, and cytochrome c, . J. Biol Chem. 255,4732-4739. 3. Bosshard, H. R. (1979) Mapping contact areas in protein-nucleic acid and protein-protein complexes by differential chemical modification. Methods Biochem. Anal. 25,273-301. 4. Bumens, A., Demotz, S., Corradin, G., Binz, H., and Bosshard, H. R. (1987) Epitope mapping by differential chemical modification of free and antibodybound antigen. Science 235,780-783 5. Kaplan, H., Stevenson, K. J., and Hartley, B. S. (1971) Competitive labelling, a method for determining the reactivity of individual groups in proteins. Biochem. J. 124,289-299. 6. Lundblad, R. L. and Noyes, C. M. (1992) Chemical reagents for protein modification. CRC, Boca Raton, FL.

Protection from Chemical Modification

95

7. Saad, B. and Bosshard, H. R. (1990) Antigenic sites on cytochrome c2 from Rhodospirillum rubrum. Eur J. Bzochem. 187,425-430. 8. Paterson, Y., Englander, S. W., and Roder, H. (1990) An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR. Science 249,755-759.

9. Paterson, Y. (1992) Mapping antibody binding sites on protein antigens. Nature 356,456,457.

10. Carbone, F. R and Paterson, Y. (1985) Monoclonal antibodies to horse cytochrome c expressing four distinct idiotypes distribute among the sites on the native protein. J. Immunol 135,2609-2616. 11. Oertle, M., Immergluck, K., Paterson, Y., and Bosshard, H. R. (1989) Mapping of four discontiguous antigenic determinants on horse cytochrome c. Eur. J Blochem. 182,699-704.

12. Chang, J.-Y. (1981) N-terminal sequence analysis of polypeptides at the picomole level. Biochem. J. 199,557-564. 13. Bosshard, H. R. (198 1) Alkaline isomerization of ferricytochrome c: Lysine is not replacing methionine at the sixth co-ordination site of the haem iron. J. Mol. Bzol 153, 1125-l 149. 14. Kaplan, H. (1972) Determination of the ionization constants and reactivrties of the amino-termim of alpha-chymotrypsin, J. Mol. Bzol. 72, 153-l 62. 15. Bosshard, H. R., Wynn, R M , and Knaff, D. B. (1987) Bindmg site on Rhodospinllum rubrum cytochrome c2 for Rhodospirillum rubrum cytochrome bc, complex. Biochemistry 26,7688-7693.

10 Epitope Mapping by Proteolysis of Antigen-Antibody Complexes Protein Footprinting Ronald Jemmerson 1. Introduction Proteolytic cleavage of antigen-monoclonal antibody (MAb) complexes can be a relatively simple and direct approach to identifying an epitope on a protein antigen. The antigen-binding domains of an antibody are resistant to proteolysis (1,2). They also confer protection against degradation of bound antigen, particularly in the immediate vicinity of the epitope (3,4). If an epitope is linear, it can be directly identified after its elution from the MAb following proteolysis of the antigen-MAb complex. Conformational epitopes can be localized, if not completely identified, by comparing the rates of peptide release from the free antigen and from the antigen-MAb complex. By analogy to DNase footprinting, a method to localize protein-binding sites on DNA by nuclease digestion of protein-bound DNA (.5j, proteolysis of antiger+MAb complexes to identify epitopes on protein antigens has been referred to as “protein footprinting” (6). Effects of bound antibodies on the proteolytic degradation of a protein antigen were first observed by Moelling et al. (7). They studied the cleavage of the avian RNA tumor virus Pr76sasprotein to the ~15, ~19, and ~27 polypeptides by the p 15 viral protease. In their experiments, rabbit antisera against each of the products were used to immunoprecipitate the precursor. Treatment of the immunoprecipitates with the p 15 viral protease resulted in different patterns of intermediate cleavage products, visualized by polyacrylamide gel electrophoresis in sodium dodecylsulfate (SDS-PAGE), depending on the specificity of the antiserum used for immunoprecipitation, i.e., which segment from Pr76saselicFrom* Methods in Molecular Biology, vol. 66: Epftope Mapping Protocols Edited by* G E Morns Humana Press Inc., Totowa, NJ

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ited the antibodies. This finding was exploited by Eisenberg et al. to compare the antigenic specrlicities of a number of mouse MAbs to the herpes simplex type 1 (HSV-1) and HSV-2 gD glycoproteins (8). Different patterns of antigen cleavage by Staphylococcal aureus V8 protease were observed depending on the MAb employed. When the MAbs were grouped according to their V8 protease cleavage patterns, within a given group, the MAbs had similar viral neutralization (HSV- 1 vs HSV-2) and binding (HSV- 1 gD and/or HSV-2 gD) specificities. These results suggested that the pattern of MAb protection of an antigen from protease cleavage was dependent on the epitope recognized by the MAb. MAb protection from proteolysis of a protein antigen was first applied to the localization of epitopes by Jemmerson and colleagues. In early studies, they found that some, but not all, MAbs specific for human placental alkaline phosphatase (PLAP) could inhibit trypsin cleavage of PLAP at the only susceptible site on the antigen, after lysine 62 (9, IO). An example of this effect is shown in Fig. 1. Removal of the amino terminal segment from PLAP by trypsin reduces the molecular weight of the protein from 67 to 57 kDa as visualized by SDSPAGE (lane 2). Binding of the MAb E5 to PLAP protects the antigen from proteolytic attack. Although both the heavy (55 K) and light (22 K) chains of the MAb are partially cleaved by trypsin (lane 4), this cleavage appears to occur in nonantigen binding regions of the MAb and does not hinder the ability of the MAb to protect the antigen. Trypsin-blocking is observed with 25-30% of the anti-PLAP MAb that have been studied (Fig. 2). Some of the MAbs that do not protect PLAP from cleavage by trypsin, e.g., MAb BlO and H7, do prevent its cleavage by another protease, bromelain (II). Bromelain removes a 2-kDa segment from the carboxy1 terminal end of PLAP (not shown). The MAbs that completely protect against trypsin cleavage (F6, E5, andC4) do not block cleavage by bromelain. Thus, by testing the affects of MAbs on the proteolytic cleavage of PLAP by trypsin and bromelain, two epitopes could be localized on the antigen: one located near the amino terminus in the vicinity of the trypsin cleavage site and the other near the carboxyl terminus in the vicinity of the bromelain cleavage site. At least one other region encompasses the binding sites for those MAbs that do not protect against either protease. The epitope assignment of anti-PLAP MAbs by protein footprinting is consistent with the grouping of the MAbs by other methods (12). Binding of the MAb BlO and H7 (bromelain-blocking MAbs) to PLAP in enzyme-linked immunosorbent assay (ELISA) did not inhibit the binding of MAbs F6, E5, and C4 (trypsin-blocking MAbs) and vice versa. Combinations of any two MAbs from within one of these two groups did not precipitate PLAP in immunodiffusion assays,indicating that within a group, the MAbs could not crosslink

Protein Foo tprin ting

99

Fig. 1. A bound MAb protectsPLAP from cleavageby trypsin. PLAP (1 mg/mL), PLAP preincubatedwith an equimolar amount of MAb E5 for 1 h, or MAb E5 only was treatedwith 2% (weight protease:weight antigen)trypsin (try) in PBS,pH 8.3, for 24 h at room temperature.Intact antigen and MAb and the trypsin-treated proteins (PLAP/try, PLAP-MAb/try, and MAb/try) were run reduced with 2% 2-mercaptoethanol in SDS-PAGE (10% gel), and the gel was stained with Coomassiebrilliant blue. The two bandsderived from the MAb correspondto the heavy and light chains. the antigen, causing it to precipitate, i.e., within a group, the MAbs appear to bind the same or overlapping site(s). However, any combination of two MAbs

from these two groups did precipitate the antigen, demonstrating that the two groups of MAbs recognize ‘distinct epitopes. The limited number of protease cleavage sites accessible on native PLAP does not allow the epitopes to be

more precisely defined by protein footprinting. Cytochrome c (CYT) is a well-characterized protein antigen with multiple proteasecleavagesites accessibleon the native protein. Application of protein footprinting to this antigen demonstratedthe usefulness of this approach in defining conformation-dependent epitopes on globular proteins (3,4). Two dif-

100

Jemmerson

Fig. 2. Most MAb specific for PLAP do not block the trypsin cleavagesite. PLAP was radioiodinated, 0.5 pg antigen incubatedwith 100 pg bovine serumalbumin and 10 pg of each of the MAb, and then treatedwith 2% trypsin for 24 h. The reaction mixtures were run in SDS-PAGE(10% gel). The gel was dried and autoradiographed. Since the MAb was not radiolabeled,the MAb heavy and light chains are not visible.

ferent sites localized on horse CYT by this method were confirmed by immunological methods, including comparative binding of MAbs to variants of CYT

(13,14) and hydrogen-deuterium exchange detected by two-dimensional nuclear magnetic resonance(15,I6). Proteasecleavage at multiple sites on CYT causesconformational changes that result in the loss of the ability to bind MAbs specific for the native protein. A bound MAb cannot completely protect proteins having conformationdependent epitopes and a number of cleavage sites, such as CYT, from proteolytic cleavage. Proteolysis at sites distal from the epitope results in conformational changes in the epitope itself and concomitant uncoupling of epitope peptides from the MAb. In contrastto the type of results observedwith PLAP, when CYT-MAb complexes are treated with trypsin, CYT is completely degradedwithin 4 h. However, partial protection from proteasecleavage can be observedwithin the first hour of proteolysis, Therefore, to examine the affect of MAbs on the proteolysis of antigens like CYT, a kinetic analysis is necessary. Results of such an analysis are shown in Fig. 3. Within 30 min during proteolysis with trypsin, peptides la (residues 2627), 18 (residues 40-53), and 20 (residues 39-53) are present in significantly higher concentrations relative to other peptides in the digest of free CYT than in the digest of the CYT-MAb C3 complex. These peptides and, to varying and lesser extents, neighboring segmentsare probably present in longer, only partially cleaved (hence, partially-protected) polypeptides that do not elute from the reverse-phasecolumn

Protein Footprinting

10.0

8.0

101

t I

cytochrome

c peptide

Fig. 3. Localization of a conformation-dependent epitope on horse CYT by protein footprinting. The CYT-MAb C3 complex (approx 1: 1 molar ratio) or free CYT, incubated with a nonspecific isotype-matched MAb, was treated with 5% (weight protease:weight antigen) (w/w) trypsin in 50 mMammonium bicarbonate, pH 8.3, for 30 min at 37°C. The digests were lyophilized, dissolved in 0.1% trifluoracetic acid, and applied to a reverse-phase HPLC column (C-18). Peptides were eluted in a gradient of O-70% acetonitrile, 0.1% trifluoracetic acid over 90 min. Peak height ratios were calculated for each eluting peptide as the peak height observed from the digest of unbound CYT divided by the peak height observed from the digest of the CYT-MAb complex. The dotted line indicates the average peak height ratio for all peptides analyzed. Peptides were identified from their amino acid compositions. The peptides with peak height ratios significantly above the average include la (a dipeptide containing residues 26 and 27), 18 (residues 40-53), and 20 (residues 39-53). These peptides appear to be involved in MAb binding. See text for further discussion. in the gradient of acetonitrile which all protease sites on mechanism in the degradation tion of certain segments from

employed. The bound MAb slows the rate at CYT are cleaved, suggesting a cooperative of the protein. However, the enhanced protecproteolytic cleavage suggests their involvement

in MAb binding. Radioimmunoassay of the binding of MAb C3 to variants of CYT had independently localized the epitope to the region around residue 44 (13). The carbony1 oxygen of residue 44 is hydrogen-bonded to the side chain of histidine 26, thus implicating the dipeptide 26-27 as well as the peptrdes 39-53 and 40-

102

Jemmerson

53 in this footprinting experiment. Peak height ratios comparable in magnitude above the background to those for MAb C3 were observed for another MAb (C7) binding thus same region on horse CYT (14). An MAb (E8) specific for another epitope on horse CYT gave different rattos (4), whtch indicated binding at a site on another surface of the antigen, consistent with immunochemical (13) and physical chemical data (15). For MAbs that bind linear epitopes, protein footprmting provides a straightforward approach to identify the MAb-binding site. An elegant example of this method is demonstrated in the study of an MAb-binding gastrin-releasing peptide (GRP) (17). Several proteases are known to cleave this 27-amino-acid long peptide at different sues, and an MAb has been shown to protect some of those sites from cleavage. When GRP was bound to this MAb (covalently coupled to agarose), and the antigen-MAb complex was treated with trypsm and then washed to remove unbound peptides, only the carboxyl terminal peptide contaming residues 18-27 was eluted from the MAb using mild, acidic conditions. This indicated that the epitope bound by this MAb 1s contained within the sequence 18-27 (Fig. 4). To define the epitope more precisely, prior to elution of the trypsm-cleaved pepttde from the insolubilized MAb, the complex was treated with aminopeptidase M. This protease removes individual residues from the amino terminus of peptides, allowing trimming of ammo acids from an MAb-bound antigen that are not protected by the MAb. Four different products eluted m differing amounts from the trypsin-degraded GRP-MAb complex after ammopepttdase treatment. These corresponded to the carboxyl terminal trypsin-cleaved fragment with O-3 of the amino terminal residues removed (Fig. 4). The smallest peptide, containing seven amino acids, appears to represent the epitope. The studies cited for epitope localization on PLAP, CYT, and GRP provide examples of the different methods that can be used for protein footprinting. The particular method one should choose will depend on the characteristics of the protein antigen being studied. If the epitopes are conformation-dependent and there are a limited number of protease cleavage sites on the native protein, then the method used to map the epitopes on PLAP would apply (Method A, Section 2.1.). For conformation-dependent epitopes on a protein that is degraded to many small peptides by protease treatment, the method used to identify epitopes on CYT should be followed (Method B, Section 2.2.). If a protein is known to have linear epitopes, i.e., they are conformation-independent, the method used to map the epitope on GRP is advrsable (Method C, Section 2.3.). For each method, there are a number of variables that need to be considered, including the protease to be employed and the conditions for proteolysts (pH, time, temperature, concentrations of reactants, and so forth). Several proteaseshave been used in protein footprinting, e.g., trypsin (4,6,9,14,17, J&20),

103

Protein Foo tprin ting tryp*itl

Gsrtr’“~Re’ess’n~ Psptlde

(GRP)

I~pdll

5 10 i 15 + 20 25 : V P L P A G G G T V L T K M Y P R G N H W A V G H L M. NH,

Peptides horn smlnopeptldase dewage of tryP&t-degraded GRP-mAb

M

i

Fig. 4. Protein footprmting of an epitope on the GRP (I 7). The antigen was incubated with MAb that hadbeencovalently attachedto agarose.The antigen-MAb complex wastreated with an equimolar amount of trypsin in PBS,pH 7.4, for 18h at 37°C. Unbound peptides were washedaway and the remaining peptide (residues 18-27)MAb complex was digestedwith aminopeptidaseM (1:4, proteaseantigen) for 48 h. Bound peptideswere analyzedby matrix-assistedlaser desorptionmassspectrometry. Four peptide products were observed in differing amounts (indicated by the relative thicknessof the bars in this figure). The epitope for the MAb appearsto be contained within the smallestsequence observed: WAVGHLM-NH2(amide), although involvement of the carboxyl terminal residue(s) was not demonstrated

chymotrypsin (6,17,21,22), V8 protease (8,18,23), bromelain (II), thermolysin (17), elastase (221, endopeptidase Glu-C (22), lysyl endopeptidase (231, and aminopeptidase M (2 7). In general, these are functional at neutral pH, so that the samebuffer may be used when testing more than one protease. For example, in the study of GRP, four proteases were tested (trypsin, chymotrypsin, thermolysin, and aminopeptidase M) using the same buffer, phosphate-buffered saline (PBS), pH 7.4 (I 7). Presented below are details of the methods that can be used for footprinting proteins having properties similar to PLAP (Method A), CYT (Method B), and GRP (Method C). These methods should serve as guidelines in the footprinting of most protein antigens. Minor variations from these protocols may be required when adapting this technique for a particular use. This technique may also apply to the identificatron of epitopes on complex carbohydrate antigens by using glycosidases instead of proteases, although such an application has not been reported. 2. Materials

2.1. Method A: Footprinting of Proteins with Few Protease Cleavage Sites 1. Proteases: High-purity

proteases are recommended, e.g., TPCK-treated

trypsin

and TLCK-treated chymotrypsin(Sigma, St.Louis, MO). 2. Purified antigen,antigen-specificMAb, andcontrol MAb having the sameisotype as the specific MAb; crude preparations of antigen and specific MAb (e.g., ascites

104

Jemmerson

fluid) and protein A- or protein G-coupled Sepharose or -coupled agarose (Sigma); or a crude preparation of antigen and purified MAb-coupled Sepharose or -coupled agarose. 3. PBS: 0.14A4 NaCl, 10 mM sodium phosphate, pH 7.4, for treatment of the antigen and antigen-MAb complexes with many proteases, e.g., trypsin and chymotrypsin. 4. Apparatus for PAGE, PAGE sample buffer containing 4 drops glycerol/ml water, 2% 2-mercaptoethanol, 1% SDS and bromophenol blue, and PAGE running buffer: 3 g Tris base and 14.4 g glycine/L, 0.2% SDS, pH 8.3.

2.2. Method B: Footprinting of Proteins Having ConformationDependent Epitopes and Mu/tip/e Protease CIeavage Sifes 5. 6. 7. 8.

Proteases, antigen, and MAbs as in Section 2.1. 50 mM ammonium bicarbonate, pH 8.3. Sephadex G-100 (optional). High-performance liquid chromatograph (HPLC) equipped with an analytical reverse-phase (C- 18) column, Columns with shorter alkyl chains or ion-exchange columns may also be used if they are adequate for the separation of short peptides. 9. Trifluoroacetic acid and acetonitrile (degassed).

2.3. Method C: Footprinting of Proteins Having Linear Epitopes 10. Proteases and buffer as in Section 2.1.) antigen, and purified MAb. 11. Matrix for covalently couplmg MAb, e.g., CNBr-activated Sepharose (Pharmacia [Piscataway, NJ]; Sigma), Affigel (Bio-Rad, Richmond, CA), or aldehyde-activated agarose (Pierce, Rockford, IL). 12. HPLC equipped with a reverse-phase (C-18) column (see Section 2.2.). 13. If available, mass spectrometer (see Chapter 13).

3. Methods

3.1. Method A: Footprinting of Proteins with Few Protease Cleavage Sites 1, Incubate purified antigen (50 pL >l mg/mL in PBS) with an equal molar amount of purified specific MAb in PBS for at least 1 h (see Note 1). As a control, incubate the antigen with a nonspecific MAb having the same isotype as the test MAb. If either the antigen or MAb is not purified, the antigen-MAb complex can be precipitated using a protein A- or G-coupled matrix, e.g., Sepharose m ref. 6. Similarly, antigen can be isolated from an impure protein mixture by adsorption to an MAb-coupled matrix. 2. Add 2-5% protease (w:w total protein) to the free antigen and antigen-MAb complex, and Incubate for 24 h at room temperature or until proteolytic cleavage is complete (see Note 2). By elevating the temperature to 37”C, the proteolysis time can be reduced substantially. If antigen-MAb were precipitated using a protein A- or protein G-coupled matrix, then it would be necessary to test several specific MAbs that bind different sites to show that the effect of the MAb on proteolytic attack is specific to the epitope recognized.

Protein Footprin ting

705

3. Run each digest in SDS-PAGE. A 10% polyacrylamide gel is usually satisfactory. If the peptides resulting from the protease digestion are smaller than, say, 20 kDa, then a higher percentage gel will be necessary. 4. Stain the gel with Coomassie brilliant blue. If smaller amounts of antigen than suggested are applied to the gel, then silver staining will be required.

3.2. Method B: Footprinting of Proteins Having Conformation-Dependent Epitopes and Multlple Protease Cleavage Sites 1. Incubate antigen (- 1 umol) with MAb (-0.5 pool) in 50 mM ammonium bicarbonate, pH 8.3, for at least 1 h (see Note 3). A volatile buffer is preferable in this method, since the antigen-MAb complexes will be lyophilized in a later step To ensure that all the epitopes of the antigen are saturated, antigen can be incubated with excess MAb, and free antigen can be separated from MAb-bound antigen by gel-filtration chromatography using an appropriate gel (e.g., Sephadex G-100 for small protein antigens like CYT). 2. Treat antigen-MAb complex and a control mixture of antigen and nonspecific MAb with 5% protease at 37“C with continuous mixing. The total volume is not crucial. At various times (30 min, 60 min, 2 h, and 4 h), remove equal volume aliquots and add 2.OM acetic acid (10% total volume/aliquot) to stop proteolysis. 3. Lyophilize the aliquots, and then dissolve them in 200 uL 0.4M acetic acid containing 0.1% trifluoracetic acid for HPLC analysis. Centrifuge and filter to remove any particulate material. Microfilters for small volumes are commercially available (e.g., Millex-GV4 syringe filter, Millipore, Bedford, MA). 4. Inject a constant volume of each solution (~200 pL) into an HPLC equipped with an analytical reverse-phase (C- 18) column. Elute peptides in a gradient of O-70% acetonitrile (containing 0.1% trifluoroacetic acid) over the course of 90 min. 5. The peptides can be identified by their amino acid composition, determined after 24 h hydrolysis of the eluting fractions in 6N HCl at 100°C. 6. The relative amounts of each peptide from the proteolysis of the free antigen vs the MAb-antigen complex can be calculated from the HPLC peak heights as a ratio of the peak height for a peptide from the proteolysis of free antigen divided by the peak height for that same peptide from the proteolysis of the MAb-antigen complex (see Note 4).

3.3. Method C: Footprinting

of Proteins Having Linear Epitopes

1. Insolubilize the MAb by covalently coupling it to a solid support (Z&l 7) (see Note 5). The chemistry for coupling one MAb while retaining antigen binding may not work for another MAb, so it may be necessary to test different conditions if the first attempt at adsorbing antigen is not successful. Instructions for coupling are usually sent by the manufacturer along with the supporting matrix (see Section 2.3., step 11). The amount of MAb-coupled matrix to be used in a footprinting experiment should be sufficient to allow detection of bound antigen in later steps. In one study, as little as 50 pg MAb was employed for coupling to

106

2.

3

4. 5. 6.

Jemmerson

aldehyde-activated agarose (Aminolink immobilization kit, Pierce) and for protein footprinting (17). Pack the MAb-coupled matrix into a small column (a disposable pipet plugged with glass wool would do), and pass excess antigen (crude or purified) through it Then wash the column in PBS to remove unbound antigen and any contammating proteins. Add the desired protease to the column (e.g , 5% trypsin). Close off the ends of the column and rotate continuously for an appropriate period of time (e.g., for several hours at 37°C). Wash the column in PBS, and elute any bound peptides in 1.OA4acetic acid. Lyophilize the eluate and prepare the drted material for HPLC analysts as in Sections 3.2., steps 3 and 4. Major peaks eluting from the HPLC can be sequenced using a gas-phase automated sequenator (Applied Biosystems), or the amino acid compositions can be determined (see Note 6).

4. Notes 1. If purified antigen is available in only small amounts, it may be radiolabeled, e.g., with 125iodine using chloramine T (24). In this case, a carrier protein e.g., serum albumin, should be added during proteolysis. The carrier protein protects from loss of the antigen as might occur by its sticking to the reaction vessel and prevents the need to work with extremely small amounts of protease in order to mamtam the specified antigen:protease ratio. The use of radiolabeled antigen also avoids the need for purified MAb. 2. To establish which protease to use and in what amount, preliminary experiments should be carried out to determine the ability of various proteases to cleave the antigen. The proteases that have been used in protein footprmting are hsted m Section 1. The antigen should be incubated with several different proteases at several concentrations relative to the amount of antigen. Those proteases that cleave the antigen into polypeptides that can be visualized by SDS-PAGE are most useful m this method. 3. The MAb can be pretreated with protease and the large antigen-binding fragment can be separated from peptides by gel-filtration chromatography using Sephadex G-100. This may allow for a cleaner background in the HPLC analysis later. 4. The peak height ratio can vary considerably between experiments. It is necessary to carry out multiple trials and to consider the average + SD in determining the sigmficance of the results. 5. With slight modifications, this method may be carried out with soluble MAb or MAb adsorbed to a protein A- or G-coupled matrix. If soluble MAb is used, following proteolyis, MAb-antigen complexes must be adsorbed to separate epitopic peptides from other products in the proteolyttc digest. 6. Mass spectrometry provides a very sensitive method for detecting peptides (2 7,20). If this instrumentation is available, it may facilitate epitope identification by detecting peptides that do not resolve well in HPLC. See also Chapter 13

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References 1 Porter, R. R. (1959) The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Bzochem. J. 73, 119-126. 2. Parham, P. (1983) On the fragmentation of monoclonal IgGl, IgGZa, and IgG2b from BALB/c mice. J Immunol. 131,2895-2902. 3. Jemmerson, R. and Paterson, Y. (1986) Mapping antigenic sites on proteins: implications for the design of synthetic vaccines. BzoTechniques 4, 18-3 1. 4. Jemmerson, R. and Paterson, Y. (1986) Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes. Science 232, 100 l-l 004. 5. Galas, D J. and Schmitz, A. (1978) DNAse footprmting: a simple method for the detection of protein-DNA binding specificity. Nucleic Aczds Res. 5,3 157-3 170. 6. Sheshberadaran, H. and Payne, L. G. (1988) Protein antigen-monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody-bound antigen: protein “footprinting. ” Proc. Natl. Acad Scz USA 85, l-5. 7. Moelling, K., Scott, A., Dittmar, K. E. J., and Owada, M. (1980) Effect of p15associated protease from an avian RNA tumor virus on avian virus-specific polyprotem precursors. J Viral. 33,680-688. 8. Eisenberg, R. J., Long, D , Pereira, L , Hampar, B., Zweig, M , and Cohen, G. H. (1982) Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1. J. Virol. 41,478-488. 9. Jemmerson, R. and Stigbrand, T. (1984) Monoclonal antibodies block the trypsin cleavage site on human placental alkaline phosphatase. FEBS Lett. 173, 357-359.

10. Millan, J L. (1986) Molecular cloning and sequence analysis of human placental alkaline phosphatase. J. Bzol Chem. 261,3 112-3 115. 11. Jemmerson, R., Millbn, J L., Kher, F. G., and Fishman, W. H. (1985) Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells. FEBS Lett 179,3 16-320. 12. Stigbrand, T., Jemmerson, R., Millan, J. L., and Fishman, W. H. (1987) A hidden antigenic determinant on membrane-bound human placental alkaline phosphatase. Tumor Biol. 8,34-44 13. Carbone, F. R. and Paterson, Y. (1985) Monoclonal antibodies to horse cytochrome c expressing four distinct idiotypes distribute among two sites on the native protein. J Immunol 135,2609-2616. 14. Cooper, H. M., Jemmerson, R., Hunt, D. F., Griffin, P. R., Yates, J. R., Shabanowitz, J., Zhu, N.-Z , and Paterson, Y. (1987) Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations, J. Biol. Chem. 262, 11,59 l-l 1,597. 15. Paterson, Y., Englander, S. W., and Roder, H. (1990) An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR. Science 249,755-759.

16. Mayne, L., Paterson, Y., Cerasoli, D., and Englander, S. W. (1992) Effect of antibody binding on protein motions studied by hydrogen-exchange labeling and twodimensional NMR. Bzochemzstry 31, 10,678-10,685.

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17. Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Epitope mapping of the gastrinreleasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. Protein Sci. 3, 1485-1492. 18. Bricker, B. J., Snyder, R. M., Fox, J. W., Volk, W. A., and Wagner, R R. (1987) Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus (New Jersey serotype): a method for preliminary mapping of epitopes. virology 161,533-540. 19. Suckau, D., Kohl, J., Karwath, G., Schneider, K., Casaretto, M., Bitter-Suermann, D., and Przybylsh, M. (1990) Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci USA 87,9848-9852. 20. Haase, E. M., Yi, K., Morse, G. D., and Murphy, T. F. (1994) Mapping of bactericidal epitopes on the P2 porin protein of non-typeable Haemophilis znfuenzae. Infect. Immun. 62,3712-3722.

21. Urayama, O., Nagamune, H., Nakao, M., and Hara, Y. (1990) A monoclonal antibody against a native conformation of the porcine renal Na+/K+-ATPase a-subunit protein. Biochim. Biophys. Acta 1040,267-275. 22. Bloom, J. W., Bettencourt, J. D., and Mitra, G. (1993) Epitope mapping and functional analysis of three murine IgGl monoclonal antibodies to human tumor necrosis factor-a. J. Immunol. 151,2707-2716. 23. Schlaeppi, J.-M., Vekemans, S., Rink, H., and Chang, J.-Y. (1990) Preparation of monoclonal antibodies to huudin and hirudin peptides-a method for studying hirudin-thrombin interaction. Eur. J. Biochem. 188,463470. 24. Hunter, W. M. and Greenwood, F. C. (1962) Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 194,495,496.

11 Proteolytic Fragmentation for Epitope Mapping Maria R. Mazzoni, Nikolai 0. Artemyev, and Heidi E. Hamm 1. Introduction Since its introduction in 1975 the methodology of Kohler and Milstein (I) for production of monoclonal antibody (MAb) from hybridoma cells has been widely used to provide antibodies with a defined specificity. One characteristic feature of this technology is that impure antigens can be used to produce monospecific antibodies that can be utilized to study the functional domains of protein molecules. In this chapter, the use of limited vs complete proteolytic digestion experiments to define the epitope on the antigen recognized by a given MAb is outlined. We describe our studies (2) with the transducin (G,) a-subunit in which proteolytic digestion, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and Edman degradation were used to determine the sequence of the fragments recognized by the MAb 4A. Therefore, reaction times and reagents presented in this chapter may require some modification when a different protein antigen is under investigation. A protein can be cleaved chemically or enzymatically to generate various internal peptides. The number of peptides that are produced depends on whether the protein is cleaved completely at many sites or at a limited number of sites. Cleavage at few sites simplifies purification because a smaller number of peptides is generated. Small numbers of large fragments are produced by limited proteolysis of native proteins. Low ratios of protease achieve efficient limited digestion of native, globular proteins because cleavages tend to occur between compact structural domains. The large fragments can be easily separated and purified by either SDS-PAGE or HPLC. Proteolytic fragments separated by SDS-PAGE can be electroblotted to an immobilizing membrane and probed with an antibody which is directed against the native protein. The antibody will recognize those fragments that contain the epitope. Therefore, this approach From

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can be useful to identify the epitope for an MAb. Proteolytic enzymes with different substrate specificity can be used to perform limited digestion of a protein antigen, and antibody binding to the fragments can be analyzed by Western blot. However, the sequential appearance and origins of proteolytic fragments should be known. When the primary sequence of the protein is known, the origins of proteolytic fragments can be easily determined after separation or purification by N-terminal sequencing of peptides using repeated cycles of the Edman degradation reaction. The use of different proteolytic enzymes appears to be important for an accurate identification of an antigenic determinant. A limitation to this procedure 1sthat the denatured protein and its proteolytic fragments bound to the immobilizing membrane may no longer contain the same conformational and structural antigemc determinant present m the native protein. This technique is not reliable to identify complex conformational epitopes, but it is useful to detect epitopes conslstmg of a specific amino acid sequence. In the case of conformational epitopes, limited proteolytic digestion of the protein antigen in combination with m-n-nunoprecipitation can be used for localization of the antibody-bindmg region. To determine the MAb 4A antigenic site, we examined limited proteolytic patterns of the transducin a-subunit (GDP- or GTP-bound state) with different proteases after Western blotting and immunoprecipitatlon protocols. After purifying the proteolytic fragments, the identity was determined by N-terminal sequencing of peptldes using repeated cycles of the Edman degradation reaction. Examples of limited proteolytic digestion of the at-subunit by different proteases and Western blot analysis of MAb 4A binding to the fragments are shown in Figs. 1-4. When several proteolytic fragments are produced during limited digestion, a time-course analysis allows a better resolution of the proteolytic pattern. Therefore, m the case of proteolytlc digestion of the a,subunit with trypsin, chymotrypsin, endoprotemase Arg-C, and Lys-C, the time-course of fragment production was exammed (Figs. 1,3, and 4). As indicated in Fig. 5, proteolytic cleavage sites of the at-subunit are located at three regions: near the amino terminus at Leu15-Lys25,at Arg204-Trp207,and near the carboxyl terminus at Arg 310. Since the cleavage sites on a, are known, the origins for most fragments are defined, and the MAb 4A epltope can be located in the amino terminal region of the protein. More specifically, the residues from Met’ to Lys17appear to be required for antibody binding. The exact location of the epitope within this 17-amino-acid residue of the q-subunit remains unidentified. Another approach to epitope mapping 1s complete proteolytic digestion of the target protein. The choice of complete vs limited proteolysis for localization of a functional epitope depends on several factors. If the unknown region is relatively big, then a limited digest with subsequent generation of large fragments is preferable. The large protein fragments are also more hkely to main-

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Fig. 1. Time-courseof limited proteolytic digestion of Gt (A) and a,-GTPyS (B) by TPCK-treatedtrypsin and Westernblot analysisof MAb 4A binding to the fragments. The proteolytic fragments(A, 35 pg of Gjlane; B, 17 pg of q/lane) were separatedby electrophoresison duplicate 16% SDS-polyacrylamidegels. One gel was stainedwith Coomassieblue (left panels),and the other (right panels)was blotted onto nitrocellulose. Molecular-weight standardsare indicated on the left side of gels. G, G,; a, cxtGTP#; T, trypsin; I, trypsin inhibitor.

tain a more native-like conformation, which may be important for epitope identification. However, many proteins do not have clear patterns following limited proteolytic fragmentation, and insteadgeneratemultiple products of partial cleavage. The complex patterns produced by limited proteolysis can be difficult to interpret, making complete proteolytic cleavage advantageous.Complete proteolytic fragmentation is also instrumental when the goal is to localize a specific epitope to the smallestpossible stretch of amino acid residues.Addi-

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Fig. 2. Limited proteolytic digestionof a,-GTPySby 5’.aureu~V8 proteaseand Westernblot analysisof MAb 4A binding to the fragment.The samples(20 pg of protein/lane)were loadedon duplicate 12.5%SDS-polyacrylarnidegels and electrophoresed.Onegel wasstainedwith Coomassie blue(left panel),andthe other(right panel)wasblottedontonitrocellulose.~1,cc,-GTPyS; CLAN, o,A22-350)-GTPyS. Lane 1, a,-GTPyS;lane2, a,(22-350)-GTPyS. tionally, completeproteolysis can be usedto identify crosslinking sitesbetween proteins, since once a crosslinkedproduct betweenproteins is formed, it can be denaturedwithout affecting the site. The method does have some limitations, including (1) that a large number of peptides may be produced after complete proteolysis even with highly specific proteases,and (2) that a complex mixture of peptides cannot always be resolved by reverse-phaseHPLC, complicating the use of peptide sequencingto identify the epitope of interest. To overcome the difficulties imposed by a large number of proteolytic fragments, matrixassistedlaser desorption ionization mass spectrometry (MALDI) may be utilized for analysis. 2. Materials 2.1. Buffer and Enzymes 1. Buffer A: 10 rnI4 3+&morpholino]propanesulfonicacid (MOPS),pH 7.5, 200 mMNaCl,2 mM MgCl,, 1 nuI4dithiothreitol(DTT). 2. L-1-Tosylamido-2-phenylethylchloromethyl ketone (TPCK)-treatedtrypsin (WorthingtonBiochem.Corp., Freehold,NJ): Stock solution: 2 mg/rnL in 0.1 rniI4HClstoredat 4°C. Diluted solution:0.08mg/mLof TPCK-treatedtrypsin in buffer A containing25%glycerol madefreshfrom stocksolution.

Proteolytic Fragmentation

Fig. 3. Time-course of limited proteolytic digestion of Gt (B) and pertussistoxincatalyzedADP-ribosylated Gt (A) by endoproteinaseArg-C andWesternblot analysis of MAb 4A binding to the fragments.(A) ADP-ribosylated G,. The proteolytic fragments (30 p&lane) were separatedon a 16% SDS-polyacrylamide gel. The gel was stained with CoomassieBlue (left panel), dried, and autoradiographed(right panel). (B) Ct. The proteolytic fragments(40 pg/lane) were separatedon duplicate 16% SDSpolyacrylamide gels. One gel was stainedwith Coomassieblue (left panel), and the other (right panel) was blotted onto nitrocellulose. Molecular-weight standardsare indicated on the left side of the gels. G, G,, I, TLCK; E, endoproteinaseArg-C.

Mazzoni, Artemyev, and Hamm

Fig. 4. Time-courseof limited proteolytic digestionof a,-GTPySby endoproteinase Lys-C and Western blot analysis of MAb 4A binding to fragments.The proteolytic fragments(10 ug/lane) were separatedon duplicate 12.5%SDS-polyacrylamidegels. One gel was stainedwith Coomassieblue (left panel), and the other (right panel) was blotted onto nitrocellulose. Molecular-weight standardsare indicated on the left side Of the gel. C%,CX,-GTPyS; a36, a,(26-350)-GTPyS; E, endoproteinaseLys-C; I, CQGTPyS + TLCK.

3. Endoproteinase Arg-C and Lys-C (Boehringer Mannheim Biochem. Corp., Indianapolis, IN): Stock solution: aliquots-of 3 mg/mL solution of either endoproteinaseArg-C or Lys-C preparedin distilled water and stored at -2OOC. Diluted solutions: 1 mg/mL of endoproteinase Arg-C and 0.01 mg/mL of endoproteinaseLys-C in buffer A made fresh from stock solutions. 4. StaphylococcusaureusV8 protease(ICN Biomedicals, CostaMesa, CA): Stock solution: 2 mg/mL made fresh in distilled water. Diluted solution: 0.012 mg/mL of S. aweus V8 proteasein buffer A made fresh from stock solution. 5. 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride (TLCK)-treated chymotrypsin (Worthington): Stock solution: 2 mg/mL madefresh in distilled water. Diluted solution: 0.08 mg/rnL in buffer A made fresh from stock solution.

2.2. Protease Inhibitors 6. Soybeantrypsin inhibitor (Worthington): Aliquots of 8 mg/mL solution prepared in distilled water and storedat -20°C. 7. TLCK (Boehringer Mannheim): Stock solution (18 mg/mL) prepared fresh in distilled water.

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Fig. 5. The major proteolytrc cleavage sites on native c+. T, trypsin; K, endoproteinase Lys-C; R, endoproteinase Arg-C; C, chymotrypsm; V, S. aureus V8 protease; Cys347, site of ADP-ribosylation by pertussis toxin.

8. TPCK (Boehringer Mannheim): Aliquots of 50-d stock solution prepared in ethanol and stored at -20°C in the dark. 9. Phenylmethanesulfonyl fluoride (PMSF) (Sigma, St. Louis, MO): Aliquots of 100-M stock solutrons prepared in ethanol and stored at -20°C. Diluted solution (5 mM in ethanol) made fresh from stock solution.

10. SDS-polyacrylamrde gels of varying percentages should be prepared according to Laemmli (3), using reagents of the highest quality available, and Nalgene (Rochester, NY) filters (0.2 urn) used to filter all electrophorests soluttons. 11. Electrophoresis buffer: 3 g Tris base (Tris ultra pure, ICN Biomedical), 14.4 g glycine (glycine electrophoresis grade, ICN Biomedical), and 2 g SDS (sodium dodecyl sulfate ultrapure, ICN Btomedical) in 1 L of drstilled water. 12. Stock solutron of sodium thioglycolate (Sigma): 0. Win distilled water: a diluted solution (0.1 mA4) of sodium thtoglycolate made fresh in electrophoresis buffer from the stock solution. 13. The electroblot transfer should be made to nitrocellulose (0.1 urn) (Schleicher and Schuell, Keene, NH) as described by Towbin et al. (4) or to PVDF-type membranes (ProBlott Membrane, Applied Biosystems, Foster City, CA) as described by Madsudarra (5).

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14. Electroblotting buffer: 25 mUTtis-base, 192 rnUglycine, 0.2% SDS, 20% (v/v) methanol, pH 6.5. 15. Immunoblot buffers: TBS (50 mMTris-HCl, pH 8.5, 150 mA4NaCl); OTBS (50 mMTris-HCl, pH 8.5, 150 m.&fNaCl, 3% ovalbumin) centrifuged at 14,000g for 20 min at 4OC. Stock solution (15%) of ovalbumin made by diluting 150 g of ovalbumin (Sigma) and 1 g of NaN, in 600 mL of distilled water. Stir overnight at room temperature, and bring the final volume up to 1 L with distilled water. Centrifuge the stock solution at 14,000g for 40 min at 4”C, collect the supernatant, and store aliquots at -20°C. 16. Stock: 3-cyclohexylamino- 1-propanesulfonic acid (CAPS) (Aldrich, Milwaukee, WI) buffer: 22.13 g CAPS in 1 L of distilled water titrated with NaOH to pH 11 .O and stored at 4°C. Electroblotting buffer (10 mMCAPS in 10% MeOH): mix 200 mL of CAPS stock solution, 200 mL of methanol, and 1600 mL of distilled water. 17. Ponceau S staining solution: 0.2% Ponceau S m 1% acetic acid. Dissolve 0.4 g of Ponceau S in 198 mL of distilled water and stir for 30 min. Add 2 mL of acetic acid to the mixture.

2.4. lmmunoprecipitation 18. Formalin-fixed Staphylococcus aureus cells (Gibco-BRL Life Technology, Gaithersburg, MD): Centrifuge cell suspension at 3000g in a mtcrocentrifuge for 10 mm. Resuspend the pellet in an equal volume of PBS containing 10% 2mercaptoethanol and 3% SDS, and boil for 30 min to reduce protein background. Afier centrifugation at 3000g for 10 min, wash the cells with NET buffer, centrifuge, and resuspend in NET buffer at a 10% (w/v) concentration. 19. Phosphate-buffered saline (PBS): 10 mMNaH2P0,, pH 7.2,0.9% NaCl, containing 10% (w/v) 2-mercaptoethanol and 3% (w/v) SDS. 20. NET buffer: 50 mMTris-HCl, pH 7.4, 150 mMNaC1,5 mA4EDTA, 0.02% NaN,, and 0.5% Nonidet P-40.

3. Methods 3.7. Llmlted Proteolytic Digestion 3.1. I, Trypsin 1. Incubate the protein in buffer A containing 25% glycerol with an equal volume of TPCK-treated trypsin (protease-to-protein molar ratio, 1:7) at 0°C. 2. Stop the reaction at specific time-points by mcubating an aliquot with trypsin inhibitor at a trypsin-to-trypsin inhibitor ratio of 1:lO (w/w) (see Note 1). 3, Incubate the aliquots at 0°C for 5 min (see Note 2), add electrophoresis sample buffer (3), and boil the samples for 5 min before analysis by SDS-PAGE.

3.1.2. Endoproteinase

Arg-C and Lys-C

1. Perform the proteolytic digestion with endoproteinase Arg-C at a protease-toprotein molar ratio of 1: 1.8 and with endoproteinase Lys-C at a protease-to-protein molar ratio of 1:40 tn buffer A at room temperature.

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2. At each time-point, remove an aliquot and stop the reaction by the addition of TLCK to a final concentration of 54 yg/mL. 3. After 5 min at O’C (see Note 2), add electrophoresis sample buffer (3) and boil the samples for 5 min before separation by SDS-PAGE (see Note 3).

31.3. S. aureus V8 Protease 1. Perform the proteolytic digestion in buffer A at a protease-to-protein molar ratio of I:32 for 2 h at room temperature. 2. Stop the reaction by the addition of TLCK and PMSF (final concentration 2.4 and 2.3 mg/mL, respectively). 3. After 5 min at O°C (see Note 2), add the electrophoresis sample buffer (3) and boil the samples for 5 min before separation by SDS-PAGE (see Note 4).

3.7.4. Chymotrypsin 1. Incubate the protein in buffer A with an equal vohune of TLCK-treated chymotrypsin (protease-to-protein molar ratio, 1:7.5) at 37V. 2. At each time-point, remove an aliquot of the mixture and stop the reaction by addition of PMSF to a final concentration of0.7 mh4. 3. After 5 min at 0°C (see Note 2), add electrophoresis sample buffer (3) and boil the samples for 5 min.

3.2. lmmunoblottlng 1. Separate proteolytic fragments by SDS-PAGE as described earlier. 2. Remove the gel from the electrophoresis cell and soak it in 100 mL of electroblotting buffer, as the nitrocellulose to be used. 3. Carry out the electroblot transfer overnight at 4OC and at a constant voltage (30 V) in a transblot tank (see Note 5). 4. After transfer, incubate the immunoblot in OTBS for 3 h at room temperature to block nonspecific binding, followed by OTBS containing the appropriate primary antibody for 3 h to overnight, with constant orbital shaking. 5. After two washes in TBS, one wash in TBS containing 0.1% Nonidet P-40, and one wash in TBS, all with vigorous shaking, incubate the immunoblot in OTBS containing [tZSI]Protein A (0.5 pCi/mL, specific activity, 30 pCi/pg) for 4 h at room temperature (see Note 6). 6. Rinse the immunoblot as described above, dry between Whatman filter paper, and then expose overnight to Kodak XAR-2 film with an intensifying screen at -7OOC.

3.3. Immunopreclpitaflon 1. Incubate protein samples (0.1 mg/mL) in 100 pL of buffer A containing 0.4% Lubrol PX and 0.1 mM DTT for 1 h at room temperature with the appropriate antibody or nonimmune rabbit sera at a 2: 1 molar ratio. 2. Add S. uureus cell suspension (400 yL), prepared as described above, and incubate the samples for an additional 1 h.

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3 Pellet the antigen-antibody-S aureus cell complex usmg a microcentrifuge (3OOOg, 10 min) and wash three times with NET buffer. 4. Elute the immunopreclpitated proteins from the S. aureus cells by resuspendmg the pellet in electrophoresls sample buffer (3) and boiling for 5 mm 5. Centrifuge the sample, and analyze an aliquot of the supernatant by SDS-PAGE.

3.4. Separation, Purification, and Analysis of Proteolytic Fragments 3.4.1. Separation of Proteolytic Fragments by SDS-PAGE and Electroblotting to PVDF-Type Membrane 1. Carry out the limited proteolytlc cleavage of the protein to completion as described above 2. Prepare SDS-polyacrylamlde gels, age them at 0°C for at least 24 h, prerun wtth 0.1 Wsodium thioglycolate (a scavenger of free radicals), for 4 h, and then load the samples (see Note 7) 3. Following electrophoresis, soak the gel in 100 mL of electroblottmg buffer (CAPS buffer) for 5 min. Meanwhile, soak the PVDF-type (ProBlott) membrane m 100% methanol for few seconds, followed by blotting buffer (CAPS buffer). 4. Carry out the electroblot transfer m a cell tank using chilled CAPS buffer at a constant voltage (50 V) and room temperature for 30 mm (see Note 8) 5 After transfer is complete, remove the membrane from the transblotting sandwich, and rinse with distilled water before staining. 6. Detect proteins on PVDF-type membrane with a conventtonal staining technique, such as Coomassle brilliant blue, Ponceau S, or Amido black (see Note 9). 7. After destaining, excise protein bands using a clean, sharp razor (see Note lo), and place them in the sequencing machme. 8. Perform amino acid sequence analysis of electroblotted peptides using a pulsed liquid protein sequencer (Applied Blosystems model 477A) according to manufacturing instructions (see Note 11).

3.4.2. HPLC Purification of Proteolytic Fragments 1. Purify the proteolytic fragments by reverse-phase HPLC (Brownlee, Aquapore RP-300, C8) using a linear acetonitrile gradient in 0.1% trlfluoracetic acid. 2. After purification, perform amino acid sequencing analysis using a pulsed liquldphase protein sequencer (Applied Biosystems model 477A) (see Note 11).

4. Notes 1. Alternatively, the reaction may be stopped by the addition of TLCK to a final concentration of 40-50 pg/mL. 2. Incubation of samples with protease inhibitors is an extremely Important step. In order to prevent extra cleavages of the protein, it is necessary to block the enzyme activity completely before the addition of electrophoresis sample buffer.

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3. When the proteolytic fragments are to be separated/purified by reverse-phase HPLC, stop the reaction by the addition of 1% trifluoroacetic acid/6M guanidine HCl (final concentration) before loading onto the HPLC column. 4. For immunoprecipitatton assays, digest the protein in buffer A containing 0.5 m&Y DTT as described above, and stop the reaction by the addition of TPCK, TLCK, and PMSF (final concentration of 5 mMeach). After 5 min at O°C, dilute the sample with buffer A containing 0.4% Lubrol PX (Sigma)/O. 1 nn’t4 DTT, and adjust the pH to 7.5. 5. Alternatively, the transfer may be carried out for a shorter time using a semidry electroblotter. An appropriate transfer buffer should be used, and transfer is usually complete m 30 mm. After transfer, the gel should be stained to check the presence of residual proteins and monitor the efficiency of transfer. In our experience, the use of a tank apparatus is preferable, since the proteolytic fragments can be produced in low amounts and quantitation is important. 6. As an alternative to 1251,antigen can be vrsualized directly on the transfer membrane using an enzyme-conjugated second antibody, directed against the IgG of the species from which the primary antibody is obtained. Using 1251-labeled protein A, we obtain clean and strong signals by autoradiography. Further information about mnnunoblotting techniques can be obtained from Tmnnons and Dunbar (6). 7 Enough protein should be loaded in a well so that at least 10 pmol of sample are m a single band on the blot. We routinely separate proteins and peptides using 0.75~mm-thick full-size gels. However, minigel systems are also suitable, For maximum separation of peptide bands, the Bromophenol blue dye should be allowed to run within 1 cm of the end of the gel. 8. The use of semidry electroblotters is also satisfactory. However, the transfer time should be determined for each protein or peptide, since the initial yield of protein and peptide sequence from electroblotted samples is affected by the efficiency of transfer. The electroblotting time varies with the gel thickness, molecular weight of the protein or peptide, and amperage of transfer. The transfer time can be judged empirically by staining the gel after electroblotting. 9. We use a 0.2% Ponceau S solutron in 1% acetic acid. Membranes are stained in the Ponceau S stammg solution with constant orbital shaking. Protein bands usually appear within 1 min, and membranes are destained by rmsing with distilled water. 10. Excess of membrane should be carefully trimmed away from the stained band to give a 2 x 4 mm segment. Stained bands can be stored dry in Eppendorf tubes at -2O’C. 11. Further information about the use of N-terminal sequence analysis of proteins and peptides can be obtained from Matsudaira (7).

References 1. Kohler, G. and Mtlstem, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256,495-497.

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2. Mazzoni, M. R., Malinski, J. A., and Hamm, H. E. (1991) Structural analysis of rod GTP-binding protein, G,. J Biol. Chem. 266, 14,072-14,08 1. 3. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. 4. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350-4354. 5. Matsudaira, P. (1989) Sequence from picomole quantities of proteins electroblottedontopolyvinylidenedifluoride membranes. J. Biol. Chem. 262,10,035-10,038. 6. Timmons, T. M. and Dunbar, B. S. (1990) Protein blotting and immunodetection. Methods Enzymol. 182,67!9-688. 7. Matsudaira, P. (1990) Limited N-terminal sequence analysis. Methods Enzymoi 182,602-613.

12 Epitope Mapping by Chemical Fragmentation Glenn E. Morris 1,

Introduction

The use of antigen fragments generated by specific chemical cleavage is a relatively simple “library” approach for epitope mapping in which overlapping fragments are screened with antibody on Western blots. It is widely applicable insofar as it is not restricted to recombinant antigens only, but the amino-acid sequence of the antigen must be known. It cannot be used for highly assembled epitopes, which will be affected by the denaturing conditions of cleavage and SDS-PAGE, as well as by fragmentation itself. Chemicals that cleave proteins at uncommon amino acids are used to produce fragments; cyanogen bromide (CNBr) cleaves C-terminal to methionine residues (I), nitrothiocyanobenzoic acid (NTCB) at Cys (21, iodosobenzoic acid (IBA) at Trp (31, and formic acid between Asp-Pro bonds (4). The fragments on Western blots can often be recognized unequivocally using M,.spredicted from the sequence. Assembly of the Western blot data into a map is illustrated by chemical cleavage results with creatine kinase (S-9). Figure 1 shows the limiting fragments expected for digestion of chick muscle creatine kinase (&fr 43,000) at Met, Cys, and Trp residues. These fragments are separated by SDS-PAGE, and the fragment reacting with any given MAb can be identified by Western blotting. The epitopes can be defined more precisely from the overlaps between the different fragments, as illustrated in Fig. 1 for three MAbs, CK-STAR, CK-2A7, and CK-JAC. The smallest fragment that the fourth MAb, CK-ART, recognizes is the 10.6~kDa C-terminal NTCB fragment E. It will also recognize the slightly larger 12-kDa C-terminal IBA fragment 2. In practice, chemical cleavage is rarely complete, and the usual result is a more complex pattern of partial digestion products on the Western blot. However, a “fingerprint,” or ladder, of bands on a Western blot can often be more From: Methods in Molecular Biology, vol. 68: Epltope Mapping Protocols Edited by: 0. E. Morris Humana Press Inc , Totowa, NJ

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122 87

IAIl

i

B

i

C

i

CK-JAC

CK-STARCK-2A7

I III

CNBr

(M)

IBA

(W)

NTCB

(C)

CK-ART

Fig. 1. Major predicted fragmentsowing to cleavageof creatmekinaseat 9 M, 4 W, or 3 C residuesand mapping of four MAbs from overlappmg fragments informative than a single product of complete digestion. This is illustrated in Fig. 2 by the mapping of MAbs against the muscular dystrophy protein, dystrophin, by NTCB cleavage (10). Figure 2 also shows that quite large antigens can be mapped successfully (a 224~kDa bacterial fusion protein in this case), provided some preliminary information 1savarlable; in this case, none of the MAbs would bind to a truncated protein (160-kDa), suggesting that all epitopes were in the last 64 l&a. The mapping results obtamed in this way were later confirmed by transposon mutagenesis (11) and use of epitope libraries of DNase fragments (12). The continuing popularity of chemical cleavage methods for epitope mapping is illustrated by refs. 23-19. 2. Materials 2.1. Digestions All chemicais are obtamable from Sigma (St. Louis, MO). 1 CNBr (toxic and may produce toxic gas; carry out all steps in an efficient fume hood): Dissolve the whole bottle to make a 100mg/mL stockin distilled water and store in a well-sealed bottle at -20°C.

2. NTCB (toxic): Preparea fresh 25 mg/mL solution in water each time. Add 5M NaOH in microliter amounts until the NTCB IS dissolved (goes completely clear).

3. IBA (toxic): Preparea fresh 5 mg/mL solution in IBA buffer each time. 4. DTE (dithioerythritol): A 100~mMsolution in water should be freshly prepared. 5. Sodium

dodecyl

sulfate (SDS).

20% (w/v)

stock solution

m water.

6. 2-mercaptoethanol(2-ME). 7. Sucrose.

8. Bromophenol blue. 9. Formic, acetic,and hydrochloric acids (analytical-grade). 10. p-Cresol (toxic andvolatile; handleit, andall solutionscontainingit, in afumehood).

Chemical Fragmentation +--

123

224kD

.

F

.

P-d e---m

E

D

,

c

,B,

A

16&D NTCB CD BC AB

DEF DE

3 43

26 18

11113

Fig. 2. Mapping of 18 MAbs against dystrophin by NTCB cleavage of a 224-kDa fusion protein of P-galactosidase (116 kDa) with a 108~kDa dystrophin fragment. Failure to bind to a shorter fusion protein (160 kDa) suggested binding to the A, B, C, or D regions, each of which should give a unique “fingerprint” in the 6-40 kDa region of a Western blot. Experimentally, 15 MAbs gave the pattern for bindmg to “C” and 3 MAbs gave the “D” pattern (10). 11. Sephadex G- 15 (Pharmacia). 12. Sephadex buffer (prepare freshly): 8A4 urea (ultrapure) in 125 mM Tris-HCl, pH 6.8. 13. NTCB buffer 1. 5Mguanidine HCl (analytical grade) in 200 mA4 Tris-acetate, pH 8.0 (pH is critical). 14. NTCB buffer 2: 8M urea in 200 mM Tris-acetate, pH 9.0 (pH critical). 15. IBA buffer: 4M guanidme HC1/80% acetic acid/2% p-cresol. The p-cresol prevents IBA cleavage at Tyr residues. 16. For gel filtration: Glass columns (12 x 1 cm, approx with 10 cm gel height) with glass sinters at the bottom and no “dead volume” below the smter.

2.2. SDS-PAGE and Western Blotting Materials are described in detail in Chapter 10, vol. 24 of this series. 1. Laemmli buffers (20) for SDS-PAGE are only satisfactory for fragments of 6 kDa

or greater,and aTns-Tricine system(22) is recommendedif resolution of smaller peptides is necessary.

724

Morris

2. Low M, range prestained protein markers are essential for identification of cleavage fragments on blots. These are available from Life Technologies (Gaithersburg, MD) or Novex (SeeBlue and Multimark, San Diego, CA). 3. Nitrocellulose (e.g., Schleicher and Schuell BA85) is a relatively inexpensive, if rather fragile, medium for blots. A 10 x 10 cm sheet can be cut into four to fit most popular types of small PAGE apparatus (e.g., Bio-Rad, Biometra).

3. Method 3.1. Digestions The protocols are for 0.5 mg of antigen in 0.5 mL reaction volume, but, if the antigen is in short supply, it may be possible to scale down both weight and volume by using a smaller Sephadex column or replacing it with a microspin column (check to ensure that the same separation 1s obtained).

3.7.7. CNBr for Cleavage of Methionine Residues All steps are performed in a fume hood. 1. Take 0.5 mg of protein antigen in 125 PL or less of distilled water (or buffer) in a glass vial and add 350 pL of formic acid. 2. Add 5 p,L of CNBr stock solution and make up to 0.5 mL with distilled water (see Note 1). Mix and leave for 18 h at 20°C (see Note 2) 3. Load onto the column of Sephadex G-15 swollen and pre-equilibrated in Sephadex buffer and precalibrated to identify the “excluded volume” fractions (see Note 3). Elute with Sephadex buffer, and collect l-n& fractions. 4. To each “excluded volume” fraction, add 50 yL of 20% SDS, 50 pL of 2-mercaptoethanol, 10 pL of Bromophenol blue, and boil for 2 min for SDS-PAGE.

3.1.2. NTCB for Cleavage at Cysteine Residues 1. 2. 3. 4.

Dissolve 0.5 mg of antigen in 0.5 mL of NTCB buffer 1, pH 8.0 (see Note 4) Add 5 pL of 100 mMDTE and leave for 30 min at 20-25°C. Add 30 pL of 25 mg/mL NTCB and incubate at 37°C for 15 min. Load immediately onto a Sephadex G- 15 column equilibrated with NTCB buffer 2, pH 9.0 and elute with the same buffer collecting 1-mL fractions (see Note 5). 5. Incubate “excluded volume” column fractions (see Note 3) at 37OC for 16 h (see Note 6). 6. Adjust fractions to pH 6.8 using a predetermined amount of 5MHCl. Add 50 pL of 20% SDS, 50 pL of 2-ME, 10 pL of bromophenol blue, 100 mg of sucrose, and boil for 2 min for SDS-PAGE.

3.1.3. ISA for Trp 1. Dissolve 0.5 mg of antigen in 450 pL of IBA buffer and add 50 yL of 5 mg/mL IBA in the same buffer. Mix and leave at 2&25”C for 24 h. 2. Follow steps 3 and 4 of Section 3.1.1.

Chemical Fragmentation

125

3. Optional: Add solid DTE (700 mMfina1 cont.) to each column fraction and incubate at 37’C for 24 h or longer (see Note 7). If this option is followed, the 2-ME may be omitted from the previous step.

3.1.4. Formic Acid for Asp-Pro Bonds 1. Dissolve 1 mg of antigen in 0.5 mL of 75% formtc acid, and incubate for 24-48 h at 37OC (see Note 8). 2. Follow steps 3 and 4 of Section 3.1.1.

3.2. SDS-PAGE and Western Blotting Methods are described in Chapter 10, vol. 24 of this series. Twenty percent of acrylamide with 0.5% bis-acrylamide is usually appropriate for the separating gel (see Note 9). 4. Notes 1. The CNBr should be in 30-fold molar excess over the Met residues in the antigen; this works out to be about 1: 1 (w/w) for a typical protein with 1 Met/3040 residues 2. CNBr cleavage is usually almost complete under these conditions, except that Met-Thr bonds are not noticeably cleaved at all. 3. The gel-filtration step removes formic acid and excess CNBr, and gets the antlgen fragments into a buffer compatible with SDS-PAGE. An important safety feature is that CNBr never leaves the fume hood and can be destroyed later by elution into bleach. The buffer described is for the Laemmli PAGE system (20) and should be replaced if other PAGE systems are used. The urea prevents any possible precipitation on the column. G- 15 allows fragments of M, > 2000 to pass unretarded in the excluded volume; the column should be precalibrated with blue dextran to identify the excluded volume fractions (usually one or two main fractions). Since the column takes only a few minutes to run, elution buffer can be applied and fractions collected manually. 4. pH is critical in this procedure. If your antigen sample contains buffer salts, make sure the final pH 1sunaffected. 5. The gel-filtration step has a twofold purpose: first, to replace GdnHCl (which is not compatible with SDS-PAGE) with urea and, second, to raise the pH to 9.0 for the subsequent cleavage step. 6. Cleavage is usually incomplete with NTCB, and a complete spectrum ofpartial diges-

tion productsis produced.It is necessaryto work out a theoretical“fingerprint” for each epitope location and compare them with the experimental result (see Fig. 1). 7. IBA oxidizes Met side-chains to the sulfoxide, which makes it difficult to map epltopes that require Met by this method. The problem can be solved by the prolonged incubation with DTE described here.

8. Although rather similar conditions areusedfor CNBr cleavage(70% formic acid instead of 75%), relatively little Asp-Pro cleavage appears to occur at the lower

temperature(20 insteadof 37°C).

126

Morris

9. The author recommends diffusion blottmg, rather than electrophoretlc blotting, for the small fragments usually produced by chemical cleavage. There are only two essential differences: (a) you can put nitrocellulose on both sides of the gel to generate two “mirror” blots (useful for mapping more than one MAb) and (b) you do not switch on the electric current!

Acknowledgments This work was supported by grants from the Muscular Dystrophy Great Britain and Northern Ireland and from HEFC (Wales) DevR.

Group of

References 1. Croft, L. R. (1980) Handbook of Protein Sequence Analyszs Wiley-Interscience, Chichester, UK. 2. Fontana, A., Dalzoppo, D., Grandi, C., and Zamboni, M. (1983) Cleavage at tryptophan with ortho-iodosobenzolc acid. Methods Enzymol. 91,3 1l-3 18. 3 Stark, G. A. (1977) Cleavage at cysteine by nitro-thio-cyano-benzoic acid Methods Enzymol. 47, 129-132 4. Sonderegger, P., Jaussi, R., Gehring, H., Brunschweller, K., and Christen, P. (1982) Peptide mapping of protein bands from polyacrylamide gel electrophoresls by chemical cleavage in gel pieces and reelectrophoresis. Anal. Biochem. 122,298-301. 5. Morris, G. E., Frost, L. C., Newport, P. A., and Hudson, N. (1987) Monoclonal antibody studies of creatine kinase. Antibody-binding sites m the N-terminal region of creatine kinase and effects of antibody on enzyme refolding. Biochem J 248,53-59. 6. Morris, G. E. (1989) Monoclonal

7.

8.

9.

10.

11.

12.

antibody studies of creatine kinase. The ART epitope: evidence for an mtermedlate in protein folding. Bzochem. J. 257,46 l-469. Morris, G. E. and Cartwright, A. J. (1990) Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatme kinase Biochlm Biophys. Acta 1039,3 18-322. Nguyen thi Man, Cartwright, A. J , Osborne, M., and Morrq G. E. (1991) Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies. Biochlm. Biophys. Acta 1076,245-25 1. Morris, G. E. and Nguyen thi Man (1992) Changes at the N-terminus of human brain creatine kmase during a transition between inactive folding intermediate and active enzyme. Blochlm. Bzophys. Acta 1120,233-238. Nguyen thi Man, Cartwright, A. J., Morris, G. E., Love, D. R., Bloomfield, J. R., and Davies, K. E. (1990) Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin. FEBS Lett 262,237-240. Sedgwlck, S. G., Nguyen thl Man, Ellis, J. M., Crowne, H., and Morris, G. E. (199 1) Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. Nucleic Aczds Res. 19,5889-5894. Nguyen thi Man and Morris, G. E. (1993) Use of epltope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy Am. J Hum. Genet. 52, 1057-1066

Chemical Fragmentation

127

13. Lin, J. J. C., Davisnanthakumar, E. J., Jin, J. P., Lourim, D., Novy, R. E., and Lin, J. L. C. (1991) Epttope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actintropomyosin filaments. Cell Motil Cytoskel. 20,95-108. 14. Vanuem, T. J. F., Swarts, H. G. P., and Depont, J. J. H. H. M. (1991) Determination of the epitope for the mhibitory monoclonal antibody 5-B6 on the catalytic subunit of gastric Mg2+-dependent H+-transporting and K+-stimulated ATPase. Biochem J 280,243-248. 15. Malouf, N. N., McMahon, D., Oakeley, A. E., and Anderson, P. A. W. (1992) A cardiac tropomn-T epitope conserved across phyla. J Biol Chem. 267,9269-9274. 16. Edwards, R. J., Sesardic, D., Murray, B. P., Singleton, A. M., Davies, D. S., and Boobis, A. R. (1992) Identification of the epitope of a monoclonal antibody which binds to several cytochromes-P450 in the CYPIA subfamily. B&hem Pharmacol. 43,1737-1746. 17. Wasserman, L., Doctor, B. P., Gentry, M. K., and Taylor, P. (1993) Epnope mapping of form-specific and nonspecific antibodies to acetylcholmesterase. J. Neurochem. 61,2124-2132. 18. Morris, C. A , Underwood, P. A., Bean, P. A., Sheehan, M., and Charlesworth, J. A. (1994) Relative topography of biologically active domains of human vrtronectin-evidence from monoclonal antibody epitope and denaturation studres. J. Blol Chem 269,23,845-23,852. 19. Rawling, E. G., Martin, N. L., and Hancock, R. E. W. (1995) Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF. Infect Immun. 63,30-42. 20. Laemmli, U. K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227,680-685. 2 1. Shagger, H. and von Jaggow, G. (1987) Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separatron of proteins in the range 1 to 100 kDa. Anal. Biochem 166,368--379.

13 Probing Antibody-Antigen by Mass Spectrometry

Interactions

Yingming Zhao and Brian T. Chait 1. Introduction Techniques that have been used for antigenic site mapping of linear epitopes in proteins include binding assays of protein components produced by synthetic chemistry (1,2) or by recombinant gene expression (34). More recently, epitope localization has been achieved through the use of synthetic and bacteriophage peptide libraries (5-9). Although effective, these methods can be costly and time-consuming. A different approach to antigenic site mapping has been reported by Suckau (IO), who compared the pattern of proteolytic digestion of free peptide antigen with the pattern produced from the antigen bound to an antibody. Alternatively, these workers subjected the peptide to proteolytic digestion and identified products that bound specifically to the immobilized antibody. In both cases,the peptides of interest were identified by 252Cfplasma desorption mass spectrometry. Recently, another approach, termed affinitydirected mass spectrometry (2 Z) has been reported by us and others (12-16) for probing antigerrantibody interactions. The basis of affinity-directed mass spectrometry is the use of direct molecular mass readout from the immune complex to determine the specific component of the protem antigen that interacts with the antibody. The strategy (12) is shown in Fig. 1. In the first step, a set of peptide fragments is produced by enzymatic digestion of the intact protein. Proteaseswith known specificity are used so that the sites of cleavage can be predicted and the resulting peptides readily identified by accurate mass measurement by matrix-assisted laser desorption mass spectrometry (MALDI-MS). This step provides an easy method for generating a set of peptide fragments that span the sequence of the protein. Parallel digestion of the protein by two or more proteolytic enzymes with difFrom: Methods In Molecukr Bfology, vol 66. Epitope Mappmg Protocols Edited by G E Morris Humana Press Inc , Totowa, NJ

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Zhao and Chait

730 Protein Antigen

Proteolytlc Digestion Step I

Imrnunoprecipitation Step II

I II

UnboundPeptldes7 WashedAway

Step III

+ Matrix-AsslstedLaserDesorphon MassSpectrometricAnalysis

Fig. 1. Strategy for defining binding sites in a protein that interacts specifically with an MAb (see text for details) The antibody is represented by the Y-shaped symbol and protein G plus protein A agarose, by two circles connected by a line. The molecular mass of the peptide in the immune complex is determined by matrix-assisted laser desorption time-of-flight mass spectrometry (Z 7). Under conditions of the mass spectrometric analysis, the epitope-containing peptide dissociates from the antibody and protein G plus protein A agarose and is measured directly as the isolated peptide.

ferent specificities enables rapid production of overlapping sets of proteinspanning peptides. In the second step, the component

peptides that contain the

binding region of interest are affinity-selected by the immobilized antibody (epitope-containing peptides bind to the antibody and are retained, whereas the remainder are washed away). In the third step, the masses of the affinityselected peptide fragments are accurately determined directly from the immune complex by MALDI-MS (see Note 1). Peptides that are specifically bound to the antibody are identified from their accurately measured molecular masses and a knowledge of the potential digestion sites in the protein antigen. The region

of the protein

involved

in antibody

binding

is deduced

from the

sequences of the peptides that are affinity-selected by the antibody. Comparison of these sequences identities a region of common sequence that contains a dominant component of the binding epitope (Fig. 2). The method uses affinity

Mass Spectrometry

731

A I

1

1 Kl

K2

1

1

K3

K4

I K5

Before Immunopreclpitation K3

KS K4

After Immunoprecipitation

Mass (dalton)

Fig. 2. A schematic illustration of affinity-directed mass spectrometry for epitope mapping. (A) The linear sequence of a hypothetical antigen protein is represented by a solid line. The antigen protein produces seven peptide fragments after digestion by a protease, whose digestion specificity is indicated by arrows above the lines. Mass spectrometric readout of digested antigen protein before (B) and after (C) immunoprecipitation. After immunoprecipitation, only two peptide fragments (K2 and K2 + K3) are identified in the immunoprecipitated complex, which indicated that the antigenic site is located inside the region K2. The position of each peak in the X-axis of the diagram represents the molecular mass of each component peptide. The height of the peak on the Y-axis represents the relative intensity of each peptide ion species, which depends on the concentration and mass spectrometric ion response of the peptide. purification in combination with mass spectrometry, and is termed affinitydirected mass spectrometry. The steps outlined above provide low-resolution definition of the binding epitope. The precise boundaries of the binding epitope are determined by affinity-directed mass spectrometric analysis (13,16) of sets of synthetic peptide ladders that span the binding region (Fig. 3). Proteolytic digestion and affinity-directed mass spectrometry can be used to determine the approximate locatton of a continuous component of a binding epitope rapidly within a protein ligand. If it is desired to explore the binding of several antibodies against a single protein, the irnmunopreclpitation step can

Zhao and Chait

132 Peptide

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 DAATGAF

PP

G

I WREP . . .

..I

. :

...*.

..a . . . . . ... . .. .. .. . :

N-terminal ladder

Leader Sequence E-NH,-caproyl-RLKLKAR

s-0

. . . -0 . . . . ..o . . . . . . . ..a

C-terminal ladder . . . . . . . . . . “0 . . . . . . . . . . ..“.o . . . . . . . . . . . . . ..**.o ..“‘................O

0Pf-J 0

Fig. 3. Precise determination of boundary residues of antigenic epitope by affinitydirected mass spectrometric analysis of synthetic ladder peptides. The scheme lllustrates design and analysis of N-terminal ladder (truncated from the N-terminal) and C-terminal ladder (truncated from the C-terminal) of a hypothetical antigenic peptlde (DAATGAFPPGIWREP) The length of the line is proportional to the number of amino acid residues in the peptide. The elliptical symbol designates the leader peptide, E-NHZcaproyl-RLKLKAR. After immunoprecipltatlon of the N- and C-terminal ladder peptides with the MAb, only certain components of the ladder mixtures are identified to bind to the antibody. The bold lines designate peptides that bind to the antibody and

dashedlines peptidesthat do not bind to the antibody. Inspectionof binding properties of peptide laddersshowsthat T“ andPgare theboundaryresiduesof the epitope. Therefore, residues T4GAFPPg are required for tight binding to the antibody.

be conveniently carried out in parallel with the set of antibodies. The rapidity of the mass spectrometric analysis (typically only a few minutes/spectrum) allows the approximate location to be determined for several such epitopes in a single day (12). Synthesis and affinity-directed mass spectrometric analysis of peptide ladders containing up to 20 amino acids can be achieved in little more time (l-2 d) than is required to make a single peptide (13,16). Hence, the present approach allows the precise definition of a linear binding epitope for a specific antibody to a short stretch of protein (typically, 6-20 residues,

133 depending on the available proteolytic digestion sites) in a single day and more accurate definition within 1 wk. For appropriate applications, the procedure should be faster than or competitive with the current approaches. It differs from library-based approaches in that (1) only the natural sequence is explored in the search for a binding epitope and (2) long epitopes can be investigated with little additional effort compared to short epitopes. We have successfully applied affinity-directed mass spectrometry for epitope mapping to several monoclonal antibodies (MAbs) with binding aftinities in the range 10-6-10-gM, including antimelittin MAb #83144 (12), antiglucagon like peptide-l 7-37 MAb #26.1 (12), antihuman basic fibroblast growth factor MAb #l 1.1 (13,16), and anti-Ad-2 MAb Dave- 1 (unpublished results). The results of these experiments demonstrate that the present method should have quite general applicability to the definition of linear epitopes.

2. Materials 1. Sequencing-grade endoproteases (e.g.,endoproteaseLys C, endoproteaaeAsp-N,and trypsin) and proteaseinhibitor PefablocSC(Boehringer Mannhelm Biochemical, Indianapolis, IN). 2. Protein G plus/A agarose(OncogeneScience,Uniondale, NY; seeNote 2). 3. a-cyano-4-hydroxycinnamic acid (Sigma, St.Louis, MO). 4. TSO buffer: 75 mMTris-HCI, pH 8.0,200 mMNaC1, 0.5% n-octylglucoside. 5. TSM buffer: 10mA4Tris-HCl, pH 8,0,200 mMNaCl,5 mM/3-mercaptoethanol. 6. MAbs were purified by protein G plus protein A agarose chromatography (OncogeneScience). 7. Digestion buffers: 50 mM sodium phosphate,pH 8.0, for endoproteaseAsp-N; 50 m/l4 Tris-HCl, 1 mM EDTA, pH 8.0, for endoproteaseLys-C. 8. MALDI-MS was carried out in our laboratory with a laser desorption time-offlight instrument constructedat the Rockefeller University (17,18). Any commercial instrumenthaving similar specificationscan also be used.

3. Method 3.1. Digestion of Proteins 1. Dissolve a 1:30 ratio (w/w) of protease and antigen protein in an appropriate buffer using a protein concentrationbetween 10and 20 uA4. 2. Incubate the resulting solution at 37OCfor 2 h. 3. Terminatethe digestionby adding l/l 0 vol of 10mMPefablock SCsolution (25’C for 10 min), followed by heating at 90°C for 15min to inactivate the protease. 3.2. lmmunoprecipitation (19) 1. Mix an MAb (2-10 pg) and digestedprotein (20-100 pmol) in TSO buffer (see Note 3). 2. After 2 h of incubation at 4°C with gentle stirring, add 2-3 yL protein G plus/A agaroseto the solution, andincubate for another 0.5-l h at 4°C. Collect the aga-

Zhao and Chait

134

rose beads by carefully aspirating the supernatant after centrifuging the solution Wash the beads three times with TSO buffer and then three times with TSM buffer (see Note 4).

3.3. Mass Spectrometry 1. To the washed beads, add 4 uL of a saturated matrix solution of a-cyano4-hydroxycinnamic acid (see Note 5) in 1% aqueous TFA:ACN (2.1) together with an appropriate amount of standard peptide and mix. 2. Measure the molecular masses of the binding peptides: Load l-2 uL of the matrix-agarose mixture onto the probe tip (see Note 6) and dry at room temperature with a stream of air The mass spectra are collected by adding individual spectra obtained from a large number (50-200) of laser shots to improve the statistics. Spectra can be calibrated either externally or internally usmg standard peptides.

3.4. Synthesis of Peptide Ladders Solid-phase peptide synthesis 1scarried out manually as described (20). 1 To synthesize the N-terminal peptide ladder pool, remove an equal portion of peptide resin from the reaction vessel after the addition of each amino acid residue. 2. Mix the resulting peptide resin samples, deprotect, and then subject to HF cleavage The resultmg peptides are used for analysis without further purification 3 To synthesize the C-terminal ladder peptides, add an equal portion of resin containing the leader peptide, a-NH+aproyl-RLKLKAR (see Note 7) after each cycle of the synthesis The mixed peptide-resin product contams peptides of all possible lengths from the C-terminal amino acid residue. 4 Deprotect and cleave with HF to produce the C-terminal peptide ladder pool.

3.5. Affinity-Directed of Peptide Ladders

Mass Spectrometric

Analysis

1. Immunoprecipitate the N-terminal ladder peptide pool with the MAb (Section 2.). 2. Identify binding peptides by mass spectrometry from the mununoprecipitated complex (Section 3.). The profile of binding and nonbinding peptides provides enough information to define the N-terminal boundary residue of the epitope.

3. The C-terminal boundary residue of the epitope is determined similarly (Fig. 3).

4. Notes 1. MALDI-MS is an analytical tool for measuring the molecular masses of peptides and proteins (17,18). The technique allows the accurate (better than 0. l%), rapid (minutes), and sensitive (~1 pmol) determination of the molecular masses of com-

ponentsof complex mixtures of peptideswithout prior separation.MALDI-MS is finding wide use for the rapid characterization of proteins, and especially the definition of posttranslational modifications and mutations.

Mass Spectrometry

135

2. Protein A agarose and protein G agarose have different binding affinities to immunoglobulins, depending on species and subclasses of the antibodies (21). Protein G plus protein A agarose provides superior performance for binding of most immunoglobulins. 3. A detergent, n-octylglucoside is used in the immunoprecipitation buffer (TSO) to reduce nonspecific binding of peptides to the antibody. This is important because nonspecific binding can be a serious problem when mapping antigenic sites of large proteins. SDS and Triton should not be used in the immunoprecipitation, because these detergents suppress the mass spectrometric response. 4. When peaks of nonspectfic binding peptides appear m mass spectrum, they can be identified by either comparing profiles of antibody-binding peptides among different digestions or using harsher conditions (more detergents) to wash away nonspecific binding peptides in the immunoprecipitation step. 5. The matrix compound, a-cyano-4-hydroxycinnamic acid (22) is used to asstst conversion of peptides from the solid phase into the gas phase. In addition, the matrix facilitates ionization of the desorbed peptides (18). 6. The antibody-antigen complex together with the protein G plus protein A agarose 1s loaded directly onto the mass spectrometer probe. The binding peptides will dissociate from the immune complex during mixmg with the acidified matrix solution and/or during mass spectrometric analysis 7. Basic amino acid residues, such as arginine, lysine, and histidine, in a peptide increase the mass spectrometric response of the peptide. To ensure good and uniform mass spectrometric response for the different components of the peptide ladders, a basic leader peptide a-NH.+aproyl-RLKLKAR is incorporated at the C-terminal of each peptide. The spacer residue a-NH* caproic acid is included in the ladder to prevent possible ambiguities that could arise at the junction between the epttope and the ladder sequence.

References 1. Wang, Z. and Laursen, R. A. (1992) Multiple peptide synthesis on polypropylene membranes for rapid screening of bioactive peptides. Pept. Res. 5,275-280. 2. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single ammo acid. Proc. Nat1 Acad. Sci. USA 81,3998-4002. 3. Dias, P., Parham, D. M., Shapiro, D. N., Tapscott, S. J., and Houghton, P. J. (1992) Monoclonal antibodies to the myogemc regulatory protein myoD 1: epitope mapping and diagnostic utility. Cancer Res. 52,643 L-6439. 4. Chen, J., Marechal, V., and Levine, A. J. (1993) Mapping of the p53 and mdm-2 interaction domains. Mol. Cell Biol. 13,4 107-4114 5. Devlin, J. J., Panganiban, L. C., and Devlin, P. E. (1990) Random peptide libraries: a source of specific protein binding molecules. Science 249,404406. 6. Lam, K. T., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierski, W. M., and Knapp, R. J. (1991) A new type of synthetic peptide library for identifying ligandbmdmg activity. Nature 354, 82-84.

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7. Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T., and Cuervo, J. H. (1991) Generation anduse of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354, 84-86. 8. Scott, J. K. and Smith, G. P. (1990) Searching for peptide ligands with an epitope library. Science 249,386-390. 9. Cwirl, S. E., Peters, E. A., Barrett, R. W., and Dower, W. J. (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc. Nutl. Acad. Set. USA 87,6378-6382. 10. Suckau, D., Kohl, J., Karwath, G., Schneider, K., Casaretto, M., Bitter-Suermann, D., and Przybylski, M. (1990) Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. Proc. Nat1 Acad. Ski. USA 87,9848-9852. 11. Hutchens, T. W. and Yip, T. (1993) New desorption strategies for the mass spectrometric analysis of macromolecules. Rapid Commun. Mass Spectrom. 7, 576-580. 12. Zhao, Y. and Chait, B. T. (1995) Protein epitope mapping by mass spectrometty. Anal. Chem. 66,3723-3726. 13. Zhao, Y., Kent, S. B. H., and Chait, B. T. (1994) Rapid anttgenic sate identification by mass spectrometry, The 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL. 14. Papac, D. I., Hyoes, J., and Tomer, K. B. (1994) Direct analysis of affinity-bound analyses by MALDI/TOF MS. Anal. Chem. 66,2609-26 13. 15. Nelson, R. W., Krone, J. R., Bieber, A. L., and Williams, P. (1995) Mass spectrometric immunoassay. Anal Chem 67,1153-l 158. 16. Zhao, Y., Muir, T. M., Kent, S. B. H , Tischer, E., Scardina, J. M., and Chait, B. T Mapping protein-protein interactions by affinity-directed mass spectrometry. Proc. Nat1 Acad. Sci. USA, in press. 17. Beavis, R. C. and Chait, B. T. (1990) Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc. Natl. Acad. Sci. USA 87,6873-6877. 18. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Matrix-assisted laser desorptiotiionization mass spectrometry of biopolymers. Anal. Chem 63, 1193A-1203A. 19. Harlow, E. and Lane, D. (1988) Immunoprecipitation, in Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 42 l470. 20. Schnolzer, M., Alewood, P., Jones, A., Alewood, D., and Kent, S. B, H. (1992) In situ neutralization in Boc-chenustry solid phase peptide synthesis. Znt. J. Pept. Protein Res. 40, 180. 2 1. Harlow, E. and Lane, D. (1988) Immunoprecipitation, in Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 6% 623. 22. Beavis, R. C., Chaudhary, T., and Chait, B. T. (1992) a-Cyano-Chydroxycinnamic acid as a matrix for matrix-assisted laser desorption mass spectrometry. Org. Mass Spectrom.

27, 156.

14 Epitope Mapping Using Multipin Peptide Synthesis Stuart J. Rodda, N. Joe Maeli, and Gordon Tribblck 1. Introduction Multiple peptide synthesis gives accessto a set of reagents that permit thorough answers to such questions as: Where are all the linear epitopes in this protein? How long is the critical part of each epitope? Which epitopes are commonly recognized and which are rarely recognized? What are the affinities for each epitope? Which amino acids in the epitope are in contact with the antibody/TCR/MHC molecule? What variants of the epitope are still recognized by the antibody/TCR/MHC molecule? Which peptides are antagonistic peptides for this epitope? Of the wide variety of methods for multiple peptide synthesis now available, the Multipin method was the first (I). From its beginning as a method for testing peptides on the same surface on which they had been synthesized (2,3), it was developed into a method for obtaining solution-phase peptides using a very mild method of cleavage that produced peptides in physiological solution ready for bioassay (4). Further development of the chemistry of grafting, synthesis, and cleavage (S-7) and scaling up of the pin size (8) have allowed a high-quality peptide on the order of 5 pmol (approx 5 mg of a decamer) to be produced from each pin, making it feasible to screen thousands of peptides in assaysrequiring high peptide concentrations. Peptides made using this technology can be applied to the search for and understanding of both linear antibody-defined epitopes (B-cell epitopes) (9, IO) and of helper and cytotoxic T-cell epitopes (I 1,22). For linear B-cell epitopes in particular, a decision must be made whether solid-phase or solution-phase peptides are to be used. Use of peptides permanently attached to the solidphase on which they were synthesized has the advantages of simplicity and sensitivity, but is subject to uncertainty about the quality of the peptides being From* Methods in Molecular Biology, vol. 66: Epttope Mapplng Protocols Edited by* 0. E. Morris Humana Press Inc , Totowa, NJ

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used, and to artifact arising from the high density of the peptide and possible interactions with the support matrix (13). For antibody-defined epitope mapping, there are many advantages in either using solution-phase peptide as a competitor of antigen-antibody binding, or in using a peptide with a “tag,” such as a biotin group, to allow capture of the antibody-antigen complex onto a plastic or gel surface (24). This removes the uncertainty about whether or not a particular peptide will passively coat onto a plastic surface. Study of T-cell epitopes with pepttdes requires solution-phase peptide, because the ternary complex among MHC molecule, peptide, and TCR is not easily formed with peptide bound on a solid-phase. For T-cell epitopes, a critical additional consideration is whether further peptide processing will occur in the assay system, since cytotoxic T-cells require a peptide of explicit length (generally an 8- or 9-mer) presented on MHC class I (25), whereas helper Tcells are less stringent in then requirements and can recognize peptides of a variety of lengths, presented in the context of the open-ended MHC class II peptide groove (I 6). This chapter deals with design of peptide sets, the Multipin synthesis of peptides, and with direct binding assays (focused on antibody binding). Description of assaysusing peptides in T-cell epitope mapping is beyond the scope of this chapter (see Chapter 30). 2. Materials 1 Synthesis kits: A Multipin kit from Chiron Mimotopes (Melbourne, Australia) or their distributors is selected accordmg to whether the peptides are: a. To remain pin-bound (MULTIPIN NCP noncleavable kit); b. To be cleaved by the mild DKP-forming reaction (MULTIPIN DKP kit) to produce peptides that all have a C-terminal cychc lysyl-prolyl dipeptide, known as a dtketopiperazme (DKP) group; c. To be cleaved by aqueous base (MULTIPIN GAP kit) to give a C-terminal glycme acid or amide; d. To be cleaved with trlfluoracetic acid (TFA) (MULTIPIN MPS kit) to give the “native” C-terminal ending of choice, i.e., any amino acid; or e. A combination of a, b, and c above (MULTIPIN 5-M-l B&T CELL kit). Each kit includes a manual, software to calculate reagent amounts and to guide the amino acid couplings in each synthesis cycle, reaction trays, wash baths, and control peptide/antibody. An IBM-compatible or Macintosh personal computer is required to run the software. Alternatively, peptides made by any of these methods can be purchased as complete ready-to-use sets. 2. Solvents: High-quality (A.R. or better) dimethylformamide (DMF) and methanol are required as reaction solvent and wash solvent, respectively. The DMF may need to be distilled or purified to reduce the amine level before use (see Note 1). Acetonitrile (HPLC-grade) may be used durmg peptide extraction from pins or to

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redissolve peptide prior to use in assays or for subsequent purification. Dtmethylsulfoxide (DMSO, A.R -grade) is also very useful for redissolving peptide sets prior to dilution into aqueous solutions. Reagents: Sets of Fmoc-protected amino acids are required. If all 20 geneticallycoded amino acids are to be used, sets can be purchased from Chiron Mimotopes or tts distributors. Individual Fmoc-protected amino acids can be obtained from many suppliers (Bachem, Novabiochem, Sigma), but care must be exercised to ensure that the side-chain-protecting groups are compatible with the reagent used for activation, and can be removed by the final deprotectionlcleavage reagent. Pipendine, a strong organic base, 1srequired for the repetitive Fmoc-deprotection step, which is a feature of each amino acid coupling cycle. Triethylamine (TEA) is needed if peptrdes are to be acetylated with acetic anhydride. An activation agent must be used to activate the Fmoc-protected amino acids before coupling can take place. A carbodiimide, such as drcyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC), can be used, or alternatively one of the more potent reagents, such as benzotriazol- 1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) or benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (8). A catalyst, such as I-hydroxybenzotriazole (HOBt), IS highly recommended Side-chain deprotection and cleavage usually requires the use of TFA, a very strong and corrosive acid. For large-scale use, TFA can be obtained in bulk from Halocarbon Inc. (North Augusta, SC). Scavengers amsole, thioanisole, and ethanedithiol can be added to the TFA. Ether and petroleum ether can be used to wash the crude peptide. The above reagents can be obtained from Novabiochem, Aldrich, Merck, Fluka Sigma, and so on Bromophenol blue (ACS indtcator standard or better), 10 m&f in pure DMF, can be diluted 1:200 into activated amino acid solutions to monitor the completeness of coupling reactions. For preparing ELISA plates for use with biotinylated peptides, streptavidin (Sigma Cat. No. S-4762), Tween-20 (Sigma Cat. No. P1379), and sodium caseinate (USB Cat. No. 12865) are needed. Alternatively, streptavidin-coated plates are available from several sources, including Chnon Mimotopes. Antispecies enzyme conjugates for ELISA and corresponding substrate can be the ones commonly used in your lab provided they are sensitive enough. For example, KPL (Gaithersburg, MD) goat antispecies H + L chain horseradish peroxidase conjugates can be used at 0.1 ug/mL. ABTWhydrogen peroxide substrate is made with 0.5 mg/mL ABTS (Boehringer Mannheim Cat. No. 122 661) and 0.01% hydrogen peroxide (lab reagent) in O.lM citrate/phosphate, pH 4.0 Phosphate-buffered saline (PBS): O.OlM sodium phosphate, pH 7 2, 0.15M sodium chloride. TPBS: 0.1% Tween-20 in PBS. TPBSA: TPBS containing 0.1% sodium azide.

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17. Plasticware: Reaction baths are supplied with the kits mentioned above, but a very convenient chemically resistant reaction bath of just the right size, which will allow economy in the use of wash solutions, is the upturned polypropylene lid of an ICN 96-pipet tip holder pack (e.g., lid from Cat. No. 77-987-H2,79-987-H2, and so forth). 18. Plates for ELISA on pins can be the cheapest flat-bottom plates available, whereas plates for ELISA on biotinylated peptides should have good binding properties for streptavidin, for example, Nunc Maxisorp polystyrene flat-bottom plates (Cat, No. 442404). 19. A sonicator is highly recommended for the final washing steps after peptide synthesis for assisting the removal of peptide from pins during cleavage, and for assisting the removal of bound antibody from pins after each cycle of ELISA testing. Common small laboratory sonicators (e.g., Branson B2200) are suitable for one block of 96 pins at a time, provided they are reasonably solvent-resistant and care is taken to avoid a buildup of solvent fumes around the sonicator. 20. Safety equipment: Dispensing, washing, and deprotection steps during synthesis should be carried out in an operating fume hood, while wearing protective gloves and safety glasses, and with emergency equipment handy in case of a spill. 21. Computer accessories: A pointing device called a PinAIDTM 1s available from Chiron Mimotopes or its distributors. It consists of an 8 x 12 array of high-intensity LEDs, mounted in a case with a glass top. The PinAID is interfaced to a computer to allow the automatic display of the location of each amino acid addition on each synthesis cycle. This takes much of the labor out of the process of adding amino acids to the correct reaction well, and greatly reduces the chance of a mistake being made.

3. Methods

3.7. Design of a Peptide Set The large numbers of discrete peptides available through Multipin technology open up opportunities for a thorough rather than a piecemeal approach to epitope mapping. Thus, it is practical to synthesize all overlapping linear peptides homologous with a sequence rather than just those areas predicted to be B- or T-cell epitopes. Likewise, it is practical to make peptides of more than one length, such as all the 8-mers and all the 9-mers for a cytotoxic epitope study, rather than just the 9-mers. If a single set of peptides is to be used for both T- and B-cell epitope mapping, it is logical to make peptides around 1318 residues in length, where one end is tagged with a biotin group for the purpose of specific capture. Such peptides can also be used for cytotoxic T-cell epitope mapping under conditions where the peptide is able to be broken down, such as in the presence of serum or cell-surface proteases (I 7). Alternatively, a family of related peptides, some on solid-phase and some in solution, each tailored for a particular purpose, can be made using the “5-in- 1” version of the Multipin synthesis kit.

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The simplest form of systematic approach is to make every possible overlapping peptide, stepping through the sequence one residue at a time (1). If an offset of more than one residue is used, careful choice of peptide length should ensure that no epitopes are missed owing to an insufficient overlap between successive peptides. Such a set contains many “internal controls”; additional random or reverse-sequence peptides can be added to provide further negative controls, or known epitopes can be added as positive controls. After identification of an antibody-binding peptide, the relevance of binding should be confirmed by a competition test with whole antigen (18,19). Resynthesis of a set of truncated versions of the active peptide found in the preliminary scan allows identification of the “minimal” epitope. Subsequent analoging of this minimal epitope is a practical way of establishing the role of each residue within the minimal epitope (21). 3.2. Synthesis

of Peptides

1. After selection of the appropriate kit type and design of the peptide set, enter the peptide sequences into the kit software to enable a peptide synthesis schedule to be generated. The printed schedule shows the layout of the peptides, including controls, on each “block” holding 96 pins, and lists the amino acid amounts and locations for each cycle of synthesis. 2. Following the methods described in the appropriate kit Synthesis Manual, prepare solutions of the Fmoc-protected amino acids, activating agent, and catalyst (HOBt) in high-quality DMF (see Note 1). Optionally, add bromophenol blue as an indicator (see step 5). 3. While the amino acid solutions are being prepared, remove the Fmoc-protecting group from the amme group on the pin surface by immersing the pins in a bath of 20% piperidine in DMF for 20 min, followed by methanol washes to remove traces of piperidine. Allow the pins to air-dry. This drying time is a convenient time to dispense the activated amino acid solutions. 4. Activate the amino acid solutions by adding an aliquot of the appropriate activating agent (DCC, DIC, BOP, or PyBOP) and HOBt. Dispense the scheduled volume of solution into the indicated wells of the polypropylene reaction tray for coupling cycle #l (see Note 2). Wells for each amino acid are indicated on the printed synthesis schedule, or if using the programmed PinAID display unit, the location is shown by the lit up LEDs under those wells. 5. Place the pins into the wells of the reaction tray, ensuring the correct orientation of each plate is observed. Place pins (in the tray) in a polyethylene bag and incubate at 25Y! for >2 h while coupling takes place. Completeness of coupling can

bejudged by observing the disappearanceof the blue staining of the pins owing to release of the Bromophenol blue indicator from the pin surface as the amino groups become unavailable after coupling to the incoming amino acid. 6. Rinse the pins in methanol to remove unreacted amino acid solution, and air-dry briefly. Rinse in a DMF bath. The pins are now ready for the next cycle of Fmoc-

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Rodda, Mae), and Tribbick deprotection/couplmg. Recommence from step 2 above, adding the ammo acid solutions to different wells corresponding to coupling cycle #2. Continue with ammo acid coupling and Fmoc-deprotection cycles until the required sequences have been synthesized (see Note 3). At the completion of all ammo acid coupling cycles, carry out the final Fmoc deprotection and include if appropriate an N-termmal acetylation step, either with acetic anhydride (DMF:acetic anhydride:triethylamine 50:5: 1) or using the same method as for amino acids, but usmg acetic acid instead of an ammo acid (Note 4) Remove the side-chain-protecting groups from the amino acid residues that have reactive side-chains. Side-chain deprotection is achieved by mrmersmg the pins mto wells of a polypropylene reaction tray containing TFA plus the selected cocktail of scavengers, such as 5% thioanisole/5% amsole/2.5% ethanedithiol (see Note 5). Incubate for 2.5 h at 25°C. (Caution: TFA is a corrosive, volatile substance and must be handled with great care to prevent contact with the fluid Do not allow TFA solutions and DMF solutions to come in contact.) For peptides that are to remain on the pm surface, this is the final “chemistry” step of synthesis For peptides that are to be cleaved from the pin, this may also be the step that removes the peptide from the pm (MULTIPIN MPS kit), or there may be another subsequent step for removal of the peptide from the pin (MULTIPIN DKP kit and MULTIPIN GAP kit). Remove the pins from the TFA. In the case of the MULTIPIN MPS kit, the peptide is now in the TFA, and the pin has no further use. In all other cases, the pins are carefully washed and readied for testing or for the cleavage of peptide from the pins. For the MULTIPIN MPS kit, dry down the TFA solutions of peptide and wash with cold ether/petroleum ether to remove as much nonpeptide material (TFA, scavengers, cleaved protecting groups) as possible (see Note 6). For permanently bound peptides (MULTIPIN NCP kit), the pm surface is neutralized, washed, and made water-permeable by somcation in aqueous detergent (1% SDS in 0 1M phosphate buffer, pH 7.2, containing 0.1% mercaptoethanol) For cleavable pins (MULTIPIN DKP and MULTIPIN GAP kits), the pins are washed by sonication m 1M aqueous acetic acid (see Note 7). Pins m MULTIPIN DKP kits are readily cleaved directly into a neutral or shghtly alkaline buffered aqueous solution of the user’s choice, because the DKP-forming linker is stable only under acid conditions and spontaneously cleaves in neutral or alkaline conditions. Choices of buffer include O.lM HEPES, pH 7.6-8.0, O.lM phosphate, pH 7.8; O.lM Tris-HCl, pH 8.0; O.lM ammonium bicarbonate, and so on. The pins are rmsed momentartly m water to remove excess acid and are then placed into cleavage buffer m wells of a microtiter tray or 96 racked Bio-Rad tubes Cleavage occurs over 1 h at room temperature when assisted by sonication. Pins in MULTIPIN GAP kits are cleaved with 0 73 mL of aqueous 0 IM sodium hydroxide, which hydrolyzes the ester link between the peptide and the pin, leaving a C-terminal glycine-free acid on every peptide Cleavage takes 30 min when assisted by somcation. The sodium hydroxide must be neutralized immediately after cleavage with 1 Eq. of, e.g., sodium dihydrogen phosphate or acetic acid, to prevent damage to the peptide (see Note 8)

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12. Regardless of the use to which the peptides are to be put, they must be stored in a way that protects then integrity. We recommend storage either dry at 4°C or colder, or wet frozen at -20°C or colder. Do not leave peptides in aqueous solution at room temperature for longer than a day, and less if the peptide solution is not sterile

3.3. Assays with Peptides 3.3.1. Direct Binding on Pins (El ISA) 1. Incubate the pms m a blocking solution of TPBS for at least 1 h. 2. Transfer the pins to a solution of diluted antibody in TPBSA (see Note 9). Incubate overnight at 4°C (see Note 10) with agitation (shaking). If all pins are being reacted with the same antibody, this IS best done m a bath rather than a 96-well microtiter plate, because the bath allows more effective agitation and a more uniform, constant antibody concentratton and temperature (Note 11) 3. Blot or flick excess antibody solution from the pins and wash the pins in one or more baths of TPBS (see Note 12). 4. Transfer the pins to a solution of excess antispecies enzyme conjugate in TPBS containing 1% (v/v) sheep serum and 0.1% sodium caseinate (see Note 13). Incubate for 1 h at room temperature with agitation. 5. Dispense an exact volume of enzyme substrate (e.g., 200 pL) mto wells of a flatbottom microttter plate. 6. Wash the pins m a bath of TPBS, and finally in PBS alone to remove Tween from the pins Place the pms mto the substrate wells. Incubate with agitation for about 30-60 min. The enzyme-substrate reaction can be stopped at any time by just removing the pms from the wells of the microtiter plate, and can be restarted just as easily by placing the pins back into the wells. 7. Read the absorbance of the colored substrate product on a mtcroplate reader (see Note 14). By use of the reading software supplied with the kits, the absorbances can be transferred to a computer, a visual check made of the agreement between the plate and its colored representation on the computer screen, and the data accepted and stored in a file. Later, retrieval of the data m the file by the software allows immediate plotting of the absorbance against the identity of the peptide that gave the data, or the data can be imported into a spreadsheet for further analysis. 8. Regenerate the peptides for further tests by disrupting the peptide-antibody association. This is achieved by sonicating the pins for 10 min in a bath of warm (60°C) 1% SDS in PBS containing 0.1% mercaptoethanol, washing m warm water (60”(Z), and rinsing in warm methanol prior to air drying. The pms can then be stored dry in the cold or reused immediately for another ELISA

3.3.2. Binding Assays on Biotinylated Peptides 1. The peptides are captured on streptavidin-coated (SA) plates. These can be obtained commerctally or can be prepared by adding 100 yL of a solutton of 5 ug/mL streptavidm m water to wells of a flat-bottom polystyrene microtiter plate and allowmg the solution to evaporate to dryness at 37’C.

Rodda, Maeji, and Tribbick 2. If the peptide set has been stored dry, dissolve the peptides in preparation for use. Owing to the varied properties of the peptides within a set, it is usually best to use a common, effective means of dissolving all of them rather than trying an individual approach with each peptide. Thus, dissolve the peptides in a small amount of good-quality DMSO to give a solution of approx 5 rnA4peptide. 3. Fill the wells of the SA plates with TPBS containing 1% sodium caseinate. After 1 h, wash the SA plates four times with TPBS and remove excess liquid, e.g., by slapping them upside down onto a bench covered with absorbent paper toweling. The toweling can be layered over a thin layer of dense foam plastic to cushion the impact so the plates are not damaged. 4. Dilute each peptide to between 1: 1000 and 1: 10,000 (between 5 piUand 500 rnV), e.g., by transferring 1 pL into 1 mL of TPBS. 5. Add 100 pL diluted peptide to each well of the SA plates, and incubate at room temperature for 1 h with agitation. 6. Wash with TPBS, and add 100 pL of antibody solution diluted in TPBS as for pins (above) (see Note 15). Incubate overnight at 4°C or for 1 h at room temperature with agitation. 7. Wash with TPBS, and add conjugate diluted as for pins (above). Incubate for 1 h at room temperature with agitation. 8. Wash with TPBS and finally with PBS alone. 9. Dispense 100 pL of substrate quickly to each well, and incubate wtth agitation for 30-60 min (see Note 16). 10. Read the absorbances as for pin ELISAs (above) (see Note 17). Save and analyze the data as mentioned for pin ELISAs.

4. Notes 1. Commercial DMF accumulates amines over time, so it must be very fresh or be tested before use. Amino acid solutions should be activated just before dispensing. To minimize deterioration of the solutions prior to adding the pins, the least stable amino acids (W, Q, N, K, C, H, R) can be activated last, in the order given. Pipeters for dispensing DMF solutions of amino acids, activation reagents and HOBt solution, and so forth, should if possible be made of polypropylene or other material that is resistant to attack by DMF. Pipet tips used must be made of such resistant plastics. 2. The volume required depends on the scale of synthesis and varies from 200 yL to 1 mL. The first amino acid added is usually the C-terminal amino acid of the peptide being made, except for the MPS kit, in which the first (C-terminal) amino acid is already on the pins as supplied. 3. If peptides of different lengths are being made, the syntheses schedule holds back the commencement of the shorter ones so that all finish on the same cycle. This naturally requires new pins to be placed on the pinholder as each shorter peptide enters the synthesis cycles. 4. N-terminal acetylation removes the “unnatural” charge on the amino group of an amino acid residue that would normally be embedded in a protein sequence.

Multipin Pep tide Synthesis

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It also prevents pyroglutamate formation with N-terminal Glu and Gln residues. However, it reduces peptide solubility, so it may be undesirable for hydrophobic peptides. Solubility is not such a major issue for peptides that are to remain pin-bound. For the MULTIPIN MPS kit, the pins are the larger “Macro” size and may be better handled in individual polypropylene tubes. The scavengers mentioned are very pungent, and particular care should be taken to avoid contaminating your clothing. Do not remove contaminated objects from the fume cupboard unless they have been swabbed down with dilute (1%) aqueous hydrogen peroxide solution to oxidize the thiols to nonvolatile compounds. When washing with cold ether/petroleum ether (danger: flammable), if the solid peptide goes into solutton in the ether/petrol, it will be necessary to dry down the solution to recover the peptide. After side-chain deprotection, the MULTIPIN DKP pins must be kept in acidic conditions to prevent premature cleavage of the peptide from the pin. The pepttdes may now be used in assays, or may be purified and characterized. The great strength of MULTIPIN peptide synthesis for initial screening is somewhat lost if each peptide has to be individually characterized. Mass methods of characterization, such as ion-spray mass spectrometry, are suitable for handling the hundreds of peptides made by this technique. Suitable working strengths for antisera to be tested on pins (bath method) are: hyperimmune sera 1:20,000, monoclonal culture supernatants 1: 100, monoclonal ascites fluids 1: 10,000-l : 100,000. Use of low temperatures during peptid-ntibody binding will help protect the peptide from the action of proteases. The sensitivity of the bath method can be lo-fold higher than the microtiter plate method, allowing a more dilute antibody to be used. This compensates for the larger volume of diluted antibody needed to fill the bath, by comparison with the wells of a 96-well microtiter plate. The number of washes is not critical, and fewer may be better. This diluent reduces nonspecific binding of the goat antispecies conjugate to the pins. Note that this diluent is totally unsuitable if the primary antibody had been sheep or goat serum, in which case the sheep serum in this diluent can be replaced with rabbit serum. A bath is again very effective, but it may be necessary to use a 96-well microttter plate to conserve expensive conjugate As with other ELISA techniques, we prefer to make all readings dual-wavelength against a reference wavelength where the substrate does not absorb. This reduces the possibility of a false positive arising from a dirty or optically imperfect well. Suitable working strengths for antisera to be tested on biotinylated peptides are: hyperimmune sera 1:2000, monoclonal culture supematants 1: 10, monoclonal ascites fluids 1: 1000-l : 10,000. Unlike pins, the reaction cannot be stopped temporarily, but can be stopped with a reagent specific for that enzyme. The stop reagent can be added in a consistent timed manner to correct for the timing delays during addition of substrate to the wells.

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17. If the software is to be relied on to record the absorbance against the peptide sequence, the position of each peptide in the plate must be kept the same as during the peptide synthesis steps.

References 1, Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid Proc Natl. Acad. Sci USA 81,399&4002. 2. Smith, J. A., Hurrell, J. G. R., and Leach, S. J. (1977) A novel method for delmeating antigenic determmants: peptide synthesis and radiomnnunoassay using the same sohd support. Immunochemis
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8. 9.

10.

11.

12. 13.

peptide synthesis strategy for T cell determmant analysis. J Immunol. Methods 134, 23-33 Maeji, N. J., Valerio, R. M , Bray, A. M., Campbell, R. A., and Geysen, H. M. (1994) Grafted supports used with the multipin method of peptide synthesis. Reactive Polymers 22,203-2 12. Valerio, R. M., Bray, A. M., Campbell, R. A., Dipasquale, A., Margellis, C., Rodda, S. J., Geysen, H. M., and Maeji, N. J. (1993) Multtpm peptide synthesis at the micromole scale usmg 2-hydroxyethyl methacrylate grafted polyethylene supports Int J Pept Protein Res. 42, l-9 Bray, A. M., Maeji, N. J., Jhmgran, A. G , and Valerio, R M. (1991) Gas phase cleavage of peptides from a solid support with ammonia vapour. Apphcation m simultaneous multiple peptide synthesis. Tetrahedron Lett 32, 6 163-6 166. Maeji, N. J., Bray, A. M., Valerio, R. M., and Wang, W. (1995) Larger scale multipin peptide synthesis. Pept. Res. 8,33-38 Rodda, S. J., Geysen, H. M., Mason, T. J., and Schoofs, P. G. (1986) The antibody response to myoglobin-I. Systematic synthesis of myoglobin peptides reveals location and substructure of species dependent continuous anttgenic determmants. Mol. Immunol. 23,603-6 10. Getzoff, E. D., Geysen, H. M., Rodda, S. J., Alexander, H., Tainer, J. A., and Lerner, R. A. (1987) Mechanisms of antibody binding to a protem. Science 235, 1191-l 196. Mutch, D. A., Rodda, S. J., Benstead, M., Valerio, R. M., and Geysen, H. M (199 1) Effects of end groups on the stimulatory capacity of minimal length T cell determinant peptides. Pept Res. 4, 132-137. Burrows, S. R., Gardner, J., Khanna, R., Steward, T., Moss, D J , Rodda, S., and Suhrbier, A. (1994) Five new cytotoxic T cell epitopes identified wtthm EpsteinBarr virus nuclear antigen 3 J. Gen Vwol. 75,2489-2493. Trifilieff, E., Dubs, M. C., and van Regenmortel, M. H. V. (1991) Antigenic crossreactivity potential of synthetic peptides immobilized on polyethylene rods. Mel Immunol 28,889-896.

Multipin Peptide Synthesis

147

14. Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J. E., Mason, T. J., Saracco, G., Bonmo, F., Crawford, K., Marion, C. D., Crawford, K. A., Brunetto, M., Barr, P. J., Miyamura, T., McHutchinson, J., and Houghton, M. (1992) Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections. Proc. Nat1 Acad. Sci. USA 89,346s3472. 15. Hunt, D. F., Henderson, R. A., Shabanowitz, J., Sakaguchi, K., Michel, H., Sevilir, N., Cox, A. L., Appella, E., and Engelhard, V. H. (1992) Characterization of peptides bound to the class I MHC molecule by mass spectrometry. Science 255, 1261-1266. 16. Rudensky, A. Y., Preston-Hurlburt, P., Al-Ramadi, B. K., Rothbard, J., and Janeway, C. A. (1992) Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs. Nature 359,429-43 1. 17. Widmann, C., Maryanski, J. L., Romero, P., and Corradin, G. (199 1) Differential stability of antigenic MHC class I-restricted synthetic peptides. J. Immunol. 147, 3745-3751. 18. Tribbick, G., Triantafyllou, B., Lauricella, R., Rodda, S. J., Mason, T. J., and Geysen, H. M. (1991) Systematic fractionation of serum antibodies usmg multiple antigen homologous peptides as affinity ligands. J Immunol. Methods 139,155-166.

19. van’t Hof, W., van Milligen, F J., Driedijk, P. C., van den Berg, M , and Aalberse, R. C. (1993) How to demonstrate specificity of antibody binding to synthetic peptides? J Immunol. Methods 161,273-275. 20 Geysen, H. M., Mason, T. J., and Rodda S. J. (1988) Cognitive features of continuous anttgemc determinants. J A401 Recognztlon 1,32-41.

15 SPOT Synthesis Epitope Analysis with Arrays of Synthetic feptides Prepared on Cellulose Membranes Ronald Frank and Helke Ovetwln 1. Introduction SPOT synthesis is an easy and very flexible technique for simultaneous parallel chemical synthesison membranesupports.This method gives immunologists rapid and low-cost accessto large numbers of peptides both as solidphase-boundand solution-phaseproductsfor systematicepitope analysis. 1.1. Prkiple of the Method When dispensing a small droplet of liquid onto a porous membrane, the droplet is absorbedand forms a circular spot. Using a solvent of low volatility containing appropriate reagents,such a spot can form an open reactor for chemical conversionsinvolving reactive functions anchoredto the membrane support, e.g., conventional solid-phasesynthesis.A great number of separate spotscan be arrangedasanarrayon a largermembranesheet,andeachof theseis individually addressableby manual or automateddelivery of the corresponding reagentsolutions (Fig. 1). The volume dispensedand the absorptive capacity of the membranedetermine the spot size. According to the area-specificfunctionality of the support, the spot size correlateswith the particular scale of synthesis.The spot size also controls the minimal distancebetweenspot positions and thereby the maximum density of the array. Synthetic stepscommon to all spot reactors are carried out by washing the whole membrane with respectivereagentsand solvents. Chemical and technical performanceof this type of simultaneousparallel solid-phasesynthesishas so far been optimized for the assemblyof arrays of From. Methods in Molecular Biology, vol 66. Epltope Mapping Protocols Edited by* G E Morris Humana Press Ino, Totowa, NJ

149

Frank and Overwin

150

Fig. 1. Schematic cartoon showing the principle of SPOT synthesis

peptide sequences up to a length of 20 residues utlhzing conventronal mild Fmoc/tBu chemistry (1,2). The membrane supports are of specially selected, pure cellulose chromatography paper, and are chemically dertvatized to carry spots of dipeptide anchors for the preparation of either immobilized @AlaPAla-anchor; entry 1 m Table 1) or solution-phase pepttdes (Lys-Pro-anchor; entry 2 in Table 1). Novel types of special safety-catch linkers have been recently developed by us to cleave also C-terminally unmodified peptide acids or amides directly into neutral aqueous buffers for the direct use m a bioassay. Arrays of spots providing suitable anchor functions for peptide assembly on cellulose membranes are most easily generated by a two-step procedure, including first, the preparation of “amino” paper through esterification of an o.N-Fmoc-protected amino acid to available hydroxyl functions on the cellulose fibers of the whole sheet, followed by Fmoc cleavage and second, spotwise coupling of another Fmoc amino acid or suitable linker compound (Table 1). During the second derivatization step, the array of spot reactors is generated and all residual amino functions between spots are blocked by acetylatron. Thts array formation step requires very accurate pipeting. During peptide assembly, slightly larger volumes are dispensed, and the spots then formed exceed those initially

formed in order to avoid incomplete

couplings

at the edges.

SPOT synthesis is particularly flexible with respect to numbers and scales that can be accomplished. The arrays are freely selectable to fit the individual needs of the experiment by variation of paper quality, thickness, specific anchor loading, and spot size (Table 2). The standard format used in manual SPOT synthesis was adapted to the 8 x 12 array of a microtiter plate with 96 spots.

Table 1 Orthogonal

Safety-Catch

Linkages Anchor/linkef

Entry peptide-RAla-R&

COO-resin

and Anchors

Used with SPOT Synthesis Peptide product

Ref.

Immobilized via C-termmus

(2)

Soluble, C-terminally modified with diketopiperazme

(6)

Soluble, C-terminal acid

(7)

Soluble, C-terminal

(s)

peptide-ml-(CIlz),

peptide-+

peptide-m

CO-fMla-resin

r”

CWkUfl-r&l

amide

Soluble, C-tennal amide (optional release after biological Immobilized,

cycl&

(8) assay) (9)

peptidcm

‘% = 0 (R = H, CHX), 1 (R = H), m = 1,2; R’ = methyl, isopropyl, [err-butyl, tert-butyl-dimethylsllyl (TBDMS), tnmethylsdylethoxymethyl (SEM), methoxymethyl (MOM), methoxyethoxymethyl (MEM), R* = ethyl, Isopropyl, &fi-butyl, &ityl bAfter selective removal of TBDMS and cychzatlon.

Table 2 Standard

SPOTS-Array

Configurations

Membrane

AnchoP wol/cm*

Format

s

(Each One Fitted to a Sheet of the Size of a Microtiter Spotted volume,C fi

Plate, 8

x

12 cm)

spot size, mm

Positional distance, mm

Synthesis scald, mm01

8x12=

96

540

0.2-0.4

0.510.7

7

9

25

7x10=

70

Cbrl

0.4-0.6

l.OA.5

8

10

50

17x25=

425

540

0.2-0.4

o.uo.15

3

4

6

40 x 50 =2000

50

0.2-0.4

0.03/0.05

1

2

1

“chromatography paper products from Whatman (Maidstone, UK). b‘rypical derivatization yields with first ~&mine. Volumes are gwen for array generation step/peptide assembly step. %kan values.

SPOT Synthesis

Fig. 2. A microtiter plate adaptedformat of a manually prepared 96-spot membrane.Dark spotsareblue, and light spotshaveturned to yellow after coupling with an Fmoc amino acid HOBt ester.

Recently, an automated SPOT synthesizer has been developed at ABIMED Analysen-Technik GmbH (Langenfeld, Germany) basedon a Gilson pipeting workstation. This instrument can handle simultaneously up to six standard membrane sheets. Moreover, automated spotting can be exploited to reduce the size of spotsand, thus, increasethe numberper areaconsiderably (Table 2), which otherwise is extremely tedious to manufactnreby hand Presently,up to 2500 spots can be generatedon a microtiter plate sized sheet.The instrumentso far performs only the pipeting work; all washing stepsare carriedout manually. Free amino functions on the spotscan be stainedwith Bromophenol blue (3) prior to the coupling reactions. This allows the visual monitoring of proper performance of all synthesis steps, such as correct dispensing, quantitative coupling, and acetylation (capping), and effective removal of piperidine from the Fmoc-deblocking steps. Thus, a standard membrane for SPOT synthesis displays an array of light blue spots on a white background. Each spot can be marked by writing a number with a pencil next to it (Fig. 2). These numbers refer to the corresponding peptide sequencesthat are assembledon them and are a guide for rapid manual distribution of the solutions of activated amino acid derivatives at each elongation cycle. An example of a respective, convenient pipeting protocol generatedwith a computer is shown in Fig. 3. For

SPOTscan Sequence Sequence Peptide 70 spots Amino

- sun Jul 09 14:18:37 1995 name: ttlpig.seq length: 378 (from 1 to 288) length: 12; Offset: 4 on 1 membrane

acid

usage:

F=34

G=Sl

B=30

P=39

Q=28

R=S3

spot ----

Mol. Wt. -----___

A=39 C-12 D=32 E=75 I=42 K=52 L=93 M=lO N=51 S=58 T=43 V=40 W=12 Y=46 Peptide sequence ---------------MYTFvvRDENss VVRDENSSWAE ENSSVYAEVSRL VYAEVSRLLLAT VSRLLLATGHWX

LLATGHWKRLRR GaWKRLRXIlNPR m NRLPFGRLGHEP FGRLGHEPGLMQ =&?:=z

2

ii761

21: 21

1311 1343 1278 1408 1460 1515 1395 EG 1213 1342 1431

LVNYYRcaDKLC YRGADKLCRKAS DKLCRXASLVXL RKASLVKLIKTS LVKLIKTSPELA IXTSPEIAEZSCT

PELAESCTWPPE ESCTWFPESWI

WFPESYVIYPTN SYVIYPTNLKTP YPTNLKTPVAPA LKTPVAPAQNGI VAPAQNGIEPPI ONGIHPPIHSSR

ttlpig.seq A: c:

D: E:

2 48 2 41

- Pxpettzng

Schedule

for

Cycle

1

for

Cycle

2

ii90 26 54 50 22 33 40 60 68 64

F: G: H: 2: 47 I: 23 27 28 35 38 K: 5 37 44 57 70 L: 11 18 31 46 58 69 M: N: 63 65 67 P: pi S: T: v: w: Y: ttlpig.seq - Pipetting Schedule A: 2 4 17 37 c: D: 2:: 56 57 E: 9 12 30 34 45 I?: 31 48 64 65 G: ;; ;; 38 47 53 63 H: I: 69 70 K: 18 33 60 61 62 L: 8 10 16 20 46 52 66 67 68 M: 13 N: 14 42 P: 7 22 26 28 55 Q: 44 R: 3 6 11 S: 1 29 36 39 49 T: 19 24 25 40 54 V: 23 43 SO w: 5 35 Y: i2 51 v

ttlpig.seq - Plpetting Schedule A: 28 42 69 C: 60 D: 32 44 E: 22 40 55 57 65 67 F: 24 33 G: 11 13 36 39 64 II: 7 14 I: 41 46 K: 18 19 21 27 35 38 50 L: 6 8 15 43 47 48 52 M: N: ;;93;04;9 61 P: 45 53 59 62 g 12 17 51 56 s: 34 63 T: :e2:031 v: 2 16 20 37 Y: 1 4 25 54 68

for

Cycle

11

tt1 .g$g;gseq - Pipetting A: D: 9 18 36 E: ;02;33;g42 49 65

for

Cycle

12

F:

G: El: I: K: L:

66 67

7 14 15 40 45 30 31 53 58 ;; 44 51 54 62 70

6. N: ;2 P: 22 . 29 :r 8 s: 25 T: 32 v: 2 W: 24 Y: 17 M:

Schedule

11 16 20

27 41 43 56

37 48 68 19 50 55 57 69 34 60 61 4 5 28 46 52 26 47 63 64

Fig. 3. Some features of a SPOT synthesis protocol form for rapid manual performance. Each peptide in the list is coded by an Arabic numeral. In this example, 70 overlapping dodecapeptrdes with a shift (offset) of four amino acid residues were generated to scan a 288 amino actd residues long part of the TTIpig protein sequence. Then, only peptide code numbers have to be followed for the distribution of amino acid derivatives during the assembly cycles.

156

Frank and Ovetwin

automated pipeting, no pencil marking is necessary. Exact fixing of the membranes is assured by the perforation for the holder pins. The dry membranes are placed in a flat, chemically inert trough or fixed on the platform of the synthesizer. As soon as the droplets of activated amino acid solutions are added to the spots, coupling proceeds with a conversion of free amino groups to amide bonds. After all amino groups have been consumed, the blue color of the spots changes to yellow, thus indicating a quantitative reaction (Fig. 2). Because the solvent within the spots is slowly evaporating over the reaction time, additional drops may be added onto the same position without enlarging the spots and risking overlap with their neighbors. In this way, difficult coupling reactions also may be brought to completion by double or triple couplings. Any series of individual peptide sequences can be freely arranged as a twodimensional (2D) array on a SPOT membrane, such as overlapping fragments derived from a protein sequence (SPOTscan) for mapping linear antigenic determinants, stepwise N- or C-terminally truncated fragments (SPOTsize) to determine epitope boundaries, substitution analogs (SPOTsalogue) to evaluate the contribution of individual amino acid residues to binding, and so forth. Examples are shown in Fig. 4. Moreover, modern peptide library screening approaches allowing the a priori delineation of peptide sequences that are recognized by antibodies and that may also mimic a conformational epitope (mimotope) have been implemented into SPOT synthesis (4,5). These approaches exploit the preparation of arrays of defined peptide mixtures (or pools) and the presentation of entire peptide libraries (e.g., all 64 x lo6 hexapeptides) as strategic sets of sublibraries. Some current strategies for the deconvolution of individual sequences by activity screening of random pools are given in Table 3. The introduction of randomized positions (X) within a peptide sequence assembled on a spot is quite reliably achieved by coupling with equimolar amino acid mixtures and applying these at a submolar ratio with respect to available amino functions on the spots. This is to allow all activated derivatives (also the slower coupling ones) to react quantitatively during a first round of spotting. All peptide elongations are then completed by two to three successive repeats of spotting. Using this coupling procedure, any position in a peptide sequence can easily be randomized without special considerations or increase in technical effort. Figure 4D gives an example of a peptide library array probed with a monoclonal antibody (MAb). Because of its hydrophilic nature, the cellulose membranes are particularly well suited for the presentation of immobilized peptides to a biological assay system. The whole membrane (or parts of it; see Note 1) is incubated with, e.g., an antiserum, and detection of antibody molecules bound to peptide spots can be achieved by conventional solid-phase ELISA or Western blot procedures.

157

SPOT Synthesis

Other labeling techniques, such as radioisotopes or fluorescent dyes, are also fully compatible. The choice of a detection system, however, should ensure that peptide spots will not become chemically or otherwise irreversibly modified, because peptide arrays on cellulose membranes are reusable many times (>20) when treated properly. Signal patterns obtained from peptide arrays on spots can be documented and quantitatively evaluated utilizing modern image analysis systems(Fig. 4) as used with other 2D analysis media, such as electrophoresis gels and blotting membranes. If solution-phase peptides are to be prepared, then synthesis is carried out on correspondingly derivatized membranes, preferably utilizing a safety-catch linkage (Table 1). These linkages are stable during peptide assembly, cleavage of protecting groups, and acidic washings to remove chemicals. After this procedure, the membrane is dried and cut into pieces carrying each one spot, or the spots are punched out with the help of a suitable device (see Note 1). For the standard 8 x 12 format, a puncher is commercially available that places the cut spots directly into the wells of a microtiter plate. Then an appropriate buffer of pH 7.0-8.0 is added to individual membrane pieces, and the peptides are released from them into solution. 2. Materials 1. A kit that includes all necessaryitems for manual SPOT synthesisis currently available from GenosysBiotechnologiesInc. (Cambridge, England). 2. Flat reaction troughs with a good closing lid made of chemically inert material (glass, Teflon, polyethylene) that have the dimensions slightly larger than the membranes used. 3. A micropipet adjustable from 0.5 to 10 pL (Eppendorf or Gilson) with corre-

sponding plastic tips. 4. Small (1-mL) plastic tubes (e.g., Eppendorf, safe twist) and appropriate racks as reservoirs for amino acid solutions. 5. A rocker table (e.g., Rockomat Type 270 from Tecnomara AG, Zurich, Switzerland). 6. Appropriate bench space in a hood! 7. -7OOC freezer.

8. A sonicationbath with temperaturecontrol (e.g.,BANDELIN Electronic, Berlin, Germany, type SONOREX SUPER RR5 10H). 9. Software: Special DOS-PC computer programs for the generation of peptide Iists

and pipeting protocols are included in the synthesiskit and the operation sot% ware of the spotting robot.

Optionally: items 10-14. 10. A spotting

robot, model

ASP 222 (ABIMED

Analysen-Technik

GmbH,

Langenfeld, Germany). 11. Two dispensers adjustable from 5 to 50 mL for DMF and alcohol containers. 12. A Teflon tubing line connected to a 5-L container that is connected to a vacuum line for collection of solvents and solutions aspirated from the reaction trough.

Frank and Overwin

158

SPOTscan Analysis Peptide Length 12, Offset 4 mouse mAb lD3

TTLPIG SEQUENCE FROM 201 TO 288

Fig. 4. A seriesof experimentsto demonstrateseveraloptions for epitope analysis exemplified with the mouseMAb lD3 raisedagainstthe tubulin-tyrosine ligase from pig (TTLpig) (13). (Top) Digital recordings of the original membranesprobed with the antibody. (Bottom) Graphical displays of quantified signals and their correlation to the peptide sequences.Dye units are directly proportional to the amount of dye precipitated on the spotsasdeterminedthrough calibration of the scanningdevice. (A) Mapping the MAb-binding site with overlappingpeptides.In such a first scan,longer peptideswith larger overlap canbe usedto reducethe number of peptidesrequired for a full scanof long protein sequences.The display showspart of the TTLpig sequence with the signals placed as bars on top of the N-terminal amino acid residue of the respective peptide fragment. (Continued) 13. A digital recording device (scanneror CCD camera)for documentationof signal patternson membranes,plus analysissoftwarefor quantificationof signals.In case of fluorescenceor chemihuninescence detection,autoradiographyfilms may beused. 14. A DotPunch (Bibby Dunn Labortechnik GmbH, Asbach, Germany). 15. Chromatography paper type Chrl, 50, 540, and 3MM (Whatman, Maidstone, England).

SPOT Synthesis

SPOTsize Anal sis MousemAb I J 3 Erperimnt NHCI

l’TLEPI4

QKEYSKNYGKYEIGNEMF

1Omer

IOmer

9mer

9mer

Smer

Smer

7mer

7mer

6mer

6mer

5mer

5mer 4mer

4mer NECI

QKEYSKNYGKYLEGNEMF

TTLpig Sequence pos.242to 263

Fig. 4(B) Defining the shortest active peptide by scanning the epitope region identified in (A) with overlapping peptides having an offset of one and a stepwise reducedlength (boundary analysis).In the spectraldiagramrepresentation,the signals again correlate to the N-terminal amino acid residue of the respective peptide fragment. The first sevenspots in the upper panel (left to right) are 4-mers, followed by 8 x 5-mers, 9 x 6-mers, and so forth, ending with 13 x IO-mers. (Continued) 16. Bromophenol blue (BPB) indicator (e.g., E. Merck, Darmstadt, Germany). A stock solution of 10mg/l mL N,N-dimethylformamide (DMF) is prepared.BPB: The stock solution is diluted to 1% with DMF. 17. DMF should be free of contaminating amines and thus of highest affordable purity, at leastpro analysi @a.) grade.Amine contaminationis checkedby addition of 10pL of BPB stocksolutionto 1mL of DMF. If theresultingcolor is yellow, then this batch can be usedwithout further purification. Check eachnew batch! 18. 1-Methyl-2-pyrrolidinone @IMP) should be of highest available purity. Amine contamination is checkedas above with DMF. If the resulting color is yellow,

Frank and Overwin

SPOTsalogue Analysis Mousendb ID3

NYGKYE ParentSequence

Fig. 4(C) Analysis of systematicsingle substitutionanalogsof the minimal epitope peptideidentified in (B) revealingthe importanceof individual amino acid residuesand the natureof someof the antibody-peptideinteractions.The first 20 spotsin the upper panel correspondto the first vertical column in the lower panel,and so on. (Continued) then the NMP can be used without further purification. Most commercial products, however, are not acceptable.Then deionize 1 L of NMP with mixed-bed ion-exchangeresinAG 501-X8 (Bio-RadLaboratories,Hercules,CA) until a l-mL aliquot gives a yellow BPB test. Filter off the resin and dry by addition of 100 g

161

SPOT Synthesis

SPOTSAnalysis BindinE of Binding ofmouse mousemAb ID3 to AcXX12XYAC*DEFGHl

Expemnent~lZXY-4 KLMNPCRSTVWY A

P 0

s : i 0 n 2

c D : : : : : Q : : :

k i : : K : :: :: : G ” AC’DEFGHI

KLYNPQRSTVWY

Position1

Fig. 4(D) A first-generationarray for the apriori delineation of an epitopepeptide; a hexapeptidelibrary in the format AC-X-X-I-~-X-X- (see Table 3, entry 1) plus 25 referencespotsin the upper lane. C* = Cys(Acm).

of molecular sieve (MS) 4A (e.g., E. Merck or Fluka). Gently agitate overnight. Decant from the MS beads and filter through a 20-pm polyethylene sinter or cellulose membrane. Divide the clear liquid into 100~mL portions and store tightly closed at -2OOC. 19. N-Hydroxybenzotriazole (HOBt) can be obtainedfrom severalsuppliersonly as hydrate. Dehydrate in a desiccatorover phosphoruspentoxide at 50°C and 1P3 bar for 3 d. Store in a tightly closedcontainer at room temperature. 20. N-Methylimidazole @MI) is distilled from solid sodiumhydroxide and storedin small aliquots (5 mL) over MS 4A at -70°C.

162

Frank and Ovetwin

Table 3 Strategies for the Deconvolution of Individual Sequences by Activity Screening of Random Peptide PooW Iterative search starting with one or more defined position&’ First generation x-x- 1-2-x-x 400 pools (each 160,000 sequences) Second generation x- 1-03-04-2-x 400 pools (each 400 sequences) Third generation 400 pools (each 1 sequence) 1-0~-03-04-05-2 Positional scanning with single fixed positronsc, one single screen 1-x-x-x-x-x 20 pools (each 3,2 x lo6 sequences) II x- 1-x-x-x-x 20 pools II x-x- 1-x-x-x 20 pools ,I x-x-x-1-x-x 20 pools II x-x-x-x1-x 20 pools II x-x-x-x-x1 20 pools Dual-positional scanningd, one single screen 1-2-x-x-x-x 400 pools (each 160,000 sequences) I‘ x- 1-2-x-x-x 400 pools II x-x- 1-2-x-x 400 pools II x-x-x- 1-2-x 400 pools II x-x-x-x- l-2 400 pools %pectal codes to describe the pool compositions are: 0, = unvarred position m a parttcular screen occupied by single amino acid residues; 1,2,3 = positions systematically varied by single amino acid residues m a particular screen; X = positton occupied by a set of (e.g., all 20 L-) ammo acid residues. b“Mimotope” approach (IO). =Wtth only 120 pools of hexapeptides, acceptor preferences for certain amino acid residues at all posmons are obtained (consensus sequence); sequences of mdwdual active pepttdes have to be identified by synthesizing and testing all possible combinations of hits from this “positional scanning library” screen (I 1). with 2000poolsof hexapeptides, acceptorpreferencefor certaindipeptidecombmations at all positionsare obtained;from matchingoverlapsof these,sequenceconnectivitiescan be directly delineated(12)

2 1. WV-Diisopropylcarbodiimide (DIC), purum (Fluka, Heidelberg, Germany, or other suppliers). 22. Fmoc amino acid derivatives are available from several suppliers in sufficient quality. In situ prepared HOBt esters of these in NMP are used throughout for spotting reactions. Side-chain protection is Cys(Acm) or Cys(Trt), Asp(OtBu), Glu(OtBu), His(Boc) or His(Trt), Lys(Boc), Asn(Trt), Gln(Trt), Arg(Pmc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). For special derivatives, see Note 2. Dissolve 1 mm01 of each in each 5 mL of NMP containing 0.25M HOBt to give 0.2Msolutions. Divide into 100~pL aliquots in correctly labeled Eppentorf tubes. Close tightly, freeze in liquid nitrogen, and store at -70°C. The preparation of derivative solutions for the assembly of peptide pools is given m Note 3.

SPOT Synthesis

163

23. Acetylation mix: A 2% solution of acetic anhydride (p.a.) in DMF. 24. Piperidine, p.a.: A 20% solution in DMF is used to cleave the Fmoc-protecting group; piperidine is toxic and should be handled with gloves under a hood! 25. Alcohol (methanol or ethanol) of technical grade. 26. Deprotection mix containing trifluoroacetic acid (TFA, synthesis-grade; e.g., Merck, Darmstadt, Germany), triisobutylsilane (TIBS; e.g., Aldrich Chemicals), water, and dichloromethane (DCM, p.a.; e.g., Merck, Darmstadt, Germany): 50% TFA, 3% TIBS, 2% water, and 45% DCM. TFA is very aggressive and should be handled with gloves under a hood! 27. Derivatized SPOT membranes: The preparation of these requires some more chemical expertise and is described in ref. 2; ready-to-use membranes m an 8 x 12 format with 96 spots of SAla-SAla anchors are available from Genosys Btotechnologies Inc., Cambridge, England; blank membranes derivatized with PAla only are avarlable from ABIMED Analysen-Technik, and for the generation of SPOT arrays with the APS-robot, follow the instructions of the supplier; commercial delivery of membranes with other types of anchors and linkers (Table 1) is in preparation, A brief outline of the derivatization procedure is included below. 28. Polystyrene plates (12 x 12 cm) with covers as used in cell culture (e.g., Greiner Labortechnik, Frickenhausen, Germany; Petri-Dish Model 688 102). 29. Flat glass tray to hold at least one membrane. 30. Tris-buffered saline (TBS): 8.0 g NaCl, 0.2 g KCl, and 6.1 g Tris-base in 1 L water; adjust pH to 7.0 with HCl; autoclave and store at 4°C. 3 1, T-TBS: TBS buffer plus 0.05% Tween-20. 32. Phosphate-buffered saline (PBS): 8.0 g NaCl, 0.2 g KCl, 1.43 g NazHP04.2Hz0, and 0.2 g KHZP04 m 1 L water; adjust pH to 7.0 with HCl; autoclave and store at 4°C. 33. Citrate-buffered saline (CBS): 8.0 g NaCl, 0.2 g KCl, and 2.1 g citric acid (lH*O) in 1 L water, adjust pH to 7.0 with HCl; autoclave and store at 4°C. 34. Membrane blocking solution (MBS): Mix 20 mL Casein Based Blocking Buffer Concentrate (No. SU-07-250; Genosys Biotechnologies, Cambridge, England), 80 mL T-TBS (pH 8.0), and 5 g saccharose; the resulting pH will be 7.0; store at 4°C (see Note 4). 35. Alkaline phosphatase (AP) conjugated secondary antibodies (see Note 5): dilute aliquots in MBS prior to use as recommended by the supplier. 36. Dissolve 60 mg 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) (e.g., BIOMOL 02291, Hamburg, Germany) in 1 mL DMF; store at -20°C. 37. MTT: Dissolve 50 mg (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (e.g., Sigma Chemicals M-2128) in 1 mL of 70% DMF/30% water; store at -20°C. 38. Color developing solution (CDS). To 10 mL CBS add 50 yL 1M magnesium chloride, 40 pL BCIP, and 60 yL MTT (see Note 6). 39. Stripping mix A (SM-A): 8Murea, 1% SDS in PBS; store at room temperature; add 0.5% mercaptoethanol prior to use, and adjust pH to 7.0 with acetic acid. 40. Stripping mix B (SM-B): 10% acetic acid, 50% ethanol, and 40% water; store at room temperature.

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Frank and Overwin

3. Method All volumes given below hold for a standard 50 or 540 paper sheet of 8 x 12 cm and have to be adjusted to more sheets, other paper qualities, or sizes. Solvents or solutions used in washing/incubation steps are gently agitated on a rocker table at room temperature if not otherwise stated, and are aspirated or decanted after the time indicated. During incubations and washings, the troughs

or trays are closed with a lid. 3.1. DerivatilatPon

of Membranes

In this step, the esterification of the first anchor component to the hydroxyl functions of the cellulose is carried out. 1. Cut out a sheet of paper plus two small of I-cm* pieces and dry overnight in a desiccator at 10e3 bar. Use only thin and acid-hardened paper qualities, such as Whatman 50 or 540, for multiple use of immobilized peptide arrays in binding assays. 2. Soak the sheet and pieces in 2 mL of a solution containing 0.2MFmoc-S-alanine or Fmoc-proline, 0.24MDIC, and 0.3MNMI in dry NMP. Keep in a closed container for l-4 h. 3. After 1 h, take out one piece and place in a small beaker. Wash with DMF, 20% piperidine m DMF for 5 min, DMF again, and then stain repeatedly with 2 mL BPB until the supematant remains yellow. Wash with alcohol, and dry with a fan. Then place in a glass vessel containing 5 mL 20% piperidine. Determine the optical density of the deep blue solution, and calculate the loading with amino functions per cm2, which is roughly the amount of dye eluted (eSs5= 95.000). If okay, proceed with step 4; typical values are given in Table 2. If too low, then take the second piece after approx 3 h and proceed as with the first piece. 4. Wash the sheet in the reaction trough twice, and then leave overnight with 20 mL of acetylation mix. 5. Wash each with 20 mL DMF (three times). 6. Incubate in 20 mL of 20% piperidine/DMF for 20 min. 7. Wash for each 2 min with DMF (four times with 20 mL), alcohol (three times with 20 mL), and then dry in a desiccator at 1t3 bar overnight. Store at -2O’C.

3.2. Generation

of the SPOTS Array

1. Generate the list of peptides to be prepared, and define the array(s) required for the particular experiment according to number, spot size, and scale (Table 2). Mark the spot positions on the membranes with pencil dots for manual synthesis, and place in the reaction trough or fix membranes on the platform of the SPOT robot. 2. Prepare a solution containing 0.2h4 of second anchor compound (e.g., FmocSAla-OH or Boc-Lys[Fmoc]-OH for the corresponding SAla- or Pro-derivatized sheets from 3. l), 0.25 M HOBt, and 0.24A4 DIC in NMP. Add 1 pL BPB. Leave for 30 min, and then spot aliquots of this solution to all positions according to the

SPOT Synthesis

3. 4. 5. 6. 7. 8. 9.

chosen array configuration. Let react for 30 min. (Repeat spotting and reaction once for the Boc-Lys[Fmoc]-OH derivative to obtain better ytelds!) Wash with 20 mL of acetylation mix for 30 s, once again for 2 min, and finally leave overnight in acetylation mix. Wash with 20 mL of DMF (three times). Incubate for 5 min with 20 mL of 20% piperidine in DMF Wash with 20 mL of DMF (four times). Incubate with 20 mL of BPB. Repeat if traces of remaining piperidine turn it into a dark blue solution. Spots should be stained only light blue! Wash with 20 mL of alcohol (twice). Dry with cold air from a hairdryer between a folder of 3MM paper, and store sealed in a plastic bag at -20°C.

3.3. Assembly of Peptides on Spots 1. Number the spot posmons on the membranes with a pencil according to the peptide list for manual synthesis, and place in separate reaction troughs (see Note 1). Alternatively, fix membranes on the platform of the synthesizer. Follow the pipeting protocol or start spotter for the respective elongation cycle (see Note 3). 2. Take one set of Fmoc amino acid stock solutions from the freezer and activate by addition of DIC (4 pL/lOO pL vial; ca. 0.25M). Leave for 30 min and then spot aliquots of these solutions or start spotter. Leave for 15 min. Repeat spotting once and let react for 30 min. If some spots stay dark blue, you may add additional aliquots. If all spots are yellow to green, then continue (see Note 7). 3. Wash with 20 mL of acetylation mix for 30 s, once again for 2 min, and then incubate for about 10 min until all remaining blue color has disappeared. 4. Wash with 20 mL DMF (three times). 5. Incubate for 5 min with 20 mL 20% piperidine in DMF. 6. Wash with 20 mL DMF (four times). 7. Incubate with 20 mL of BPB. Repeat if traces of remainmg piperidine turn it into a dark blue solution. Spots should be stained only light blue (see Note 7)! 8. Wash with 20 mL of alcohol (twice). 9. Dry with cold air from a hairdryer in between a folder of 3MM.

Then go back to step 2 for the next elongation cycle. After the final cycle, all peptides can be N-terminally acetylated by additionally carrying out steps 3,4, 8, and 9 (see Notes 2 and 8).

3.4. Side-Chain Deprotection This must be performed under a hood! TFA is very aggressive! 1. Prepare 40 mL of deprotection mix. 2. Place the dried paper in the reaction trough, add 20 mL deprotection mix, close the trough tightly, and agitate for 1 h. Replace the deprotection mix with the remaining 20 mL, and leave again for 1 h. 3. Wash for 5 min with 20 mL of DCM (four times).

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166 4. 5. 6. 7.

Wash Wash Wash Wash

for 5 min with for 5 min with for 5 min with with 20 mL of

20 mL 20 mL 20 mL alcohol

of DMF (three times). of alcohol (three times). of 1M acetic acid in water (three times) (three times).

The sheet may now be dried with cold air and stored at -20°C or processed further as described in the next section. 3.5. Antibody-Binding This procedure

Assay

is worked out for use with AP-conjugated

secondary anti-

body detection (see Note 5). Remember, the peptides are linked to the cellulose by ester bonds, which are labile to alkaline hydrolysis in aqueous media of pH > 7.0! All buffers used must be of pH 7.0 or below! 1. Place the membrane in a polystyrene plate, and wet with a few drops of ethanol. This is to enhance solvation of hydrophobic peptide spots. 2. Wash for 10 min with 10 mL of TBS (three times) 3. Incubate overnight with 10 mL of MBS. 4. Wash once for 10 min with 10 mL of T-TBS. 5. Incubate for 2-4 h with test antibody or antiserum diluted in S-10 mL of MBS. 6. Wash for 10 min with T-TBS (three times). 7. Incubate for l-2 h with AP-conjugated secondary antibody diluted m MBS 8. Wash for 10 min with T-TBS (twice). 9. Wash for 10 min with CBS (twice). 10. Transfer the membrane to a flat glass tray, and add 10 mL of CDS Incubate without agitation until good signals are obtained. For individual peptides on spots, this usually takes 10-30 min, peptide pools may require longer incubations (2 h to overnight). Stop reaction by washing twice with PBS Keep membrane wet For storage, leave at 4°C in a container with PBS or cover with plastic wrap. If dried out, proteins may denature and become difficult to remove! After successful documentation of signals, continue with membrane stripping (see Note 6). 11. Wash for 10 min with 20 mL of water (twice). 12. Incubate with 20 mL of DMF until the blue color of spot signals is dissolved (usually about 10 mm; incubate in a sonication bath if necessary). Remove the solution, and wash once again for 10 min with 20 mL of DMF 13. Wash for 10 min with 20 mL of water (three times). 14. Wash for 10 min at 40°C with 20 mL of SM-A in a somcation bath (three times). 15. Wash for 10 min with 20 mL of SM-B (three times). 16. Wash for 10min with ethanol (three times). 17. Repeat from step 2 for the next antibody-bmdmg assay, or dry with cold au from a hairdryer in between a folder of 3MM and store at -20°C sealed m a plastic bag.

3.6. Peptide Elutlon This procedure is applicable for syntheses on safety-catch linkers of types 2,3, and 4 of Table 1.

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167

1. Follow steps l-3 of Section 3.4. 2. Wash for 20 min with each 20 mL of methanol/water (1: 1) containing 0.1% HCI (three times). 3. Wash for 20 min with 20 mL of 1M acetic acid in water (three times). 4 Dry in vacua (1 Om3bar) in a desiccator over solid NaOH overnight 5. Cut the membrane into pieces of each individual spots, or punch out with a suitable device. Place the pieces into separate plastic tubes or wells of a microtiter plate. 6. Add a buffer of pH 7.0-8.0 and sufficient capacity, e.g., O.lMphosphate. You may add an organic solvent, such as ethanol or dimethylsulfoxide, to dissolve more hydrophobic peptides. Final concentrations of peptide and other buffer constituents must be compatible with the bioassay to be performed! Check pH with a reference piece of membrane. 7. Close or seal the tubes, and shake overnight at 30-37”C, so that the membrane pieces are gently agitated. 8. Remove aliquots and dilute as required for the assay. Store remainder at -20°C.

4. Notes 1. Membranes can be easily cut into parts or pieces with scissors prior to, during, or postsynthesis as required for the particular experiments. Simply mark the cutting lines with a pencil. Spot positions should be also marked with a pencil dot if peptides are to be eluted individually after the synthesis (see Section 3.6.). The pencil marking is sufficiently stable during the synthesis procedure 2. Special chemical derivatives: Free thiol functions of cysteine may be problematic because of postsynthetic uncontrolled oxidation. To avoid this, you may replace Cys by serine (Ser), alanine (Ala), or a-aminobutyric acid (Abu). Alternatively, choose the hydrophilic Cys(Acm) and leave protected. For the simultaneous preparation of peptides of different sizes with free ammo terminus, couple the terminal ammo acid residues as o.N-Boc derivatives, so that they will not become acetylated durmg the normal elongation cycle Boc is then removed during the final side-cham-deprotection procedure (Section 3.4.). 3. Assembly of peptide pools on spots: This procedure follows essentially the steps of Section 3.3., except that the amino acid stock solutions are diluted to 50 mM with NMP and activated with only one-quarter of DIC. For X-couplings, an equimolar mixture of the amino acid derivatives to be presented at random positions is prepared by combining equal aliquots of the diluted stock solutions (e.g., all 20). This mixture is activated and applied in the same way as the other individual amino acid solutions. Spotting per elongation cycle is repeated up to four times. 4. Usually, proteins do not tend to stick to the cellulose support. However, some peptide spots can efficiently trap proteins, such as the secondary antibody conjugates. Thus, an effective blocking of these unspecific interactions is required. So far, optimum results are obtained with the recipe of Genosys, but you may try other recipes. Always check for unspecific binding of your secondary antibody first by omittmg steps 5 and 6 in the assay procedure of Section 3.5.

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5. The reporter enzyme of the secondary antibody conjugates was chosen to allow the mildest reaction conditions possible in order to protect the peptides for multiple reuse of the membranes. P-Galactosidase, imtially used, gave considerable unspecific signals; peroxidases were excluded to avoid hydrogen peroxide. Alkaline phosphatase performs very well, despite the color development bemg carried out at pH 7.0! 6. For color development, a number of mild oxidative reagents were tested to convert rapidly the indoxyl generated by hydrolysis of the BCIP substrate into the unsoluble indigo dye. Tetrazolium salts are perfect, since they are simultaneously converted to water-insoluble dyes. MTT was identified as optimum, particularly with regard to its good solubility in organic solvent, which is required for a mild stripping of the membranes. Steps 11 and 12 in the antibody-binding assay protocol (Section 3.5.) are omitted if other detection systems are being used that do not precipitate a dye compound. 7. Bromophenol blue staining is a very helpful visual aid to momtor the efficiency of the steps during peptide assembly. It is, however, no quantitative means. Some peptide sequences and terminal amino acid residues, such as Cys, Asn, or Asp, give a particularly weak staining. The indication of free amino functions is quite sensitive, and a color change to green is sufficient for a good couplmg. 8. Synthetic peptides mimicking B-cell determmants represent fragments of a longer continuous protein chain. To account for this, these peptides should be N-terminally acetylated. Alternatively, special labels can be attached to the N-termmi of peptides by spotting respective derivatives. We have successfully added biotm via its in situ formed HOBt-ester (normal activation procedure) or fluorescem via its isothiocyanate (FITC) dissolved in DMF. Synthetic peptides to be used m T-cell eprtope mapping, particularly when MHC class I restricted, are required with free amino- and carboxy-termini in solution (see also Note 2).

References 1. Frank, R., Giiler, S., Krause, S., and Lindenmaier, W. (1991) Facile and rapid “spotsynthesis” of large numbers of peptides on membrane sheets, in Peptides 2990 (Giralt, E. and Andreu, D., eds.), ESCOM Science Publishers B. V , Leiden, pp. 15 1,152 2. Frank, R. (1992) Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48,92 17-9232. 3. Krchnik, V., Vagner, J., Safar, P., and Lebl, M. (1988) Noninvasive continuous monitoring of solid phase peptide synthesis by acid-base indicator. Collect. Czech. Chem. Commun. 53,2542-2548. 4. Kramer, A,, Volkmer-Engert, R., Malin, R., Reineke, U., and SchneiderMergener, J. (1993) Simultaneous synthesis of peptide libraries on single resm and continuous cellulose membrane supports: examples for the identification of protem, metal and DNA binding peptide mixtures. Pept. Rex 6,3 14-3 19. 5. Frank, R. (1994) Spot-synthesis: an easy and flexible tool to study molecular recognition, in Innovations and Perspectives In Solid Phase Synthesis 1994 (Epton, R., ed.), Mayflower Worldwide Ltd., Birmingham, pp. 509-5 12.

SPOT Synthesis 6. Bray, A. M., Maeji, N. J., and Geysen, H. M. (1990) The simultaneous multiple production of solution phase peptides; Assessment of the Geysen method of srmultaneous peptide synthesis. Tetrahedron Lett. 31,581 l-58 14. 7. Hoffmann, S. and Frank, R. (1994) A new safety-catch peptide-resin linkage for the direct release of peptides into aqueous buffers. Tetrahedron Lett. 35,7763-7766. 8. Hoffmann, S. and Frank, R. (1995) in preparation. 9. Kramer, A., Schuster, A., Reineke, U., Malin, R., Volkmer-Engert, R., Landgraf, C., and Schneider-Mergener, J. (1994) Combinatorial cellulose-bound peptide libraries: screening tools for the identification of peptides that bind ligands with predefmed specificity. METHODS (Comp. Methods Enzymol.) 6,9 12-92 1. 10. Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986) A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Immunol. 23, 709-715. 11. Dooley, C. T. and Houghten, R. A. (1993) The use of positional scanning synthetic peptide combinatorial libraries for the rapid determination of opioid receptor ligands. Life Sci 52, 1509-l 5 17. 12. Frank, R,, Kieb, M., Lahmamr, H., Behn, Ch., and Gausepohl, H. (1995) Combinatorial synthesis on membrane supports by the SPOT technique, in Peptides 1994 (Mara, H. L. S., en.), ESCOM, Leiden, pp. 479,480. 13. Ersfeld, K., Wehland, J., Plessmann, U., Dodemont, H., Gerke, V., and Weber, K. (1993) Characterization of the tubulin-tyrosine ligase. J. Cell Biol. 120,725-732.

16 Tea Bag Synthesis of Positional Scanning Synthetic Combinatorial Libraries and Their Use for Mapping Antigenic Determinants Clemencia

Pinilla, Jon R. Appel, and Richard A. Houghten

1. Introduction Positional scanning synthetic combinatorial libraries (PS-SCLs) are useful in identifying the amino acid sequences of antigenic determinants recognized by monoclonal antibodies (MAbs). These libraries are typically composed, in total, of tens of millions of nonsupport-bound peptides, and can be readily screened using enzyme-lmked immunosorbent assay (ELISA) to determine specrfic sequences that bind to a target antibody. From our studies using peptide libraries, we have found that MAbs exhibit a broad range of specificities, ranging from those recognizing only conservative substitutions at one or two positions in the antigenic determinant to antibodies that recognize sequences completely unrelated to the parent antigen while having comparable affinities (1-10). The information derived from the screening of PS-SCLs is similar to that of “fingerprint” profiles using individual substitution analogs at each position of the antigenic determinant (II). For example, the more specific a position is, the fewer amino acids in the peptide mixtures that will be found to be effective. On the other hand, if there are many active peptide mixtures for a given defined position, then that position can be considered redundant or replaceable. In other instances, there may be several amino acids that are conservative replacements of each other that have equal activities. Additionally, since the diversities of PS-SCLs consist of millions of sequences, the extent of the multiple binding specificities of a given antibody can be readily addressed in a systematic manner. Finally, it should be noted that a single PS-SCL can be used to identify antigenic determinants and high-affinity sequences for many different antibodies. From

Methods m Molecular Biology, vol 66 Epltope Mapprng Protocols Edlted by G E Morris Humana Press Inc , Totowa, NJ

171

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Pinilla, Appei, and Houghten

Antibodies that have been raised against proteins often recognize discontinuous antigenic determinants. Since peptide libraries are composed of linear hexapeptide sequences, one may assume that such libraries are not applicable for identifying discontinuous determinants. In one example, two different sequences were independently identified from a PS-SCL along with an iterative synthesis and selection process that are recognized by a MAb against the surface antigen of hepatitis B virus (20). The two sequences were synthesized as one linear peptide and found to have comparable affinity with the proteirrantibody interaction. The PS-SCL method is very rapid for the identification of high-affinity peptide ligands. Screening of the PS-SCL against one or more antibodies takes a single day. If the results indicate good inhibition for one peptide mixture for each of the six positions, then the peptide sequence can be determined directly from the ELISA data. If more than two or three peptide mixtures are found to have good activity at any of the six positions of the PS-SCL, then individual peptides, representing the combinations of the most effective amino acids in the defined positions, need to be synthesized. These individual peptides are then tested by competitive ELISA in order to identify the most effective peptide resulting from the screening of the PS-SCL. The positional scanning concept can also be extended to at least a decapeptide format. Highly active sequences recognized by an MAb have been identified from a decapeptide PSSCL composed of four trillion sequences (6). This protocol describes the synthesis and use of PS-SCLs for the identification of individual peptide sequences that inhibit the binding of a target antibody to its respective antigenic peptide or protein. This protocol includes methods for the synthesis of a hexapeptide PS-SCL, standardization of the ELISA conditions of the antigen-antibody interaction, and the screening of the PS-SCL. Owing to the enormous number of peptides in each peptide mixture of PS-SCLs, the specific ELISA conditions used for each antigen-antibody interaction study must be optimized to achieve the best level of sensitivity. 2. Materials

2.1. Library Synthesis 1. Methylbenzyhydrylamine (MBHA) polystyrene resin (Peninsula, Belmont, CA). 2. Protected amino acids (either Boc or Fmoc chemistries; Bachem, Torrance, CA). 3. Appropriate solvents and reagents for solid-phase peptide synthesis (Fisher, [Pittsburgh, PA], Aldrich [Milwaukee, WI]). 4. Polypropylene mesh for tea bags (McMaster Carr, Los Angeles, CA).

Positional Scanning Libraries

173

2.2. ELBA 5. 96-Well microtiter plates (high-binding polystyrene, l/2 area, Costar [Cambridge, CA] #3690 or Dynatech Immunolon 4 [Chantilly, VA]). 6. 1-mL (for peptide mixture dilutions) and 50-mL (for antigen and antibody dilutions) polypropylene tubes. 7. 8- or 1Zchannel pipeter and repetitive pipeter (Brinkman Instruments, Westbury, NY). 8. Peptide or protein antigen and corresponding MAb. 9. PBS: 0.3M bicarbonate, pH 9.3, buffer, 1% w/v BSA/PBS as blocking buffer. 10. Antimouse-peroxidase conjugate antibody (or another appropriate antibody detecting reagent; Calbiochem, La Jolla, CA). 11. Enzyme substrate and developing reagents. For peroxidase, use 3% Hz02 and o-phenylenediamine (OPD) tablets (Sigma [St. Louis, MO] #P-8287). For each plate, dissolve one tablet of OPD in 6 mL of deionized water, and add 25 pL of 3% hydrogen peroxide. 12. 4N sulfuric acid. 13, Microplate spectrophotometer.

3. Method 3.1. Synthesis of PS-SCL The peptrde mixture

resins making

up either a nonacetylated

or an N-acety-

lated hexapeptide PS-SCL are prepared using the chemical mixture approach (i.e., a specific ratio of a mixture

of amino acids) (12) in conjunction

with

simultaneous multiple peptide synthesis (SMPS) (13) using MBHA resin and t-Boc chemistry. A hexapeptide PS-SCL consists of six positional libraries, each of which has a position defined with a single amino acid and a mixture of amino acids at the remaining five positions (Table 1). Thus, ahexapeptide PS-SCL is made up of 120 different peptide mixtures. In total, the PS-SCL contains nearly 50 million different hexapeptides. This synthesis protocol is designed for a person trained and experienced in solid-phase peptide chemistry. 1. Number 120 polypropylene tea bags, and add 100 mg MBHA resin to each. 2. Use SMPS methodology to synthesize peptide mixtures. For the first coupling step, couple the 20 L-amino acids individually to bags 10 l-120. (We use a singleletter code for amino acids, in alphabetical order) To bags l-100, couple a 19-amino acid mixture as shown in Table 2. Cysteine is excluded in the mixture positions to avoid disulfide aggregates. 3. At the second step, couple 20 amino acids individually to bags 81-100. Couple the 19-amino acid mixture to bags I-80 and 101-120. 4. Repeat through the sixth coupling. 5. Acetylate the N-terminal of the peptide mixtures, if desired (see Note 1). 6. Deprotect and cleave the peptide mixtures from the resin using the low-hi hydrogen fluoride method (14,15). Note: Hazardous.

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174

Table 1 Positional Scanning Synthetic Combinatorial Library (PS-SCL) #l-20 #2l-40 #4l-60 #6 l-80 #8 l-l 00 #lOl-120

0, XxXxX-NH2 X02XXXX-NH2 XXOsXXX-NH2 XXX04XX-NH2 XXXXOSX-NH2 XXXXX06-NH2

0 = single L-ammo acid. X = mixture of 19 L-amino acids.

7. Extract peptide mixtures from the resin with water or dilute acetic acid. 8 Lyophilize solutions twice, and reconstitute material in water at 10-20 mg/mL. Sonicanon 1soften used for peptide mixtures containing hydrophobic amino acids m the defined positions. 9. Aliquot in 1-mL polypropylene tubes. Store for l-2 wk at 4’C while m use or indefinitely at -20°C

3.2. Direct ELBA Antigen and antibody are titered against each other using direct ELISA to determine the optimal concentrations of both reagents. These concentrations will be used to determine fixther the appropriate library screening conditions using competitive ELISA. 1 Coat microtiter plates with 50 pL/well antigen (l-10 pg/mL) m PBS or bicarbonate buffer (try both buffers separately to see which might give better results) Perform twofold serial dilutions of the antigen across the plate, starting with a high concentration (10 pg/mL). Incubate for either 2 h at 37°C or 18 h (overnight) at room temperature in a moistened box to avoid evaporation of reagents. 2. Shake out lrquid from wells and wash plates 10x with deionized water. Remove residual water from wells by rapping plates upside down over paper towels. Avoid complete drying of wells. Repeat washing step after each incubation. 3. Block plates for nonspecific binding by adding 100 pL/well of 1% (w/v) BSA/ PBS to microtiter plates, and incubate plates for 1 h at 37°C. 4. Add 50 FL/well of antibody, performing twofold serial dilutions in 1% BSA/PBS down the plate. Incubate plates overnight at 4’C (see Note 2). 5. Add 50 pL/well of secondary antibody+rzyme conjugate (goat-antimouse peroxidase) at manufacturer’s specified dilution in 1% BSAIPBS. Incubate plates for 1 h at 37°C. 6. Add 50 pL/well of developing solution to plates, and develop in the dark for lO15 min. Terminate developing reaction with 25 pWwel1 of 4N sulfuric acid. 7. Read plates on a microplate spectrophotometer at 492 nm.

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Table 2 Ratio of Amino Acids Used for the Synthesis of PS-SCLs Amino acid Alanine Aspartic acid Glutamic acid Phenylalanine Glycine Histidine Isoleucine Lysine Leucine Methionine Asparagine Proline Glutamine Argmine Serine Threonine Valine Tryptophan Tyrosine

Mol”ha 3.4 3.6 3.7 2.6 2.9 3.6 55 6.3 5.0 2.3 5.4 4.4 5.4 6.6 2.8 4.8 5.0 3.8 4.2

aMoloh of eachamino acid derivative necessaryfor equlmolar coupling when using a lo-fold excess of amino acid denvatrve over resm. 8. Choose the concentrations for antibody and antigen that give the optimum results, i.e., lowest antigen and antibody concentrations that still give high OD values (1 S-2.0). Use these conditions for competitive ELISA (see Note 3).

3.3. Competitive ELBA 1. Coat microtiter plates with the control antigen at the predetermined concentration (see Section 3.2., step 8), and incubate plates in a moist box for 2 h at 37°C 2. Wash plates 10x with deionized water and after each subsequent mcubation. 3. Block for nonspecific binding as in direct ELISA. 4. Add 25 uL/well of blocking buffer to each plate. Add 25 pL/well of control antigen (10, 100, and 1000 times the amount of control antigen on the plate) to the top row, and perform twofold serial dilutions down the plate. A fixed dilution of MAb (25 pL/well) is added to each well. Do the same for antibody concentrations two to five times higher and lower than the selected concentration. Incubate plates for 18 h at 4°C.

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5. Add goat-antimouse peroxidase conjugate, and develop and read plates as m direct ELISA. 6. Determine the concentration of antigen that inhibits 50% antibody binding (IC,,) of the control antigen for the three antibody concentrations. Choose the antlbody concentration that gives the lowest KS0 while maintaining an acceptable slgnalto-noise ratio (10: 1). Use these conditions to screen the PS-SCL.

3.4. Screening

PS-SCL

The hexapeptide PS-SCL is screened by competitive ELISA to identify specific peptides that inhibit the binding of an MAb binding to its antigen. It is important to screen the library at a high concentration (5-10 mg/mL) owing to the enormous diversity of peptides within each peptide mixture (see Note 3). If there are many active peptide mixtures

on the initial

screening,

those active

peptide mixtures can be serially diluted and tested again to select the most active peptide mixtures

at each position

(see Note 4).

1. Perform steps l-3 of Section 3.3. using six plates. 2. Add the 120 peptide mixtures of the PS-SCL according to the scheme in Fig. 1 The first column of each mlcrotiter plate is used for 100% antibody binding to the antigen on the plate (no Inhibitor). The second column is used for the antigen m solution serially diluted as a competitive control to ensure the assay is working in a sensitive manner. The remaining 80 wells on each plate are used to test 20 peptide mixtures using the configuration shown in Fig. 1. We use 1-mL polypropylene tubes to aliquot the peptide library. The peptide mixtures are then arranged starting with tubes #l-10 (or Al-Ll) across the top of the plate in wells A3-A12, and tubes #l l-20 (Ml-Y 1) in wells E3-E12. Each peptide mixture (50 yL at 20 mg/mL) is diluted twofold four times using a multichannel plpeter. 3. Once the PS-SCL has been added to the plates, use a repetitive multichannel pipeter to add antibody at a fixed dilution (25 pL/well) prevtously determined to compete effectively for binding of the control antigen m solution with the control antigen on the plate. 4. Incubate plates for 18 h at 4°C. 5. Perform steps 5-7 of Section 3.2. 6. Express inhibitory activity ofpeptide mixtures as optical density (OD) values (inhibition = low OD), or convert peptide mixture a&iv&y to percentage of inhibition relative to the binding of antibody to the control antigenfrom column 1 of each plate. 7. Retest peptide mixtures that were found to have good inhibitory activities (> 50% inhibition) at lower peptide concentrations. 8. Determine inhibitory concentrations at 50% of antibody binding (ICsO) for each peptide mixture in order to select the most active peptide mixture(s) from the peptlde library (see Note 4). 9. Synthesize individual peptides that correspond to the combinations derived from the most active peptide mixtures at each position, and assay them by competitive ELISA in order to confirm the screening of the PS-SCL (see Note 5).

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Fig. 1. Layout for the screeningof 20 peptide mixtures (see Section3.4.2.). 4. Notes 1. A nonacetylated PS-SCL will be a better choice for screenmg those antigenantibody interactions that require a free amine on the N-terminal of the peptide for antibody recognition. For example, a nonacetylated PS-SCL was successfully used for the identification of the antigenic determinant of P-endorphin as recognized by MA\, 3E7, since the first residue of the antigenic determinant is the first residue of P-endorphin, namely tyrosine, which has a free amine group (7). Likewise, we recently identified calcium-independent sequences that were 100 times more active than the known antigen for a calcium-dependent MAb using a nonacetylated PS-SCL (9). 2. For most of the antigeHntibody systems we have examined, better assay sensitivity is obtained when the antibody incubation step is carried out at 4°C. However, incubation at 37’C for 1 h may give reasonable results in some cases. We have found that the lowest antibody concentration taken from the top of the linear portion of the saturation binding curve gives the best sensitivity for the competitive ELBA. 3. Standardization and optimization of ELISA and screening conditions for each antigerrantibody interaction are essential for the successful identification of antigenic determinants using PS-SCLs owing to the number of peptide sequences in each peptide mixture. When tested at 5 mg/mL, each peptide in the mixtures of the PS-SCL is present at a concentration of 3 nM. However, positional redundancy is often found in most antigen-antibody interactions. In this manner, the relative concentration of the family of antigens recognized by the antibody would be much higher

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Pinilla, Appel, and Houghten

4. It is also important to generate dos+response curves, and determine IC,, values in order to select the most active amino acids at each position. The combinations of amino acids are then used to make the individual peptides. If the sequence of the umnunogen is known, the screening results can be used to locate the antigenie determinant recognized by the antibody quickly. 5. The number of amino acids selected from each position that will be used to synthesize the individual peptides should be minimized. For example, if two amino acids are selected from each position, one would need to synthesize 64 pepttdes. It should be noted that smce the PS-SCL IS composed of six separate positional SCLs, each one can be considered independent of the others. Therefore, each positional SCL can be independently screened and pursued using the iterative synthesis and selection process (1,2).

References 1. Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T., and Cuervo, J. H. (199 1) Generation and use of synthetic peptide combmatorial libraries for baste research and drug discovery. Nature 354, 84-86. 2 Houghten, R. A., Appel, J. R., Blondelle, S. E., Cuervo, J. H., Dooley, C. T , and Pinilla, C. (1992) The use of synthetic peptide combinatorial libraries for the tdentitication of bioactive peptides Biotechnzques 13,4 12-42 1 3. Pinilla, C., Appel, J. R., Blanc, P., and Houghten, R. A (1992) Rapid identitication of high affinity peptide ligands using positional scanning synthetic pepttde combinatorial libraries. Biotechniques 13,901-905 4. Appel, J. R., Pinilla, C., and Houghten, R. A. (1992) Identification of related peptides recognized by a monoclonal antibody using a synthetic peptide combinatorial library. Zmmunomethods 1, 17-23. 5. Pmilla, C., Appel, J. R., and Houghten, R. A. (1993) Synthetic pepttde combmatorial libraries (SCLs): identification of the antigenic determmant of l3-endorphin recognized by monoclonal antibody 3E7. Gene 128,71-76. 6. Pinilla, C , Appel, J. R., and Houghten, R. A. (1994) Investigation of antigenantibody interactions usmg a soluble nonsupport-bound synthetic decapeptlde hbrary composed of four trillion sequences. Biochem. J 310, 847-853. 7. Pinilla, C., Appel, J. R., Blondelle, S. E., Dooley, C. T., Eichler, J., Ostresh, J. M., and Houghten, R. A. (1994) Versatility of positional scanning synthetic combmatorial libraries for the identification of individual compounds. Drug Dev Res. 33,133-145. 8. Pinilla, C., Buencamino, J., Appel, J. R., Houghten, R. A., Brassard, J. A., and Ruggeri, Z. M. (1995) Two antipeptide monoclonal antibodies that recognize adhesive sequences in fibrinogen: identification of anttgenic determinants and unrelated sequences using synthetic combinatorial libraries. Biomed. Pept. Prot Nucleic Acids 1, 199-204. 9. Pinilla, C., Buencamino, J., Appel, J. R., Hopp, T. P., and Houghten, R. A. (1995) Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: identitication of potent calcium-independent antigens. Mol. Diversity 15,21-28.

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10. Pinilla, C., Buencamino, J., Houghten, R. A., and Appel, J. R. (1995) Detailed studies of antibody specificity using synthetic peptide combinatorial libraries, in Vaccines 95 (Chanock, R. M., Brown, F., Ginsberg, H. S., and Norrby, E., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 13-17. 11. Pinilla, C., Appel, J. R., and Houghten, R. A. (1993) Functional tmportance of amino acid residues making up peptide antigenic determinants. Mol. Immunol. 30,577-585.

12. Ostresh, J. M., Winkle, J. H., Hamashin, V. T., and Houghten, R. A. (1994) Peptide libraries: determination of relative reaction rates of protected ammo acids in competitive couplings. Blopolymers 34, 1681-1689. 13. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Nat1 Acad. Sci. USA 82,5 13 l-5 135. 14. Tam, J. P., Heath, W. F., and Merrifield, R. B. (1983) SN2 deprotection of synthetic peptides with a low concentration of HF in dimethyl sulfide: evidence and application in peptide synthesis. J, Am. Chem. Sot. 105,6442-6455. 15. Houghten, R. A., Bray, M. K., De Graw, S. T., and Kirby, C. J. (1986) Simplified procedure for carrying out simultaneous multiple hydrogen fluoride cleavages of protected peptide resins. Int J Pept. Protein Res 27,673-678

Epitope Mapping Using Phage-Displayed Peptide Libraries Diane Dottavlo 1. Introduction Monoclonal antibodies (MAbs) play a key role in defining structural, functional, and regulatory aspects of complex protein-protein interactions (1-4). The ability to identify the epitope recognized by an antibody and to understand how it relates to the primary or tertiary structure or the function of the protein has proven to be a difficult process. Epitope mapping experiments have shown that antigenic determinants fall into two major classes. Conformational or discontinuous epitopes consist of residues widely spaced in the primary sequence, yet brought in close proximity of one another by protein folding. These determinants are present on native protein, but are lost on denaturation or fragmentation. Linear or continuous epitopes contain at least four to six adjacent ammo acid residues of the primary sequence and can be identified on denatured as well as native protein (5-9). The first attempts to identify a recognition sequence bound by an MAb involved screening a collection of antigen-derived peptide fragments (6). This approach served to localize the antibody-binding site to a specific region on the antigen (6-8). Subsequently, automated systems were developed to generate and probe larger collections of random, synthetic peptides, which could include, but were not exclusively, antigen-derived (10-23). These technologies have been employed to define more precisely smaller (minimum) linear epitopes (ZO), to identify conformational epitopes (I I) and “mimetopes” (reactive with the antibody, but unrelated to the antigen sequence [12-141). From* Methods in Molecular Bology, vol 66: Epifope Mapping Protocols Edlted by G E Morns Humana Press Inc , Totowa, NJ

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In the past five years, a novel molecular btological approach has been exploited to map antibody epitopes and to probe protein-protem interactions. This technology mvolves the construction of random peptide libraries displayed on the surface of filamentous bacteriophage (1549). The most widely used libraries are composed of peptides fused to either the amino-terminal domain of the phage coat protein pII1 (1618), or pVII1 (19). Vast numbers of unique peptide sequences (11 08) can be generated and screened simultaneously through a process called “biopanning.” Each round of competitive selection of the displayed peptides is followed by amphflcation of the binders, which results m the isolation of one or more subsets of related peptides. Each subset may define a consensus sequence; the degree of homology between the consensus sequence and contmuous stretches of ligand enables one to. 1. Localize the epitope; 2. Determine the amino acids most important for bindmg, and 3. Identify allowable substitutions m the varrable (nonhomologous)

posmons

(20-22). A collection of sequences that lacks consensus or similarity to the antigen sequence may indicate that the antibody recognizes a conformational epitope (23-25). The uttltty

of phage-displayed

pepttde

ltbrartes

was first demonstrated

through epitope mapping studies in which known, linear epitopes, recognized by certain MAbs, were selected from millions of peptide sequences (1618). A variety of peptide libraries have now been generated that incorporate structural features, such as “loops,” cysteine-flanked cychc peptides of random sizes, or “scaffolds,” rigid tertiary structures for presentation of various peptide randomers. These constramed libraries have enabled several groups to identify conformational epitopes and to study spatial requirements for binding and specificity (25-29). Construction of the first phage-displayed peptide libraries involved the mtroduction

of random oligonucleotides

into the genomic DNA of filamentous

bacteriophage by a variety of methods. Protocols often included restriction digests, which eliminate certain sequencesfrom the library, and intermolecular ligations, which reduce transformational efficiency. Moreover, these systems resulted in the multimeric display of peptides on the phage surface, allowing selection and enrichment based on avidity as well as affinity. In this chapter, we describe a phage-display system that utilizes phagemid technology and in vitro mutagenesis (30) to generate libraries of random peptides on the surface of bacteriophage (31). Expression of a modified gene III protein is placed under control of the Zac promoter on the phagemid vector, pGEM-3Zf+. With this system,helper phage supply the genes necessaryto pack-

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age the phagemid DNA and produce viable phage particles. The progeny contain phagemid DNA encoding the modified coat fusion protem in low copy number, in combination with wild-type coat proteins derived from the helper phage genome. Several reports have shown that monovalent expression can be achieved, and that it contributes significantly to the selection of high-affinity variants of the displayed polypeptides (32-34). The primary advantage of this system (31) is the ability to control the expression level of peptides on the phage surface through induction of the lac promoter, which may lead to the selection of higher affinity peptide ligands. Section 3.1. describes the preparation of the modified Ml 3 gene III fusion gene as it appears in the pGEM-3Zf+ phagemid vector. The assembly of the modified Ml 3 gene III introduces an 18nucleotide spacer between the leader and the second domain, which contains the enzyme restriction sites SpeI and X401, separated by 10 bp to ensure cleavage at both positions. The spacer was subsequently replaced by a 15mer lmker containing a Not1 restriction site, as described in Note 3, to aid in the selection of phagemid, which contained random oligomer inserts after mutagenesis. Preparation of peptide libraries using the Ml3 pIII.PEP vector is described. 2. Materials The following list of instruments, bacterial strains, helper phage, and btotechnology kits has been used for convenience in the protocols below. Other reagents can be substituted, but may need to be optimized to ensure successful results. For phage-related procedures, it is important to use autoclaved or disposable sterile flasks, tubes, bottles, and so on, and cotton-plugged pipets and prpet tips to avoid contamination of uninfected cells and crosscontamination of phage library stocks. 2. I. Vector Preparation I. Oligonucleotide synthesizerModel 392 (Applied Biosystems, Foster City, CA). 2. OPC Sepak columns #40077 1. 3. Wizard PCR “clean up” kit #A7170 (Promega, Madison, WI). 4. Ml3 RF, restriction enzymes, and T4 DNA ligase (Gibco/BRL, Grand Island, NY). 5. Perkin Elmer thermocycler and PCR kit (Perkin Elmer). 6. pGEM3Zf+ plasmid vector (Promega). 7. Supercompetent Escherzcia coli strain DHlOB (Gibco/BRL). 8. Electrocompetent XLI-Blue #200236 (Stratagene, La Jolla, CA). Store at-80% 9. LB broth: 10 g bactotryptone, 5 g yeast extract, and 5 g NaCl, pH 7.5, supplemented with 30 ug tetracycline/ml.

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2.2. Phagemid Library Preparation 10. Muta-Gene Phagemid In Vitro mutagenesis kit #I703582 with T7 DNA polymerase (Bio-Rad, Hercules, CA). Store at -20°C. 11. E. coli CJ236 (dut-,ung-) bacteria in agar stab. (Stratagene). Store m dark at room temperature. 12. Prep-a-gene kit (Bio-Rad). 13. Interference-resistant helper bacteriophage R408 (Invitrogen Corp., San Diego, CA). Store in lOO-pL aliquots at -80°C. 14. Modular heating block or water bath. 15. Small-scale CPG columns (0.2 @l4, 1000-A pore size) for oligonucleotide synthesis (Applied Biosystems). 16. Applied Biosystems 373A DNA sequencer and PRISMTM Dyedeoxy terminator sequencing kit. 17. TE buffer: 10 mMTris-HCl, pH 8.0; 1 mMEDTA. 18. Electroporator (Bio-Rad) and 0.2-cm cuvets (Gibco/BRL). Store cuvets at -20°C. 19. 2X YT broth (16 g bacto-tryptone, 10 g yeast extract, 5 g NaCl/L), pH 7.5, containing 70 &mL chloramphenicol or 50 pg/mL ampicillin.

2.3. Screening Phagemld Libraries 20. 2 1. 22. 23. 24. 25.

Antibody coating buffer: 50 mMNaHC03 buffer, pH 9.6. Phagemid resuspension buffer (TBS buffer): 50 mMTris-HCl, pH 7.4,150 mA4NaCl. Wash buffer: TBS buffer + 0.5% Tween-20. Elution buffer for phage: O.lM glycine buffer, pH 2.2. LB plates contain ampicillin (100 pg/rnL). X-gal (40 pg/mL) and 1 mM isopropyl-thiogalactoside (IPTG), where mdicated.

3. Method 3.7. Preparatlon of Phagemid Vector pIlLPEP Using Ml 3RF as template and the oligonucleotide pairs l/2 (oligo 1:5’-AAG CTT TAA GAA GGA GAT ATA CAT ATG AAA AAA TTA TTA TTC GCA, oligo 2:5’-GCC ACC ACT AGT GAA ATG AGG ACT CGA GAG CGG AGT GAG AAT AGA) and 3/4 (oligo 3:5’-GAT CTC ACT AGT GGT GGC GGT GGC TCT CCA TTC GTT TGT GAA TAT CAA, oligo 4:5’-GAA TTC TTA TTA AGA CTC CTT ATT ACG CAG), respectively, DNA encoding the gene III leader sequence (110 bp) and gene III second domain (65 1 bp) is amplified by PCR. The oligonucleotide 5’- and 3’-overhangs incorporate appropriate restriction

sites and complementary

overhangs for joining

and subcloning

the

fragments. A short IO-bp spacer between the Hz01 and 5”eI restriction sites separates the cleavage sites and is needed for SOEing (splice overlap extension) the fragments together. Following the QeI site, 15 nucleotides encoding the amino acid sequence GlyGlyGlyGlySer provide a flexible tether between the random peptide and the second domain of gene III.

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3.1.1. Generation of the Two Fragments Needed to Construct the Gene III Minigene 1. Set up two separate PCR reactions as follows (final volume, 100 pL): 100 ng M13RP template, 10X PCR buffer, Tug DNA polymerase (0.5 U), 1.5 mM MgCl*, 1.25 mMeach dNTP (final concentration). Add 10 pmol oligonucleotides 1 and 2 to generate the leader sequence of gene III and 10 pmol oligonucleotides 3 and 4 to amplify the second domain of gII1. (See Note 1 for purification of oligonucleotides.) 2. Set the PCR program for both reactions (35 cycles) as follows: 94°C for 30 s; 55’C for 30 s; and 72’C for 1 min. 3. Isolate the two PCR products on 2.0 and 0.6% agarose gels, respectively (35). 4. Purify the DNA using the Wizard DNA cleanup kit. 5. Elute the DNA from the matrix in 25 pL of TE, pH 7.5, previously heated to 55OC. 6. Determine the concentration DNA in each sample.

3.1.2. Preparation of the Gene III Fusion Gene by (SOEing) and PCR Amplification 1. Mix equimolar amounts (0.5 pmol) of each fragment together in an initial volume of 50 pL containing the following: Ml 3 leader fragment (18 ng), gII1 second domain fragment (100 ng), 10X PCR buffer (1.5 mMMgCl,, 1.25 mikfeach dNTP [final concentration]), and Taq DNA polymerase (0.5 U). 2. The PCR program links three different reactions; for the first 5 cycles (see Note 2) set the program at 94’C for 30 s, 50°C for 30 s, and 72’C for 1 min; follow with a 5-min hold at 4O’C. Add 50 pL containing the same salts, dNTP concentrations, and 10-20 pmol each of oligonucleotides 1 and 4 to the reaction. Set the PCR program for the next 40 cycles as follows: 94°C for 30 s; 65OC for 30 s; and 72OC for 1 min. 3. Isolate the 743-bp fragment on 0.6% low-melt agarose in TAE buffer, and purify using Wizard DNA cleanup kit. 4. Restrict the fragment with Hind111 and&&I, and ligate into pGEM3Zf+ (which had been similarly digested) to position the gene III fusion gene under control of the lac promoter.

3.7.3. Insertion of Specific Sequences in Place of the Spacer A final modification in the pIII.PEP vector was introduced by the insertion of a ‘Not1 linker,” which replaces the spacer and adds a unique Not1 restriction site between the 27~01and SpeI sites. In this way, phagemids that do not undergo mutagenesis could be identified by Not1 digestion. However, any coding sequence may be spliced into the gII1 fusion gene by this protocol. 1. Synthesize AAT ACT TCA GGA Biosystems

the oligonucleotide, 5’-G TCC TGA CGC TCG GCG GCC GCT AG-3’, and its complement, 5’-T ATT AGC GGC CGC CGA GCG CTC GA-3’, which encode the “Not1 spacer region,” using an Applied Model 392.

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2. Purify the ohgonucleotides on an OPC Sepak column equilibrated in triethanolamme bicarbonate buffer, according to the manufacturer’s protocol. 3. Vacuum-dry, resuspend the residue in 200 pL H,O, and determine the concentration. 4. Phosphorylate each oligonucleotide using 200 pmol of purified material, according to the protocol outlined in the Bio-Rad Muta-Gene kit. 5. Heat-inactive T4 polynucleotide kinase at 6YC. 6. Precipitate the reaction mix, using l/IO vol of 7.8M ammonium acetate and 2 vol ethanol. 7. Wash the pellet wtth 70% ethanol to remove traces of salts, dry, and resuspend at 10 pmol/pL in water. 8. Anneal the primers (10 pmol each) m 10 pL annealing buffer (Muta-Gene ktt) by placing the tube in a small heating block at 94”C, and allowing it to cool to 37”C, such that the ends formed 5’-overhangs complementary to XhoI and +eI restricted sites 9. Ligate the “linker” into the pGEM-3Zf+/pIII cassette vector, which had been restricted with XhoI and SpeI. 10. Use 1 pL of the ligation mix to transform supercompetent XL1 -Blue cells, using standard protocols (35). Alternatively, the in vitro mutagenesis protocol described below could be used to replace the linker regton with a sequence encoding a specific pepttde.

3.2, Preparation of Phagemid Peptide Libraries 3.21. Preparation of Random Oligonucleotides

for Library Synthesis

The oligonucleotides for library construction consist of random nucleotides encoding five or more amino acids, flanked by 30 “fixed” residues, which are complementary to the template on either side of the Not1 linker sequence. For example, libraries of (-) strand oligonucleotides were synthesized containing the sequence [5’ TGG AGA GCC ACC GCC ACC ACT AGT (SNN),CTC GAGAGCGGAGTGAGAATA3', whereS=GorC,andN=A,G,TorC] for linear peptides, and [5’ TGG AGA GCC ACC GCC ACC ACT AGT TGT (SNN), TGT CTC GAG AGC GGA GTG AGA ATA 3’1 for cyclic peptides. Varying the number of nucleotides encoding the random peptide region (SNN)x from 18 to 30 bases (X = 6-10 amino acids) has not affected the yields of dsDNA or transformation efficiencies. 1. Purify oligos as described in Notes 1 and 3. 2. Phosphorylate the oligos (200 pmol) according to the protocol outlined in the Bio-Rad Muta-Gene kit for use as primers in the in vitro mutagenesis protocol with (+) single-stranded, uracil-containing phagemid template. 3. Heat-inactivate T4 kinase. 4. Precipitate the reaction mix and wash the pellet as above. 5. Resuspend at 10 pmol/pL and store at -20°C.

Phage-Displayed

Peptide Libraries

187

3.2.2. Preparation and Purification of Template SSDNA The single-stranded,uracil-containing DNA packaged in the phagemid particles servesas the template for second-strand synthesis(seeNote 4). Therandomoligonucleotide pool described above is used collectively to prime second-strand synthesis. To isolate ssDNA template, follow the protocol outlined in the Bio-Rad Ml3 in vitro mutagenesiskit, using the bacterial strain CJ236 (dut-,ung-). 1. Inoculate 4 x 125 mL flasks containing 25 mL 2X YT broth (50 yg/mL ampicillin and 70 &mL chloramphenicol) with 2.5 m.L C3236 (dut-,ung) infected with the phage prepared from ~111.PEP in Section3.1. (seeMuta-Genekit instructions) and grown to approx 0.5 Abm.

2. Culture the cells for 8 or 16h (overnight). 3. Centrifuge twice at 12,000 rpm at 4’C for 30 min to remove cells and debris. 4. Precipitate the phage by adding l/4 ~0120% PEG 8000/3.5Mammonium acetate to the clarified supematant, and let stand on ice for 1 h. 5. Collect phage by centrifugation at 12,000 rpm at 4°C for 30 min. 6. Carefully pour off the supematant from the pellet and drain dry on paper towels. 7. Resuspend the pellet in 0.2-l .O mL TE buffer and extract twice with equal volumes of TE-saturated phenol, followed by equal volumes of chloroform until the interface is clear. 8. Precipitate with 3M sodium acetate and ethanol as previously described. 9. The DNA is diluted to 200 ng/yL and stored at -2O’C in 100~pL aliquots. Puri-

fied ssDNA is approx 20-100 pg, dependingon the growth interval. 3.2.3. Preparation of Phagemid Libraries from Synthesized dsDNA 1. Just prior to use, purify 2 pg ssDNA (10 pL) on 10 pL resin according to the BioRad protocol in the Prep-a-gene kit (see Note 4). 2. Elute the template in 25 pL TE buffer and use directly in the annealing reaction. 3. Using the Bio-Rad in vitro mutagenesis kit, mix 1.5-2 pg (approx 1 pmol) of template ssDNA, 20-30 pmol of primers, and 10 pL annealing buffer (10X) in 100 pL annealmg reaction (see Note 5). 4. On completion of the synthesis reaction (200 yL containing 20 pL synthesis buffer [ 1OX], 10 U T7 polymerase, and 30 U T4 ligase), run 10 pL on an agarose gel to determine if the reaction is complete. 5. Extract the double-stranded DNA twice with an equal volume of phenol (equilibrated with 10 mM Tris-HCl, pH 8.0, 1 mA4 EDTA). 6. Extract twice with chloroform and then precipitate with l/10 vol 7.8MNH40Ac and 2 vol ethanol (see Note 6).

7. Centrifuge the DNA pellet, rinse with 70% ethanol, vacuum dry, and resuspend in water (10 pL).

8. Electroporate 1 pL DNA (36) into eachof 10aliquots of electrocompetentXLlBlue (500 ng DNA, 80 pL cells, 0.2 cm cuvet, 2.5 kV/cm, 200 R, 25 pF) using a Bio-Rad Gene Pulser apparatus. Electrocompetent bacteria may be purchased or

prepared in the laboratory (seeNote 7). Eachaliquot is immediately diluted in 1 mL of ice-cold SOC medium.

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188 3.2.4. Ampification of Phagemid Peptide Libraries

1. Combine the aliquots of transformed cells (10 mL) and shake (200 rpm) at 37OC for 1 h to let the cells recover.

2. After 1 h, remove 10JJLfrom the culture andplate dilutions onto LB agar plates containing ampicillin

(100 yg/mL) to score the total number of independent

transformants. 3. Add R408 helper bacteriophage to the remaining culture at a multiplicity of infection (MOI) of 10, and incubate with shaking at 50 rpm for 1 h at 37°C. Alternatively, to generate multivalent phagemid libraries, add IPTG (1 mM) to the culture for 1 h, prior to the addition of helper phage. 4. Dilute the culture into 1 L (2 x 500 mL) of 2X YT (in 2-L flasks) containing ampicillin (100 ug/mL) and tetracycline (30 pg/mL), and shake at 200 rpm for 15 h (20). 5. Remove cells from the culture by centrifugation (twice at 12,000g for 10 min)

andtransfer the cleared supematantto sterile flasks. 6. Add II4 vol of 3.5iWammonium acetate and 20% polyethylene glycol, PEG 8000 to the supernatantto precipitate the phageand chill on ice for 1h. 7. Harvest the phage by centrifugation at 17,000g for 20 min and discard the super-

natant. 8. Resuspend the pellet in a final volume of 5 mL TBS (50 mM Tris-HCl, pH 7.4,

150m&f NaCl). Titer the phage (transducing units) by infecting 100 I.~Lof E. coli XL 1-Blue cells (A6s0=l .O)with 100 I.~Lof the phage resuspension (l/l 0 serial dilutions). Plate on LB agar plates containing ampicillin (100 I&&) and count colonies (CFU). Typically the yield is at least 10’ I-10t2/mL. 3.3. Panning Phage-Displayed

Peptide Libraries

Several strategies and rationales for screening phage-displayed peptide libraries have been used to identify the epitope recognized by an antibody or receptor. In our system, the initial screen may be performed with a library displaying a high copy number of peptides (IPTG-induced) on the phage surface to select peptides, which bind with a broad range of affinities. In subsequent pans, higher-affinity motifs might be distinguished from low-affinity (avidity) binders by panning with an uninduced, amplified phagemid subset, which displays a reduced number of peptides on the phage surface. Similar variation in screening conditions has been achieved by diluting the concentration of the antibody during subsequent screens. However, our system remains versatile in cases where the receptor density cannot be easily recombinantly expressed, regulated on, or isolated from the cell surface. In the panning protocol given below, IPTG-induced or uninduced phage-displayed peptide libraries may be used.

Phage-Displayed Peptide Libraries

189

When more information about the ligand is known, alternative strategies may be employed to determine the epitope more quickly or efficiently. For example, phage libraries were panned by competitive elution with somatostatin-14 (Sigma), the NH%-terminal 14 amino acids of the ligand known to crossreact with this mouse antihuman somatostatin antibody (SOM- 14; BiosPacific, Emeryville, CA), as follows: 1, Coat microtiter plates (96 well) with 100 pL of the same antibody (20 pg/rnL in 50 mMNaHCO,, pH 9.6) for 2 h at 37’C. 2. Wash the plates twice with TBS/O.S% Tweet+20; rinse twice with TBS, and block with bovine serum albumin (3% in 50 mMNaHCO,, pH 9.6) for 2 h at 37’C. 3. Wash and rinse plates as before and incubate overnight with the library phage (lOlo transducing units in 100 pL) at 4’C. 4. Wash wells 10 times with 250 pL of TBS/OS% Tween-20 to remove unbound and nonspecifically bound phage, then rinse twice with 250 pL of TBS. 5. Elute bound phage first for 1 h at room temperature with 100 pL of 100 r.Ln/r somatostatin-14. 6. Transfer eluates to mlcrofuge tubes and carry out a second elution for 24 h at room temperature with an additional 100 pL of 100 fl somatostatin-14. 7. Following the second elution, incubate each 100 U 1 mL of E coli Xl 1-Blue cells (A,,, = 1.0 in LB broth with 30 pg tetracycline/ml) for 30 min at 37°C. 8. Plate samples (10 pL) of infected cells onto LB plates containing ampicillin (100 pg/mL) to determine the number of phage eluted at each step. The remaining phage are amplified and harvested as described above. 9. Use recovered amplified phage from the 24 h elution for the next round of panning, and repeat the entire process for a third round. After the third pan, pick 50100 colonies from each library from the final 24-h elution plates, and sequence the phagemid DNA using an Sp6 oligonucleotide. Synthesize several of the deduced peptides for binding evaluation.

4. Notes 1. Synthesize oligonucleotides bearing random sequences on an Applied Biosystems Model 392, using the 0.2~pmol synthesis scale and the 1000-A pore size ABI columns. Cleave the oligonucleotides from the synthesis column and deprotect in ammonium hydroxide (55°C for 8 h). Isolate the oligonucleotides on base-resistant OPC columns in triethylamine bicarbonate (TEAB) buffer, according to the manufacturer’s protocol. Collect aliquots (8 x 500 pL) and dry down in a Savant Speed-Vat. At this point, the oligonucleotide preparation is approx 80% pure. 2. No oligonucleotides were added to the initial reaction. During the first five cycles, the 3’-end of the M 13 leader fragment (110 bp) anneals to the complementary overlap (18 bp) added to the 5’-end of the DNA fragment encoding the gene III second domain (651 bp), and primes the extension of the overlapping ends. Addition of oligonucleotides 1 and 4 during the subsequent cycles amplifies a 743-bp fragment.

190

Dottavio

3. Further purify the random oligos on polyacrylamide (20%)/urea gels (3.5) to remove contaminants (see Note l), which interfere with second-strand prrmmg and synthesis. Resuspend the oligonucleotides in 50 uL TE, add an equal volume of sample buffer, and electrophorese the samples at 150 V until the Bromphenol blue runs about two-thirds of the way into the gel. Visualize the oligonucleotrde band by UV absorption, cut out, and elute overnight in TE buffer at 4°C. After preciprtation usmg l/10 vol7.8Mammonium acetate and 2 vol ethanol, wash the pellet in 70% ethanol and resuspend in water (50 uL). Adjust the concentration to 10 pmol/uL based on the absorbance determined at 280 nm. 4. Purification of the ssDNA and the oligonucleotides prior to performing secondstrand syntheses 1s of utmost importance. Conversron of template ssDNA to covalently closed dsDNA is required for high-efficiency transformation of bacteria and generation of vast repertoires of peptide sequences. The additional purity achieved by the Prep-a-gene (Bio-Rad) purification step dramatically improves dsDNA synthesis and subsequent transformation efficiency. Promega (Wizard) and BioRad (Prep-a-gene) kits no longer contain the same DNA-binding resin; the recovery of ssDNA from the Promega resin 1snot recommended by the manufacturer. If another kit is used, it is important to check whether ssDNA can be recovered from the resin supplied. 5. Heating the annealing mixture of random primers and ssDNA template to 95°C in a water bath (or heating block), and then allowing the bath to come to 37°C increases the efficiency of priming with the “randomer” ohgonucleotides. 6. In vitro mutagenesis protocols generally predict a 50-80% conversion of “wildtype” template to mutant sequence That suggests that as many as 50% of the transformants carry the Not1 template sequence. To reduce this background, the synthesis reaction is purified as described above and restricted with Not1 in the appropriate digestion buffer. The material that does not restrict is gel-purified and used in the electroporation protocol. 7. Electrocompetent XL-1 Blue cells can be purchased or made m the laboratory using the following protocol. All steps should be carried out m the cold room, using prechilled prpets and tubes, and the cells transported on ice for centrrfugation. a. Prepare an overnight culture from frozen cells (XL-l Blue, and so on) in 20 mL LB broth and tetracycline (35 ug/mL) at 37’C. b. Dilute 20 mL overnight culture (l/100) into 2 L LB broth containing tetracycline. c. When the culture reaches an ODeoOequivalent to 1.O, set culture on ice for a minimum of 1 h d. Centrifuge in 4 x 500 or 8 x 250 mL flasks at 4000 rpm for 15 mm. e Wash by resuspending cells in 1 vol (2 L) ice-cold water (1 mM HEPES, pH 7.5, may be used in place of water). f. Collect cells by centrifugation at 4000 rpm for 10 mm. g. Resuspend pellet in l/2 vol(1 L) water. h. Centrifuge as before and resuspend in l/50 vol(40 mL) 10% glycerol/water (v/v). i. Collect cells by centrifigation and resuspend in l/100 vol(20 mL) glycerol/ water ( 10% v/v).

Phage- Displayed Peptide Libraries

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j. Aliquot cells (100 pL) and freeze in liquid nitrogen. Quick-freezing in liquid nitrogen is important for long-term stability. Store at -8O“C freezer. Check efficiency by electroporation the following day.

References 1. Stephen, C. W., Helmmen, P., and Lane, D. P. (1995) Characterization of epttopes on human p53 using phage-displayed peptide libraries: insights mto antibodypeptide interactions. J. Mol. Biol. 248,58-78. 2. Lane, D. and Stephen, C. W. (1993) Epitope mapping using bacteriophage peptide libraries. Curr Opin. Immuol. 5,268-271. 3. Sioud, M., Dybwad, L., Jesperson, L , Suleyman, S., Natvig, J. B., and Forre, 0. (1994) Characterization of naturally occurring autoantibodies against tumour necrosis factor-alpha (TNF-a): in vitro function and precise epitope mapping by phage epitope library. Clw. Exp. Immunol. 98,520-525. 4. Orlandi, R., Menard, S., Colnaghi, M. I., Boyer, C. M., and Felini, F. (1994) Antigemc mimicry of the HER2/neu onto protem by phage-displayed peptides. Eur. J. Immunol. 24,2868-2873. 5. Atassi, M. Z. and Smith, J. A. (1978) A proposal for the nomenclature of antigenie sites in peptides and proteins. Immunochemistry 15,609,610. 6. Sela, M. (1969) Antigenicity: some molecular aspects. Science 166, 1365-1374. 7. Berzofsky, J. A. (1985) Intrinsic and extrinsic factors in protein antigemc strncture. Science 229,932-940. 8. Haaeim, L. R., Maskell, J. P., Mascagni, P., and Coates, A. R. (1991) Fine molecular specificity of linear and assembled antibody binding sites in HIV-l ~24. Stand. J. Immunol. 34,341-350. 9. Lin, J., Fendly, B. M., and Wells J. A. (1992) High resolution functional analysis of antibody-antigen interactions. J A401 Biol 226, 85 l-865. 10. Geysen, H. M. (1985) Antigen-antibody interactions at the molecular level: adventures in peptide synthesis. Immunol. Today 6,364-369. 11. Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986) A priori delineation of a peptide which mimics a discontinuous antigenic determinant. Mol. Zmmunol. 23,709-7 15 12. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) Strategies for epitope analysis using peptide synthesis. J Immunol. Methods 102, 259-274.

13. Houghten, R. A. (1985) General method for rapid solid-phase synthesis of large numbers of peptides: specificity of antibodyantigen interaction at the level of individual amino acids. Proc. Natl. Acad. SCL USA 82,5 13 l-5 135. 14. Pinilla, C., Appel, J. R., and Houghten, R. A. (1993) Functional importance of amino acid residues making up peptide antigenic determinants. Mol. Immunoi 30,577-585.

15. Parmley, S. F. and Smith,G. P. (1988) Antibody-selectablefilamentous phage: fd vectors: affinity purification of target genes. Gene 73,305-3 18. 16. Scott, J. K. and Smith, G. P. (1990) Searching for peptide ligands with an epitope library. Science 249,386-390.

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17. Devlin, J. J., Panganiban, L. C., and Devlin, P. E. (1990) Random peptide libraries: a source of specific protein binding molecules. Science 249,404-406. 18. Cwirla, S. E., Peters, E. A., Barrett, R. W., and Dower, W. J. (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc Nat1 Acad Sci USA 87,6378-6382.

19. Felici, F., Castagnoli, L., Musacchio, A., Jappelli, R., and Cesarem, G. (1991) Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 222,301-3 10. 20. Stephen, C. W. and Lane, D. P. (1992) Mutant conformation of ~53: precise epitope mapping using a filamentous phage epitope library. J. Mol. Biol. 225, 577-583.

21, Bottger, V. and Lane, E. B. (1994) A monoclonal antibody epitope on keratin 8 identified using a phage peptide library. J. Mol. Biol 235,61-67. 22. Bottger, V., Bottger, A., Lane, E. B., and Spruce, B. A. (1995) Comprehensive epitope analysis of monoclonal anto-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes. J Mol. Biol. 247,932-946. 23. Meola, A., Delmastro P., Monaci, P., Luzzago, A., Nicosia, A., Felici, F., Cortese, R., and Galfre, G. (1995) Derivations of vaccines from mimetopes: immunologic properties of human hepatitis B virus surface antigen mimetopes displayed on filamentous phage. J. Immunol. 154,3 163-3 172. 24. Balass, M., Heldman, Y., Cabilly, S., Givol, D., Katchalski-Katzir, E., and Fuchs, S. (1993) Identification of a hexapeptide that mimic a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library. Proc Natl. Acad. Sci USA 90, 10,638-10,642. 25. Koivunen, E., Wang, B., and Ruoslathi, E. (1995) Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. BioRechnology 13,265-270. 26. Lener, D., Benarous, R., and Calogero, R. A. (1995) Use of a constrained phagedisplayed library for the isolation of peptides binding to HIV-I nucleocapsid protein (Ncp7). FEBS Lett. 361, 85-88. 27. Hoess, R. H., Mack, A. J., Walton, H., and Reilly, T. M. (1994) Identification of a structural epitope by screening a peptide library displayed on tilamentous bacteriophage. J. Immunol. 153,724-729. 28. Connell, A., Kendall, M. L., Reilly, T. M., and Hoess, R. H. (1994) Constrained peptide libraries as a tool for finding mimotopes. Gene 151, 115-l 18. 29. Bianchi, E., Folgori, A., Wallace, A., Nicotra, M., Acali, S., Phalipon, A., Barbato, G., Bazzo, R., Cortese, R., Felici, F., and Pessi, A. (1995) A conformationally homogeneous combinatorial peptide library. J. Mol. Biol. 147, 154-160. 30. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient sitespecific mutagenesis without phenotypic selection. Methods Enzymol. 154,367-382. 3 1. Wright, R., Gram, H., Vattay, A., Byrne, S., Lake, P., and Dottavio, D. (1995) Binding epitope of somatostatin defined by phage-displayed peptide libraries. Bio/ Technology 13, 165-169.

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32. Lowman, H. B., Bass, S. H., Simpson, N., and Wells, J. A. (1991) Selecting high-affinity binding proteins by monovalent phage display. Biochemzstry 30, 10,832-10,838. 33. Bass, S., Greene, R., and Wells, J. A. (1990) Hormone phage: an enrichment method for variant proteins with altered binding properties. Protein Struct. Funct. Genet. 8,309-3 14. 34. Garrard, L. J., Yang, M., O’Connell, M. P., Kelley, R. F., and Henner, D. J. (1991) Fab assembly and enrichment in a monovalent phage display system. Bio/Technology 9,1373-1377. 35. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Clomng: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 36. Dower, W. J., Miller, J. F., and Regsdale, C. W. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nuclezc Acids Res. 16,6127-6145.

18 Reiterative Screening of Phage-Display Peptide Libraries with Antibodies Alexander Pereboev and Glenn E. Morris 1. Introduction Filamentous bacteriophage can tolerate the insertion of a foreign DNA into their gene encoding phage minor coat protein III. Such recombinant phage particles display on their surface foreign peptide sequencesfused with the gene III product, which is a protein represented by five copies on the top of the virion. The N-terminal part of this coat protein (cp III) is involved in binding phage to the F-pilus of a bacterium at an early stage of the infection process. The exposed peptide region exists, therefore, in a form accessible for immune recognition (I). Modem gene engineering allows the creation of phage libraries in which every possible combination of amino acid sequence is expressed as fusion proteins (2-s). An important difference between random peptides and specific peptides (e.g., PEPSCAN) or antigen fragmentation methods is that the former not only localizes the epitope in the antigen sequence, but also provides information on which amino acid residues are most important for antibody binding. Phage-display systems based on another coat protein, cpVII1, have also been developed, but the length of the inserted fragment is restricted to six amino acids (6”. Filamentous phage do not kill their host. In the course of infection, the cells continue to grow, and phage progeny are released from the cells without cell lysis. When thefd-tet phage-based system is used to create an epitope library, the cells infected with the phage become resistant to tetracycline, so the ability of the cells to grow in the presence of tetracycline is a selective marker. If the phage contains on its surface a foreign sequence identical or similar to a natural epitope, this sequence can be recognized and bound by an appropriate ligand, e.g., antibody. This allows affinity selection (biopanning) when the From

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ligand is immobilized on a solid surface. Therefore, only specifically bound phage particles are isolated from a vast number of peptide sequences. The enrichment of the initially bound phage is achieved by several rounds of biopanning with phage amplification in Escherichia coli between rounds. The phage progeny bearing specific sequencescan then be cloned in bacteria. Phage from a single colony can be propagated, and its DNA isolated and sequenced. The established amino acid sequence can then be compared with the known sequence of the antigen. The following protocol is based on the detailed methods of George P. Smith (University of Missouri), who also supplied the 15mer peptide library we have used in our studies (7). There are three ways for attaching the antibody to the solid phase (Petri dishes or microtiter plates): direct attachment of purified Ig, attachment via protein A, or attachment via second antibody (e.g., rabbit anti[mouse Ig] for mouse MAbs). Some directly attached antibodies could lose their binding properties because of conformational alterations taking place when the antibody interacts with the plastic surface. In our experience, however, there has been little difference between two ways of antibody attachment: directly and via second antibody, but the latter method has the great advantage that unpurified MAb (culture supernatant containing 20% fetal serum or ascites fluids) can be used. We have developed a systematic method for mapping panels of up to 20 MAbs. The procedure is first carried out with a whole mixture of antibodies. Colonies of E. coli infected by the eluted phage are screened first with the corresponding mixture of MAbs. The positive clones obtained are then screened with each individual MAb to determine their AB specificity. MAbs that are able to react with these clones are then removed from the mixture, and the biopanning is repeated again with the remaining antibodies. If an MAb mixture no longer selects peptides from the library, a different library might be tried (e.g., longer peptides or constrained [5/ peptides) before abandoning the MAb epitopes as unsuitable for the peptide approach. Finally, we recommend testing 3040 clones for MAb binding after the second, or even the first, biopanning, since further rounds of panning are often unnecessaryand could, in theory, reduce the diversity of peptides selected in favor of higher-affinity sequences. As an example of screening a 15-mer peptide library, we will describe the characterization of the binding epitopes for a panel of MAbs created against human utrophin (8.. The panel consists of 12 MAbs obtained against a recombinant fragment of utrophin (N-terminal part containing ammo acids 113-37 1 of utrophin). In the first cycle, a mixture of all 12 MAbs captured with rabbit anti-(mouse Ig) antibodies was used. Two rounds of biopanning were performed. The whole phage library was first preincubated with rabbit anti-(mouse Ig) antibodies attached to a plastic plate to remove phage capable of interaction

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with these antibodies themselves. Single colonies of E. coli cells infected with phage eluted on each round of biopanning were grown on a nitrocellulose membrane (NCM), which was then treated with the mixture of 12 MAbs and peroxidase-labeled second antibodies to find out which colonies are positive for the whole mixture. As shown in Fig. 1, after the first round of biopanning, none of 60 clones tested were positive. After the second round, 21 clones out of 60 were positive. These positive clones were screened against 12 individual MAbs. Most of the clones (20 out of 21) were specific for MAb MANNUT 4. One clone reacted with another MAb MANNUT 3. The MAb MANNUT 4 was removed from the mixture, and a second cycle of biopanning was performed with the rest of the MAbs. Three rounds of biopanning were done at this stage. Figure 2 shows that only one positive clone was found after the first round, but most of the clones tested after second and third rounds were strongly positive (80 clones were tested for each round). Screening for individual MAbs revealed three more MAbs (in addition to MAb MANNUT 3 already known as being positive; see Fig. l), which interacted with these positive clones (lines 5,7,9, 10; MAb MANNUT 8, MANNUT 13, MANNUT 3, and MANNUT 12, respectively). A third cycle of biopannmg was performed with the remaining seven MAbs, but no more positive clones were identified (data not shown). Thus, the improvement of the method we propose allows simultaneous mapping of a whole panel of different antibodies. Immunoassay of phage clones on NCA4 reduces the possibihty of accidental selection of nonspecific clones for further sequencing. Moreover, even before sequencing, this additional procedure can be very helpful in terms of MAb specificity. As Fig. 2B shows, three different MAbs interact with the same set of clones (lines 5, 7, and lo), so that we can consider them as recognizing the same epitope. 1.1. Brief Outline of the Protocoi A phage library containing random 15-mer peptide sequences is allowed to interact with a mixture of the antibodies immobilized on a plastic surface (biopanning). Unbound phage are washed away. Bound phage are eluted and amplified by infection of E. coli. Amplified phage are purified by PEG precipitation. The biopanning procedure is repeated two more times. At each stage, E. coli cells are infected with the eluted phage and grown on agar plates to obtain single colonies. The colonies are screened by Western blotting with the whole mixture of antibodies. Positive colonies are grown again as discrete columns on a nitrocellulose membrane, so that each column contains replicas of all of the clones being tested, and are screened with individual antibodies to determine their specificity. These positive colonies are propagated, phage DNA 1sisolated, and the sequence of each specific insert is determined.

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1st biopanning

2nd biopanning

Clones l-60

1

2

3

4

Clones 61-l 20

5

6

7

8

9

101112

Fig. 1. Screeningfor positive clones of E. coli cells infected with phage, using a mixture of all 12 MAbs. (A) Nitrocellulose membranetreatedwith whole mixture of 12 MAbs. Clones l-60 were obtained from biopanning round I; clones 61-120 were obtained from biopanning round II. (B) Positive clones identified in (A) were grown on replicate strips of nitrocellulose that were then treatedwith eachMAb individually. Lines 1-12 representMAbs MANNUT 6, MANNUT 2, MANNUT 7, MANNUT 9, MANNUT 8, MANNUT 5, MANNUT 13, MANNUT 10, MANNUT 3, MANNUT 12, MANNUT 4, and MANNUT 11, respectively. The last (lowest) clones on every line are negative controls (a clone that was negativein [A]).

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1 st biopanning Clones l-80

2nd biopanning Clones 81-l 60

3rd biopanning Clones 161-240

B

123456

7

8

9

10 11

Fig. 2. Screeningfor positive clones of E. coli cells infected with phage using a mixture of MAbs with the exception of MAb MANNUT 4. (A) Nitrocellulose membranetreatedwith mixture of 11 MAbs. Clones l-80 were obtained from biopanning round I; clones8 l-l 60 were obtainedfrom biopanning round II; clones 16l-240 were obtained from biopanning round III. (B) Sixty of the positive clones identified in (A) were grown in an array on replicate strips of nitrocellulose that were then treatedwith each MAb individually. Lines l-l 1 represent MAbs MANNUT 6, MANNUT 2, MANNUT 7, MANNUT 9, MANNUT 8, MAN-NUT 5, MANNUT 13,MANNUT 10, MANNuT3,MANNuT 12,andMANNUT 11,respectively.The last (lowest)clonesin eachgrouparenegativecontrols(a clonetbat wasnegativein [A]).

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2. MaterDaIs 2.1. Biopannlng and Phage Amplification 1. E. coli strain K91Kan: The strain was supplied together with the 15mer library by G. P. Smith. 2. TBS: 50 nil4 Tris-HCI, pH 7.4,50 mMNaC1. Autoclave. 3. TBST: TBS autoclaved and 0.5% Tween-20. 4. TBS/gelatin: 0.1 g gelatin in 100 mL TBS. Autoclave to dissolve. 5. 1M Tns-HCl, pH 9.1: Filter-sterilize. 6. Elution buffer: O.lM glycine-HCl, pH 2.2,O. 1% BSA, 0.01% phenol red. 7. Blocker: 4% BSA in sterile TBS. 8. 80 mMNaC1: Autoclave. 9. NAP buffer: 50 mMNH4H2P04, pH 7.0, with ammonia, 80 mMNaC1. 10 PEG/NaCl: 10 g PEG8000, 11.7 g NaCl, 47.5 mL water Autoclave to dissolve. 11 Stock solutions of antibiotics: 10 mg/mL kanamycin in water; filter-sterilize; 5 mg/mL tetracycline in ethanol; store at -20°C. 12. LB broth: 10 g bacto-tryptone, 5 g yeast extract (both from Difco, Detroit, MI), 5 g NaCl, water to 1000 mL: Autoclave. 13. LB agar plates: LB broth + 16 a of bactoagar (Difco) Autoclave, allow to cool down to 45-5O”C, and pour into Petri dishes. 14. LB agar plates + 100 pg/mL kanamycin. 15. LB agar plates + 40 pg/mL tetracycline + 100 pg/mL kanamycin: Antibiotics are added after autoclaving, just before pouring into Petri dishes. 16. Plasticware: sterile 35- and 90-mm Petri dishes, autoclaved yellow and blue pipet tips, 1.5- and 0.5~mL Eppendorf tubes. 17 Autoclaved toothpicks (antibiotic free).

2.2. Colony Screening 1. Nitrocellulose membranes (Schleicher and Schuell, pore size 0.45 pm). 2. Peroxidase-labeled antimouse Ig antibodies (DAKO, Carpmteria, CA). 3. Incubation buffer (IB): TBST supplemented with 0.1% BSA, 1% fetal calf serum, and 1% horse serum. 4. Blocking solution: 3% low-fat dried milk (from any supermarket) dissolved in TBST. 5. Substrate buffer: mix 25.7 mL of 0.2M Na2HP04, 24.3 mL of 0. 1M citric acid, and 50 mL of water (final pH 5.0). 6. Diaminobenzidine dihydrochloride (DAB) (Sigma, St. Louis, MO) stock solution (80X: 32 mg/mL in water). This should be handled as possible carcinogen. 7. Hydrogen peroxide (30% v/v).

2.3. DNA Sequencing 1, Primer for dideoxy sequencing: 5’ HO-TGAATTTTCTGTATGAGG-OH 3’. 2. Electrophoresis equipment (e.g., Sequi-Gen with 3000xi Power Pack and Model 583 Gel Dryer, all from Bio-Rad Labs, Hercules, CA). 3. Phenol/chloroform saturated with 10 mMTris, pH 8.0, and 1 mA4EDTA (Sigma).

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4. Dideoxy sequencingkit (Sequenasev.2.0 from Amersham International, Little Chalfont, UK). 5. CZ-~~,.S-~ATP (> 1000Ci/nnnol; 10pCi/pL; AmershamInternational). 6. Kodak X-Omat AR X-ray film; Kodak GBX developer and fixer (Sigma); Saran Wrap for gel drying (Genetic ResearchInternational, Great Dumnow, UK), 7. Solutions for sequencinggels: a. Acrylamide Stock:38 g acrylamide (Bio-Rad, 99%+) and 2 g NJ’-methylene-bis-acrylamide (Bio-Rad). Make up to 100 mL in deionized water and filter. Caution: Unpolymerized acrylamide solutionsare neurotoxic. b. Acrylamideiurea mix: 15 mL acrylamide stock solution and 48 g Urea (98%+). Make up to 90 mL in deionized water. Warm to dissolve. Filter. c. 25X TBE buffer: 27 g Trizma base, 13.75 g boric acid, and 2.33 g EDTA. Make up to 100mL. d. TE buffer: 10 mA4Tris-HCl, pH 8.0, and 1 mMEDTA. 8. Agarose,ethidiumbromide, andstandardequipmentfor agarosegel electrophoresis. 3. Method The procedure for epitope mapping by the phage display method involves: biopanning (Section 3.1.), amplification (Section 3.2.), assayfor positive colonies (Section 3.3.), DNA isolation (Section 3.4.), and sequencing (Sections 3.5. and 3.6.). Some steps of the procedure must be carried out at the same time. Therefore, we describe the protocols in Sections 3.1. and 3.2. in a dayby-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible.

3.1. BCopannhg 1. Day 1: a. Coat 35-n-m sterile Petri disheswith 1mL of rabbit-anti-(mouse Ig) antibodies diluted l/1000 in TBS (seeNote 1.). b. Incubate overnight at 4OCon rocker. 2. Day 2: a. Wash plateswith TBST. b. Add blocking solution (4% BSA in sterile TBS) and rock for 1 h at room temperature. c. Wash plates six times with TBST. Add 1 mL of MAb mixture (each MAb is diluted l/50 in TBST; see Note 2). Incubate for 1 h at room temperature on rocker, d. Wash six times with TBST. Add phagelibrary (10’ * transducingunits in 0.75 mL TBST). Incubate for 4 h at 4’C on rocker. e. Washthoroughly 10 timeswith TBST. Elute bound phagefor 5-10 min with 400 PL of elution buffer. Wash the plate surface thoroughly during this elution using a pipet tip to remove all phage.Add 75 pL of ZMTris-HCl, pH 9.1, and mix (seeNotes 3 and 4).

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1. Day 0: Streak out K91Kan from stock on LB/Kan plate. Grow cells overnight at 37°C to obtain single colonies. 2. Day 1: Grow a single colony overnight in a 1.5~mL Eppendorf tube in 1 mL LB/ Kan. 3 Day 2 (early): a. Inoculate 20 mL LB in 100&L bottle with 20 PL K91Kan culture Grow to midlog (3-4 h) with vtgorous shaking at 37°C (OD600 = 0 45). Leave for 5 min without shaking at 37°C. b. Pellet the cells at 8OOg for 10 min. Resuspend in 20 mL of 80 mM NaCl. Shake gently for 45 min at 37’C. c Pellet the cells at SOOg for 10 min. Resuspend in 1 mL of cold NAP buffer. This bacterial suspension may be stored at 4°C for up to 5 d. 4. Day 2 (later): a. Mix 100 pL of phage eluate with 100 pL of K91Kan cells in NAP in an Eppendorf tube. Rock at room temperature for 10-30 min b. Transfer to 20 mL LB containing 0.2 &mL tetracycline. Shake at 37°C for 1 h. c. Add 80 pL of tetracycline 5 mg/mL stock solution. Continue shaking overnight at 37°C. 5. Day3: a Centrifuge cells at 1OOOgfor 15 min. b. Transfer supernatant into another centrifuge tube, and centrifuge at 10,OOOg for 10 mm. c Transfer supernatant into a fresh tube containing 3 mL of PEG/NaCl. Mix by inverting 100 times and leave overnight at 4°C (see Note 5). 6. Day 4: a. Centrifuge at 10,OOOgfor 15 mm. Remove supernatant. Resuspend pellet m 1 mL TBS. b. Transfer to an Eppendorftube and microfuge for 1 min at 8000g. c Transfer supernatant to 150 JJL of PEG/NaCl in a second Eppendorf tube. Mix by inverting 100 times. Leave on ice for at least 1 h. d. Microfuge for 10 min at top speed. e. Remove supernatant, recentrifuge brtefly, and remove supernatant. f. Resuspend pellet in 200 pL of TBWazide. g. Use 100-200 PL of this purified phage and 400 pL of TBST for next panning. This is now ready for the second round of biopanning, becomes d 2 again (see Note 6).

3.3. Assay for Positive

so that d 4 now

Colonies

1. Prepare serial dilutions of the “red eluate” in TBS/gelatin (see Note 7). 2. Mix 10 l.tL diluted phage and 10 pL K91Kan cells in NAP (see Section 3.2.3., step 5). 3. Rock for 10 min at room temperature.

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13. 14.

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Add 1 mL LB containing 0.2 pg/mL tetracycline and shake at 37°C for 30 min. Spread 200 pL on LB/Tet/Kan agar, and grow overnight at 37’C (see Note 8). Cut a nitrocellulose membrane (NCM) to fit a 90-mm Petri dish (see Note 9). Put NCM onto LB/Tet/Kan agar, and let it soak. Using the same toothpicks, transfer single colonies onto both an LB/Tet/Kan agar master plate and to an NCM on LB/Tet/Kan agar. Incubate plates overnight at 37OC. Put the master plates in refrigerator (these can be stored for several weeks). Remove the NCM from agar surface, and transfer to another Petri dish with 10 mL of TBST. Using a piece of sponge, remove all the cells from the NCM. Wash six to eight times with TBST. Block NCM for 30 mm at room temperature in 3% low-fat dried milk in TBST. Incubate for 1 h with MAb (hybridoma supernatant, l/50 in IB, see Note 10) at room temperature on rocker. Wash five times with TBST. Incubate for 1 h with peroxidase-labeled rabbit anti(mouse IgG) antibodies diluted l/1000 in IB at room temperature on rocker. Wash five times with TBST and two to three times with distilled water. Add 5 mL of DAB solution m substrate buffer supplemented with 0.012% hydrogen peroxide. Traces of the positive colonies should be bright-brown-colored (see Notes 11 and 12). Make replicas of positive clones with sterile toothpicks on another NCM placed on an LB/Tet/Kan agar surface, so that the replicas would form the columns containing the same clones in the same place. Grow the cells overnight at 37OC. Repeat steps 1 l-14 wtth the exception that incubation with antibodies is performed mdlvidually with each MAb.

3.4. DNA lsolation 1. Inoculate 3-mL portions of LB containing 20 pg/mL tetracycline with posttive clones identified in Section 3.3. Shake the tubes vertically for 16-24 h at 37°C. 2. Microfuge each culture briefly to pellet cells. 3. Pipet supematant into a vessel containing 450 yL of PEG/NaCl. Mix by inverting 100 times and incubate on ice for at least 4 h. 4. Microfuge for 15 mm, remove supernatant, recentrifuge briefly, and remove supernatant. 5. Dissolve pellet in 500 pL TBS by vortexing. These phage are already pure enough for preparing sequencing templates. 6. Put 500 pL of PEG-purified phage suspension into Eppendorf tube. Add an equal volume of phenol/chloroform. Vortex vigorously. Microfuge to separate phases. Collect the upper aqueous phase (-400 pL) trying to avoid any traces of mterphase and lower phase. 7. Transfer aliquot to a second Eppendorf tube with 40 yL of 3M sodium acetate and 1 mL of ethanol. Allow DNA to precipitate for at least 1 h on ice. 8. Micromge for 15 min. Remove supematant, recentrlfuge briefly, and remove supematant again.

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9. Wash pellet (usually invisible) with 1 mL of 70% ethanol. Remove supernatant, recentrifuge briefly, and remove supernatant. 10. Dissolve pellet in 8 pL of water. 11. Run 1 pL of sample in 1% agarose gel to control purity of DNA. In order to obtain good sequencing results, a sharp band of approx 9000 bp should be visible. The rest of the DNA can be stored at -20°C for a few weeks.

3.5. DNA Sequencing This consists of three steps: annealing of the primer with the DNA template, building up the complementary DNA chain in which the 35S-dATP is incorporated, and termination of the chain building by adding ddNTPs to the reaction mix. Adequate results have been obtained using the Sequenase v.2.0 sequencing kit in accordance with the manufacturer’s instructions.

3.6. Gel Electrophoresis

and Autoradiography

1. Wash the glass plates and dry with ethanol-moistened paper tissue. Drop SigmacoteTM on the back plate and polish with tissue. Assemble the plates. 2. Prepare 6% acrylamide gel: 1.6 mL 25X TBE, 36 mL acrylamide/urea mix, 2.4 mL water, 190 pL ammonium persulfate, and 34 pL TEMED. 3. Pour gel between the plates laid almost flat on the bench. Insert shark’s tooth comb at the top with the flat surface facing the acrylamide. When the gel is completely set (about 30-60 min), assemble the apparatus and pour 1X TBE into both reservoirs. Prernn the gel at 50 W (approx 2000 V) to warm it to 50°C Invert the comb and allow the teeth to just penetrate the gel. 4 Heat the samples up to 8OY!, and load onto the gel (3-5 pL/track). 5. Run at 50 W. Stop running when the green dye reaches about three-fourths of length of the gel. 6. Cool plates with tap water for 10-l 5 min. Separate plates and wash the gel with 10% acetic acid until the dye has disappeared. Rinse the gel with water. Dry the gel on thick filter paper at 80°C for 40 min. 7. Expose the film directly in close contact with film (2&36 h, depending on halflife of isotope). 8. Develop the film with GBX developer for 6 min. Stop: 2% acetic acid for 1 min. Fix with GBX fixer for 5 min. 9. Reading the film from the bottom, find the sequence CCCCAGCGGCCCC. All sequences below this are identical. Read 45 nucleotides upward. Note that it is a complementary chain reading. Translate the original sequence to amino acids (5’ + 3’). Compare the peptide sequence with the known one of the antigen.

4. Notes 1. The rabbit antibodies used for capturing MAbs may also interact with some phage from the library. This might result in selection of many non-MAb-specific clones. Thus, the library should be preincubated with anti-(mouse Ig) antibodies alone in

order to prevent nonspecific binding of phageby rabbit Ig.

2. Plates might be coated at the same time with MAbs of different specificitiesindividually or in combination with others. The principle of MAb combination depends on whether there is some preliminary information about their mutual relationship, e.g., competitive assay or peptide-binding assay. Afterward, the plates would be sequentially incubated with the same library preparation. 3. Phenol red is added to elution buffer and is yellow at pH 2.2. When lMTris-HCl, pH 9.1, is added, the elution buffer turns red, hence the name “red eluate.” 4. Because the phage containing the desired peptides might be present in the main library as only a few particles, it is recommended to concentrate the first “red eluate” to 100 yL using a membrane concentrator (e.g., Vivascience, VSO 132) before amplifying. 5. We have found that this first step of PEG precipitation aimed at concentrating the phage-containing supematant can be replaced by concentration with a Vivaspm 15 (Vivascience) unit. Pour 15 mL of supematant into the unit. Centrifuge at SOOg for 15 min. Add the remaining supematant, and centrifuge for another 15 min at 8OOg. Collect filtrate (about 0.5 mL), and add stertle TBS to 1 mL. Then go to step 3 of Section 3.2.6. 6. On further rounds of panning, the MAb mixture is reacted only with the corresponding amplified eluate. 7. Tentatively, suitable dilutions of “red eluate” (in TBS/gelatin) appear to be A: 1 yL in 1000 pL of TBSG; B: 100 pL of A plus 900 pL of TBSG; C:lOO pL of B plus 900 j.tL of TBSG. 8. It is useful to spread the cells remaining after amplification (Sectton 3.2.5., step 1) to obtain single colonies for screening. Add 2 mL of LB broth to the pellet of overnight culture. Make serial dilution (IO”‘, 10m5, lo6 should be fine) in LB medium. Use 200 yL to spread on a plate. 9. To tit a 90-mm Petri dish, an NCM might be cut as a rectangle -5.0 x 6.5 cm. 10. Hybridoma SN should preferably be used for colony screening, because there is always the possibility of false-positive colonies being selected using ascites Ascites contain a lot of antibodies with unknown specificity, which could bind phage from the library 11. For some antibodies, positive clones were found even after the first round of panning. It seems that further enrichment is undesirable in these cases, because there is the possibility that this might lead to isolation of fewer clones with higher affinity for the antibodies. 12. If quantitative analysis of antibody-phage binding is desirable, it is possible to perform an ELISA procedure using purified phage as antigen. There are many descriptions of the procedure (e.g., Chapter 16 and ref. 2).

Acknowledgments We thank George Smith (University of Missouri) for supplying the phage library kit. This work was supported by a DevR award from the Higher Education Funding Council and by grants from the Muscular Dystrophy Group of Great Britain and Northern Ireland.

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References 1. Kishchenko, G , Batliwala, H , and Makowski, L. (1994) Structure of a foreign peptide displayed on the surface of bacteriophage M 13. J Mol. Biol. 241,2082 13. 2. Smith, G. P. (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the vnion surface. Science 228, 13 15-l 3 17. 3 Scott, J K. and Smith, G. P (1990) Searching for pepttde ligands with an epttope library. Science 249,386-390. 4 Barbas, S. M. and Barbas, C. F. (1994) Filamentous phage display Fzbrinolysis 8 (Suppl. l), 245-252. 5. McConnell, S. J., Kendall, M. L., Reilly, T. M., and Hoess, R. H. (1994) Constrained peptide libraries as a tool for finding mimotopes. Gene 151, 115-l 18 6. Eroshkin, A. M., Miner&ova, 0. O., Fomin, I., Ivanisenko, V. A., and Ilichev, A. A. (1993) Analysis of peptide inserts mto Bacteriophage-M13, fl, and fd major coat protein-relation between protein structural characteristrcs and mutant phage viability. Mol Blol. 27, 843-849 7. Smith, G. P. (1992) “Cloning in fUSE vectors” available directly from G. P. Smith, Division of Biological Sciences, University of Missouri. 8 Nguyen, thi Man, Helliwell, T. R., Simmons, C., Winder, S. J., Kendrick-Jones, K., Davies, K E., and Morris, G. E. (1995) Full length and short forms of utrophin, the dystrophin-related protein. FEBS Lett. 358,262-266.

19 Homolog

Scanning

Lin-Fa Wang 1. Introduction The high specificity of monoclonal antibodies (MAbs) enables them to dlscriminate subtle sequence and structural differences among homologous proteins. They have thus become important tools for serotyping of viruses and bacteria (I-3), and for typing of homologous proteins from multigene families, such as subtypes of different human interferon molecules (4) or HLA markers (5). With the advances in rapid DNA sequencing, more homologous genes are being discovered that share extensive structural and functional similarities. Although the biological implications for these homologs may not be easy to define, it is often possible to detect their subtle structural differences using MAbs. These observations have led to the development of the homolog-scanning strategy (671, which is useful in identifying sequences that cause functional variation among homologous proteins, such as enzyme activity and epitope antigenicity. Epitope mapping by homolog scanning is based on the systematic or random replacement of sequence segments in an MAb-binding homolog by cognate sequence segments from homologs known not to bind to the MAbs or vice versa. By testing the binding ability of the recombinant hybrid protein and comparing its sequence with those of the parent molecules, rt is possible to identify the sequence segment or amino acid residues essenttal for MAb binding. Although the homolog-scanning approach can, in theory, be used in mapping linear epitopes, it is designed mainly for mapping conformational epitopes, which are difficult to map by other means. Basically, the homologscanning approach can be divided into three steps: From.

Methods m Molecular Biology, vol. 66’ Epitops Mappmg Protocols Edtted by 0. E Morris Humana Press ho, Totowa, NJ

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Wang

1. Construction of hybrid genes; 2. Expressionof recombinantproteins either in VIVOor in vitro; and 3. Determination of the capacityof the recombinantproteins to bind MAb There are several methods reported for constructing hybrid genes by in vitro gene manipulation (S-12) or by in vivo homologous recombination (13,14). The method to be described in this chapter is a PCR-based approach, termed template-coupled PCR (7), which is convenient and very efficient for creating hybrid genes with either defined or random crossover points. As shown in Fig. 1, this approach uses two homologous genes cloned in two different vectors: one coding for a protein reactive with the MAb of interest and the other nonreactive. Of the two vectors, one is an expression vector suitable for production of recombinant hybrid proteins to be used in MAb binding studies. The PET vector system (24) is ideal for this purpose, since the T7 RNA polymerase-directed expression makes it possible to produce proteins both in vlvo and m vitro. The other vector is usually a pUC-based plasmid (15) or any general cloning vector frequently used in the laboratory. Four “universal primers” (A, a, B and b) are synthesized that anneal to the regions flanking the cloning sites of the two vectors. After cutting one template with an appropriate restriction enzyme (e.g., X for template I or Y for template II), the other template is added together with either pair of the primers (A + b or a f B). The whole mixture is then subjected to a standard PCR amplification to obtain the hybrid gene. In the first cycle of PCR, the coupling template (the digested template) actually functions as an “elongated primer” to form the first hybrid molecule, which is further amplified in successive cycles by the flanking PCR primers. The final configuration of the hybrid gene 1sdetermined by the pair of primers used as well as by the nature of the coupling template(s). Figure 2 illustrates an example of mapping a conformation-dependent epitope using homolog-scanning strategy. MAb I-4-A reacts with human interferon-a4a (IFN-a4a), but not with IFN-al4 (7,161. I-4-A reacted with intact IFN-a4a molecules in Western blotting, but failed to react with truncated recombinant polypeptides expressed from the cloned IFN-cL4a gene. This prevented us from mapping the epitope using a serial deletion approach. Using homolog scanning in conjunction with other methods, we were able to show that the N-terminal 23 aa residues were essential for the binding of the MAb (7). It should be pointed out that the strategy illustrated in Fig. 1 is not the only method for generating the coupling templates. It is also possible to use PCR and an internal primer to produce a coupling template using any of the internal amino acid residues as a crossover point (7). Further more, it is also possible to generate coupling templates with random crossover points along the molecule by using partial digestion with enzymes, such as DNase I, exonuclease III, or

Homolog Scanning

209

1

Cut with X

B

I (A+b)

(a+B) A

C-B

i -

-

Fig. 1. Generationof homolog hybrid genesby a single-steptemplate-coupledPCR amplification. The coupling templates shown here are produced by digestion with restriction enzymes.The solid and shadedbars representtwo templatesI and II (i.e., two homologousgenescloned in two different vectors),whereasthe double and single lines indicate the different vector sequences.Vector-specific primers are labeled asA, B, a, and b, respectively.X and Y are restriction enzymecleavagesiteslocatedwithin the genecoding regions. (A) Generationof hybrid genesusing templateI (cut with X) as coupling templates.(B) Generationof hybrid genesusing template II (cut with Y) as coupling templates.

210

Wang I L

40 /

Bo I

120 I

160 (aa) I

S-S S pET - IFN4

’ s BLOT +

ACT +

PET-4-84 PET-4-46 PET-4/14-46 PET-4/14-23 PET-IFNl4

Fig. 2. Antigenic andbiologic propertiesof recombinantINF proteinsexpressed from the pET system.Thenumbersat the top areaminoacidresidue(aa)numbersof the IFN-a protein with the diagramunderneathrepresentingthe two disulfide bonds betweenresidues1 and99, and29 and 139,respectively.At the right is a summaryof the resultsfrom Westernblotting (BLOT) and antiviral activity (ACT) analysis:+, positive result;-, negativeresult.The solid barsrepresentthe coding sequencesfor IFN-o4a, whereasthe shadedbarsarefor IFN-a14 (seeref. 7 for moredetails).

Ba13 1, thus creating a library of random hybrid molecules. If hybrid genescan be expressedand displayed on the surface of filamentous phagesas described (in ref. 17 and 18 and also in Chapter 24 of this volume), it is possible to construct a phage-display random library of hybrid molecules. The phagedisplay expression will make the screening process much more efficient and will also make it possible to examine a large number of hybrid molecules simultaneously. 2. Materials All reagents should be of AR grade. All solutions and buffers should be autoclaved or filter-sterilized where appropriate. Sterile tubes and tips should be used. Unless otherwise stated, all molecular biology reagentsare obtained from Promega(Madison, WI). 2.1. General 1. Antibodies:MAbs of interestand alkaline phosphatase(AP)- and horseradish peroxidase(HRP)-conjugated antimouseantibodies. 2. Recombinantgenes:At leasttwo clonedhomologgenesare requiredfor this mappingapproach.It is preferableto engineerthetwo homologgenesto havethe

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Homolog Scanning

3. 4.

5.

6. 7.

same cloning sites at each end so that they can be conveniently moved from one vector to another. Bacterial strains and plasmids: Eschemhza colz BL21[DE3] (14), vectors PET3a (14), and pUC18 (ZS). Oligonucleotide primers. a. Flanking primers for pUC 18, USP (5’ GTA AAA CGA CGG CCA GT 3’) and RSP (SAAC AGC TAT GAC CAT G 3’); b. Flanking primers for PET-Sa, ET5 (5’ CCT CTA GAA ATA ATT TTG TTT 3’) and ET3 (5’ CAG CCA ACT AAG CTT CCT TTC 3’). Equipment: power supply, horizontal agarose gel electrophoresis tank, MiniPROTEAN II SDS-PAGE system, Mini Trans-Blot Module, sequencing gel apparatus, gel dryer, and Gene Pulser for electroporation were all purchased from Bio-Rad (Hercules, CA). Dark room facilities for Polaroid photography and for X-ray film developing. Rocker and orbital shaker.

2.2. Generation 8. 9. 10. 11. 12. 13.

of Hybrid Genes

Appropriate restriction enzymes. Taq polymerase and PCR reagents: use as recommended by supplier.

PCR machine: Hybaid OmniGene or any simtlar thermal cycler. GeneClean DNA purtfication kit from BIO-101 (San Diego, CA). T4 DNA ligase and ligation buffer: use as recommended. Competent cells. Electrocompetent cells are prepared according to the method provided with the Bio-Rad Gene Pulser. Forth-microliter aliquot tubes are quickly frozen in liquid nitrogen and kept at -80°C until use. 14. LB medium: 1% bacto-tryptone, 0.5% yeast extract, 1% NaCl 15. LB/Amp plates: LB medium containing 1.5% agar, autoclave to sterilize. Cool to 50°C before adding ampicillin from a 50 mg/mL stock to a final concentration of 50 pg/mL. Pour approx 20 mL/9Omm plate.

2.3. Production of Recombinant 2.3.1. Expression in E. coli

Protein Molecules

16. Isopropyl-/3+thiogalactoside (IPTG): 100-m stock solution in water, kept at -20°C for up to 6 mo. 17. MTPBS buffer: 150 m.A4NaCl,l6 mMNa2HP04, 4 mMNaH2P04, pH 7.3. 18. Sonieator: Vibra Cell High Intensity Ultrasonic Processor, 50-W Model, from Sonics & Materials (Danbury, CT).

2.3.2. In Vitro Translation 20. 2 1. 22. 23. 24.

35S-Met, 1 Ci/mmol, ICN (Costa Mesa, CA). TNT T7 Coupled Reticulocyte Lysate System, Promega. SDS-PAGE reagents: see vol. 32 of this series for detailed rectpes. X-ray film: for example, Kodak X-Omat AR5 Autoradiography cassettes.

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2.4. Antibody Binding Assays 2.4.1. ELBA 25. Multichannel pipet, ELISA plates, and microplate shaker: all from Titertek Flow Laboratories (McLean, VA). 26. Microplate reader: Multiskan MS, from Labsystems (Helsinki, Finland). 27. Coating buffer: 50 mM Tris-HCl, 150 mMNaC1, pH 9.0. 28. PBST: Dilute 10X PBS (per liter: 10.7 g Na2P04, 3.9 g NaH2P04, 80 g NaCl, pH 7.2) to 1X with distilled water, and add Tween-20 to a final concentration of 0.05% (v/v). Store at room temperature for up to 6 mo. 29. Blocking solution: PBST containing 1% skim milk powder; make fresh before use. 30. Citrate acetate buffer: Make up 100 mL of 1M sodium acetate and 10 mL of 1M citric acid. Adjust the sodium acetate solution to pH 5.9 with approx 1.5 mL of the citric acid. 31. TMB substrate: Dissolve 100 mg of 3,3,5,5,-tetramethylbenzidine (Sigma, St. Louis, MO) in 10 mL dimethyl sulfoxide (DMSO) to make a 42-&solution. Store at 4°C in small aliquots (0.5 mL) for up to 12 mo. Prewarm at 37°C for 10 min before use. 32. Substrate solution: Make fresh by mixing 18 mL of distilled water with 2 mL of citrate acetate buffer and 0.2 mL of the TMB substrate. Add 2.5 pL 30% H202 just before use. 33. Stopping solution: 1M HzS04.

2.4.2. Western Blotting 34. 35. 36. 37. 38.

Nitrocellulose membrane: 0.45 pm, Schleicher & Schuell (Dassel, Germany). Whatman 3MM filter paper. Plastic bag and heat sealer. Container with flat bottom (e.g., square Petri dishes). Tris-glycine transfer buffer: Make fresh by mixing 100 mL 10X transfer buffer (250 mM Tris and 1.92M glycine, pH 8.3) sequentially with 700 mL distilled water and 200 mL methanol. 39. TBST: Dilute 10X TBS (per liter: 90 gNaCl,60 g Tris base, adjust pH to 7.9 with HCl) to 1X with distilled water and add Tween-20 to a final concentration of 0.05% (v/v). Store at room temperature for up to 6 mo. 40. Blotto solution: TBST containing 5% skim milk powder, make fresh before use. 41. AP substrate buffer: 100 mMTris-HCl, pH 9.5, 100 mMNaC1,5 mMMgCl*.

2.4.3. Magnetic lmmuno Capture 42. Streptavidin magnetic beads (SMB): 1 mg/mL suspension, Promega. 43. Magnetic separation stand (two-hole), Promega. 44. Biotinylated sheep antimouse antibodies, Amersham (Buckinghamshire,

UK).

Homolog Scanning

213

3. Methods 3. I. Generation of Hybrid Genes For convenienceof discussion, we assumethat homolog I (see Fig. 1) is reactivewith the MAb, whereashomolog II is not, andthat homolog I is cloned in pET vector with flanking primers A and B, whereashomolog II is cloned in pUC with flanking primers a and b. Both homolog genescan be excised from the vectors as a BarnHI-EcoRI gene cassette.The proceduresbelow are for template I as the coupling template (seeNotes 1 and 2). 1. Digest 100 ng pET plasmid containing homolog gene I with 5 U of restriction enzyme X (see Note 3) in 20 pL reaction mixture. Incubate at 37“C for 60 min, and then at 65OC for 15 min (see Note 4). 2. Take 2 ltL of the digested template I, mix with 5 ng of undigested template II plasmid DNA, and adjust volume to 20 pL with water. Boil the mixture for 2 min, followed by rapid cooling on ice. 3. Set up two PCR reaction mixtures with the following components: PCR-1: 50 pmol each of primers A and b, 1 yL of the denatured template mixture prepared above in step 2, 10 PL 10X PCR buffer, 10 PL 25 r&I 16 l.tL 1.25 mM dNTPs, 2.5 U of Taq polymerase. Adjust volume to 100 pL with water.

MgCl,,

PCR-2: same as above except that primers a and B are used instead of primers A and b. PCR reactions are carried out for 25 cycles at 94’Wl min, 5OW2 min, and 72”C/2 min.

4.

5.

6.

7.

(The PCR product from PCR- 1 can be named I/II-X, whereas that from PCR-2 is named II/I-X to distinguish the two different hybrid molecules obtained as shown in Fig. 1. For the remaining steps, the two will be treated exactly the same.) Total PCR products are separated on a 1% agarose-TAE gel, and the corresponding PCR band is excised and purified using the GeneClean kit following procedures provided by the supplier. The purified PCR fragment is eluted in 20 l.tL water. Digest 10 pL of the purified PCR product in a total volume of 20 pL containing 2 PL 10X reactlon buffer and 5 U each of BamHI and BcoRI. The reaction mixture is incubated at 37OC for 60 min, followed by heating at 65’C for 15 min. Ten microliters of this digestion mixture are ligated with 50 ng PET-3a vector DNA, which has been digested with BumHIIEcoRI and treated with calf intestine alkaline phosphatase. The ligation is carried out overnight at 14’C in a total volume of 20 PL containing 2 pL 10X ligation buffer and 2 U of T4 DNA ligase. The ligation reaction is terminated by heating at 65’C for 15 min. Take 2 pL of the above ligation mixture to transform E. coli strain BL21 [DE31 by electroporation using the Bio-Rad Gene Pulser following the supplied instructions (see Note 5). The transformed cell suspension is immediately transferred to a 10-n& culture tube containing 1 mL LB medium, and incubated at 37°C for

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60 min with gentle shaking. Aliquots of 50, 100, and 200 pL of cells are plated onto LB/Amp plates, followed by overnight incubation at 37’C 8. Two to four single colonies from each transformation mixture are picked for

minipreps, followed by restriction enzymedigestion to confirm the presenceof the expected insert (see Note 6).

3.2. Production

of Recombinant

Protein Molecules

Depending on the solubility of the recombinant protein and the quantity of the protein required, one can choose to express the hybrid gene either tn E colz or by in vitro translation m the presence of 35S-Met. It is sometimes necessary to try both methods to achieve optimal results. 3.2.1. Expression in E. coli 1. Pick a single colony from a fresh plate to inoculate 1 mL LB/Amp medmm (i.e., LB containmg 50 pg/mL ampicillm), and incubate the culture at 37°C overnight with shaking. 2. On the next morning, transfer the 1 mL of overnight culture to a lOO-mL flask containing 9 mL of prewarmed LB/Amp medium, and shake at 37°C for 60 min. 3. Add 100 pL of 100 mA4 IPTG (final concentration of 1 mM) to induce the expression and continue the incubation for a further 3 h. 4. Harvest the cells by centrifugation at 5000g for 5 min, and resuspend the cell pellet in 0.5 mL of MTPBS buffer. Transfer the cell suspension to a 1.5-mL Eppendorf tube. 5. Lyse the cells by sonication (5 x 30 s) using the output control setting at 50. Hold the tube on ice m a 100~mL beaker during sonication, and leave the tube on ice for 1 mm between each somcation. Save 100 pL as the “total lysate fraction.” 6. Spin the remaining lysate for 5 mm. Transfer the supernatant to a clean tube, and save as the “soluble fraction.” 7. Resuspend the pellet in 0.4 mL MTPBS buffer, and save as the “insoluble fraction ”

8. Examinethe size,solubihty, and level of expressionfor the recombinant hybrid proteins by SDS-PAGE following standard methods (see Note 7)

3.2.2. In Vitro Translation 1, Isolate plasmid DNA using standardalkaline lysis method (19). 2. Further purify approx 5 pg plasmid DNA using the GeneClean kit following given procedures for liquid-phase purification. 3. Use 2 pg of the purified DNA for m vitro translation using the Promega TNT T7 Coupled Reticulocyte Lysate System and the supplied instructions. The total reaction volume will be 100 pL using the ratio of 1 pg DNA/SO pL as suggested by the supplier. 4. Take 5 uL (i.e., 5% of the total reaction mixture) to examinethe translation efficiency by SDS-PAGE, followed by autoradiography (see Note 8).

275

Homolog Scanning 3.3, Antibody-Binding

Assays

Depending on the nature of the epitope(s) in study and the solubility of the expressed recombinant proteins, one may have to try different assaysto optimize the detection of antibody binding. Below are three of the most frequently used assays for monitoring antibody binding. The magnetic immunocapture assay is designed for use with the labeled proteins produced by in vitro translation. ELISA is more suitable for detection of soluble proteins produced in E. co/i, whereas Western blotting can be used with any form of the expressed proteins described in Section 3.2. 3.3.1. ELlSA All incubations, except for substrate development, are carried out at 37OC with gentle shaking on a microplate shaker at setting 6. 1. Use the soluble fraction produced in E. coli (see Section 3.2.1.) to make 1:2 serial dilutions in coating buffer from 1:20 to 1:2560 (see Note 9). 2. Use 50 I.~L each of the diluted solutions to coat an ELISA plate m triplicate. Incubate the plate for 60 min with gentle shaking. 3. Wash the plate three times (5 min each) with PBST. 4. Add 100 pL blocking solution to each of the wells, and incubate for 30 min. Discard the solution after incubation. 5. Add 50 & MAb solution, diluted in blocking solution at 1: 10 for tissue culture or 1: 100 to 1: 1000 for ascetic fluid (see Note lo), followed by incubation for 60 min. 6. Wash as in step 3. 7. Add 50 uL HRP-conjugated sheep antimouse IgG diluted m blocking solution at 1:2000, followed by incubation for 60 mm. 8. Wash as m step 3. 9. Add 50 pL substrate solution, and incubate at room temperature for 10 min. 10. Stop the reaction by adding 50 PL stopping solution. 11, Read the absorbance at 450 nm.

3.3.2. Western Blotting All incubations are carried out at room temperature. 1. Prepare denatured protein samples for loading by mixing equal volumes of 2X SDS-PAGE sample loading buffer and protein samples at a concentration of approx 3-4 mg/mL (see Note 11).

2. Separatethe proteins by SDS-PAGE using appropriate gel concentration (see vol. 32 of this series for detailed instruction). 3. Carry out electrophoresis at constant voltage of 200 V until the front dye reaches the bottom of the gel. This usually takes about 45 mm when the Bio-Rad MiniPROTEAN II apparatus is used.

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4. Remove the gel assembly, separate the glass plates, and cut off the stacking gel. Assemble the filter paper/gel/membrane/filter paper “sandwich” for electroblotting (see vol. 32 of this series). 5. Carry out transfer for 60 min at a constant current of 250 mA. 6. Remove the assembly from the Mini Trans-Blot apparatus, and cut away excess nitrocellulose membrane from around the gel. Carefully peel away the gel from the membrane, and place the membrane in a square Petri dish containing 30-50 mL blotto solution. Incubate for 15 mm with gentle rocking (see Note 12). 7. Seal the membrane inside a plastic bag with one side open, add l-2 mL of MAb solution diluted in blotto at 1:5 for tissue-culture supernatant or 1: 100 for ascetic fluid, and finally seal the remaining side. Incubate the bag for 30 min with gentle rocking (see Note 13). 8. Wash three times (3-5 min each) with approx 50 mL TBST. 9. Continue as in step 7, except that an AP-conjugated sheep antimouse antibody is used at 1: 1000 dilution instead of MAb. 10. Wash as m step 8. 11. For color development, incubate the membrane with 10 mL of AP substrate buffer containing 33 pL 5-bromo-1-chloro-3-indolyl phosphate (BCIP) and 66 pL nitro blue tetrazolium (NBT), mixed just before use. Dark purple signals should appear within 10-30 min (see Note 14).

3.3.3. Magnetic lmmunocapture All incubations

are carried out at room temperature.

1, Take 50 pL suspension (1 mg/mL) of SMB, and wash twice with 0.5 mL of the same blotto solution as used in Section 3.3.2. 2. Resuspend the washed SMB in 100 pL blotto containing 5 p,L biotinylated antimouse antibodies, and incubate the mixture for 30 min with gentle rocking (see Note 15). 3. Wash the SMB three times (5 min each) with 0.5 mL TBST. 4. As in step 2, except that 5 pL MAb solution are used (this can be either 5 JJL tissue-culture supernatant or 5 PL ascitic fluid at 1: 100 dilution). 5. Wash as in step 3. 6. As in step 2, except that 10 PL 35S-labeled protein mixture (produced in Section 3.2.2.) is used (see Note 16). 7. Wash as in step 3. 8. Resuspend the SMB in 20 pL of 1X sample loading buffer, and boil for 2 min before taking 5- and lo-p.L aliquot for SDS-PAGE analysis. 9. Separate the protein samples using standard SDS-PAGE (e.g., see vol. 32 of this series), and transfer the gel onto a precut 3MM filter paper for drying. 10. Dry the gel under vacuum for 60 min at 60°C. 11. Autoradiography using standard X-ray film and developing methods to reveal the signals on the gel.

Homolog Scanning 4. Notes 4.7. Generstlon

of HyMd

217 Genes

1. For simplicity, the protocols presented in this chapter only describe the procedures for the generation of two hybrid genes using the X-digested homolog I as the coupling template. As indicated in Fig. I, the procedures used for the production of the other two hybrid genes using the Y-digested homolog II as coupling template are identical to those presented in this chapter. 2. The template-coupled PCR method presented in this chapter is certainly one of the most efftcient strategies available for construction of homolog hybrid genes. However, when there are common restriction enzyme sites present in both of the homolog genes, gene splicing using restriction enzymes may still be a preferred method since the downstream characterization of the recombinant hybrid genes will be much simpler. 3. Restriction enzyme cleavage site X (or Y for homolog II) need not be a unique site in the plasmid, as long as there is no internal cut between the flanking primer and the desired crossover point. 4. Most restriction enzymes can be inactivated by heating. However, for certain heat-resistant restriction enzymes, it might be necessary to inactivate the enzyme activity by phenol extraction. 5. Transformation by electroporation is a highly efficient process and will certainly enhance the overall performance of this mapping experiment. However, for laboratories lacking electroporation equipment, use of the conventional CaC&-mediated transfotmation method is adequate. 6. The appropriate methods of characterizing the recombinant hybrid genes will vary depending on the sequences of the parental genes and on the nature of the antibodyassay method. If there are gene-specific restriction enzyme sites available in the parental molecules, they can be conveniently used in analyzing the hybrid genes (e.g., see ref. 7). If such restriction sites are also present in the vector DNA, one may wish to simplify the digestion pattern by carrying out the diagnostic restriction digestion on the insert DNA only. This can be easily achieved by PCR amplification of the insert DNA using two flanking primers, followed by direct digestion of the PCR product using appropriate enzyme(s). We found this approach very efficient, because high-quality insert DNA can be produced by direct colony PCR (e.g., see ref. 28), eliminating the need for plasmid minipreps and generating results within 4-5 h. If there are gene-specific internal primers available from other studies (e.g., primers made during the initial sequencing analysis of the genes), they can be directly used for hybrid gene analysis by PCR amplification using one vector-specific primer (i.e., one of the two flanking vector primers) and one gene-specific primer. Finally, since the hybrid genes are generated by PCR, the best characterization of these genes can only be carried out by DNA sequencing. A practical approach is to do a preliminary analysis by restriction enzyme digestion or diagnostic PCR using gene-specific primers, then carty out antibody-binding studies on the expressed proteins, and finally sequence the hybrid gene or selected gene segment to confirm the sequence change responsible for the observed antigenic variation.

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4.2. Production of Recombinant Protein Molecules 7. For some “partially soluble proterns,” it might be useful to add Trtton in the MTPBS buffer to a final concentration of 1% (v/v) to mcrease the solubthty during sonication and subsequent centrifugation. 8. If overnight exposure gives an easily visible or strong signal from a 5-uL sample (i.e., 5% of total translated product), the m vitro translation reaction 1s considered to be successful, and 10 yL of the translation product should be enough for immunocapture assays detailed in Section 3.3.3. If the signal is invisible or very weak after overnight exposure, it may be worth repeating the in vitro translation reaction before proceeding to the next step.

4.3. Antibody-Binding

Assays

9. Since the ELISA is carried out using unpurified protem samples, it is essential to include proper controls m this type of assay. It 1srecommended to use the following three controls m all assays. a. Protein sample from E. colz containing vector alone (e.g , pET3a) as a negative antigen control; b. Protein sample from E. colt containing the expression plasmid for the MAbreactive homolog (e g., PET-IFN4) as a positive antigen control; and c. An unrelated MAb as a negative antibody control. 10. The antibody dilution given is only to be used as a general guide. It is found that certain MAbs give better results when diluted m PBST m the absence of skim milk proteins. If the supply of antibody is not a major hmitmg factor, it is recommended to carry out a serial dilution for the antibody as well as the recombinant antigen to determine the optimal assay conditions 11. Since most conformation-dependent epitopes are sensitive to treatment by heat, SDS, and/or reducing agents, such as B-mercaptothanol and dithtothreitol (DTT), one may wish to try different conditions for sample treatment to increase the chance of epitope detection by Western blotting. One starting point will be to take out the reducing agent from conventional SDS-PAGE sample buffer and not to boll the sample before loading. 12. Membrane left in the blotto solution can be kept at 4°C for up to 48 h without sigmficant impact on the overall performance. 13. We find that a convenient way of keeping the membrane flat is to put the bag m the middle of a thick heavy book (e.g., a telephone directory readily available m every laboratory), which is m turn placed on top of a rocker. The 30-min mcubation time is the minimum time required, but can be extended to incubation at 4°C overnight to fit in with other ongoing experiments. This 1salso true for the incubation with conjugated antibody in Section 3 3.2 , step 9. 14. It is sometimes necessary to carry out an overnight mcubation in a hght-protected area for very weak signals to appear. For extremely high sensitivity, one may use HRP-conjugated secondary antibody and a chemiluminsecence substrate (e.g., the Chemilummscence Blotting Substrate [POD] Kit from Boehringer Mannheim) to reveal weak signals.

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15. It should be pointed out that the use of a biotinylated antimouse antibody is optional in this assay. It is possible to biotinylate the MAb of interest so that it can be used directly without the bridging antibody.

References 1. Robert-Hebmann, V., Emiliani, S., Resnicoff, M., Jean, F., and Devaux, C. (1992) Subtyping of human immunodeficiency virus isolates with a panel of monoclonal antibodies: identification of conserved and divergent epitopes on p17 and p25 core proteins. Mol. Immunol. 29, 1175-l 183. 2. Haijimorad, M. R., Dietzgen, R. G., and Francki, R. I. B. (1990) Differentiation and antigenic characterisation of closely related alfalfa mosaic virus strains with monoclonal antibodies. J. Gen Virol. 71,2809-2816. 3. Gabelish, C., Harbour, C., Beard-Pegler, M. A , Stubbs, E., Steffe, R., Large, M., Vickery, A., and Benn, R. (1991) Serological typing of coagulase-negative staphylococct using monoclonal antibodies. Epidemzol. Infect. 106,23 1-237. 4. Kwok, A. Y. C., Zu, X., Yang, C., Alfa, M. J., and Jay, F. T. (1993) Human interferon-gamma has three domains associated with its antiviral function: a neutralizing epitope typing scheme for human interferon-gamma. Immunology 79, 131-137. 5 Hansen, T. and Hannestad, K. (1989) Direct HLA typing by rosetting with immunomagnetic beads coated with specific antibodies. J, Immunogenet. 16, 137-139. 6. Cunningham, B. C., Jhuram, P., Ng, P., and Wells, J. A. (1989) Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis. Science 243, 1330-1336. 7. Wang, L., Hertzog, P. J , Galanis, M., Overall, M. L., Wame, G. J., and Linnane, A. W. (1994) Structure-function analysis of human IFN-a. Mapping of a conformational epitope by homologue scanning. J, Immunol. 152,705-7 15. 8. Wang, L.-F., Scanlon, D. B., Kattenbelt, J. A., Mecham, J. O., and Eaton, B. T. (1994) Fine mapping of a surface-accessible, immunodommant site on the bluetongue virus major core protern VP7. Virology 204, 8 11-814. 9. McClelland, A., deBear, J., Yost, S. C., Meyer, A. M., Marlor, C. W., and Greve, J. M. (1991) Identification of monoclonal antibody eprtopes and critical residues for rhinovirus binding in domain 1 of intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 88,7993-7997.

10. Horton, R. M., Hunt, H D., Jo, S. N , Pullen, J. K., and Pease, L R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77,6 l-68. 11. Keil, A. and Wagner, R. R. (1989) Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector. Vzrology 170,393407. 12. Caramori, T., Albertini, A. M., and Galizzi, A. (1991) In vivo generation of hybrids between two Bacillus thuringienesis insect-toxin-encoding genes. Gene 98, 3744.

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13. Gritz, L., Destree, A., Cormier, N., Day, E., Stallard, V., Caiazzo, T., Massara, G., and Panicali, D. (1990) Generation of hybrid genes and proteins by vaccima virus-mediated recombination: application to human immunodeficency virus type 1 env. J. Viral. 64,5948-5957. 14. Studier, F. W., Rosenberg, A. H., Dunn, J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89.

15. Yanisch-Perron, C., Vietra, J., and Messing, J. (1985) Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-l 09. 16. Overall, M. L. and Hertzog, P. J. (1991) Functional analysis of interferon-a subtypes using monoclonal antibodies to interferona4a: subtypes reactivity, neutralisation of biological activities and epitope analysis. A401 Immunol. 29, 39 l-399. 17. Gram, H., St&matter, U., Lorenz, M., Gluck, D., and Zenke, G. (1993) Phage display as a rapid gene expression system: production of bioactive cytokine-phage and generation of neutralizing antibodies. J. Immunol. Methods 161, 169-176. 18. Wang, L.-F., Du Plessis, D. H., White, J. R., Hyatt, A. D., and Eaton, B. T. (1995) Use of a gene-targeted phage display random epitope library to map an antigemc determinant on the bluetongue virus outer capsid protein VP5 J. Immunol. Methods 178, 1-12. 19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

20 Epitope Mapping Using an Oligonucleotide Replacement Method Hannah Alexander 1. Introduction Two of the major aims of immunology are to understand the molecular basis of antibody-antigen interaction and the elicitation of the immune response. Epitope mapping techniques are useful in defining these processes. Epitope mapping is aimed at defining small regions of a protein that are reactive with antibodies. Some epitope mapping techniques go further and identify the amino acids within the epitope that are critical for immune recognition, such that their substitution results in the loss of antigenicity. Generally, this is accomplished by testing the role of the critical amino acid residues within the context of short (generally synthetic) peptides. To assessthe true relevance of these techniques for determining the molecular mechanisms of antigenic recognition and immunogenicity, the results obtained with isolated peptides must be tested within the context of the entire folded protein. To this end, it is necessaryto be able to substitute specific amino acids within the protein of interest, allowing the production of the protein with antigenic epitopes containing specifically altered amino acid residues. The basic procedure that was used was to construct a synthetic gene, in modular form, that allowed the easy replacement of segments encoding epitopes, and then to perform site-directed alterations of specific amino acid residues within the epitopes. This method can be adapted easily to segments of larger proteins as well. The method is designed to test the antigenic role of short peptides within the expressed protein and, thus, relies on previous knowledge of proposed antigenic sites and some idea about which residues within the site are critical to immune recognition. From

Methods m Molecular Bology, vol. 66. Epitope Mappmg Protocols Edlted by. G E Morns

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Press Inc , Totowa,

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The protein that was studied is myohemerythrin (MHr) from the marine worm Themiste zostericola (I). Examples throughout this protocol will refer to this protein, and will be detailed in Section 4. The antigenic profile of the protein has been studied in detail using the epitope scanning technique (2), and the contribution of specific amino acid residues to the chemistry of the binding was determined using replacement studies (3). To assessthe validity of these techniques for identifying the antigenic epitopes of the MHr protein, the results obtained were tested with isolated peptides within the context of the entire folded protein. Based on the available protein sequence of the MHr protein (4), a synthetic MHr gene was constructed from a series of oligonucleotides, and as many restriction enzyme recognition sites as possible were engineered in to allow facile substitutions of segments of the protein, A recombinant protein was then produced and its physical, chemical, and immunologrcal properties were assessed(5). Critical amino acids within the protein were substituted by replacing the oligonucleotides encoding the proposed antigemc site. The mutant proteins were expressed and purified. By analyzing the binding of monoclonal antibodies (MAbs) to recombinant wild-type and mutant proteins, the role of individual critical residues in the highly antigenic site (MHr 79-84) within the context of the folded protem could be tested. The results directly showed that short peptides that were identified as antigenic sitesby vntue of binding to exlsting antibodies are indeed antigenic in the folded protein, and that protein antigenicity can be significantly reduced by alteration of single critical amino acid residues without destroying the biological activity of the protein (6). 2. Materials

2.1. Deriving the Nucleic Acid Sequence of the Gene and the Design of Oligonucleotkfes This requires a computer program that will perform DNA mampulations.

2.2. Construction of a Synthetic Gene 1. All DNA manipulations,

such as phosphorylatron of the oligonucleotrdes,

hga-

tion reactions,restriction enzymedigestions,transformations,andplasmid puri2. 3.

4. 5. 6.

fication, were performed according to conventional procedures (7). Buffers for DNA modification reactions are included with the enzymes by the manufacturers. T4 polynucleotide kinase (PNK). 5X PNK buffer: 350 mM Tris-HCl, pH 7.6, 50 mM MgCl,, 25 miI4 dithiothreitol (DTT). Phosphorylationreaction: 1.5 yL 10nuI4 ATP, 4 pL 5X PNK buffer, 1 pL PNK m a total of 20 PL HzO. 1MNaCl. T4 ligase and 10X ligase buffer: 200 rrGI4 Tris-HCl, pH 7.6, 100 rnJ4 MgClz, 100 mMDTT, 6 mA4ATP

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7. Oligonucleotides (30-40 nucleotides long; see Section 3.1.). 8. Restriction enzymes (according to the specific sites within your gene) and suitable buffers. 9. Expression vector (we recommend pT7-7; see Note 1). 10. Competent bacterial cells for transformations (see Note 2) 11. Ampicillin stock: Make a solution of 50 mg/mL ampicillin in H,O, filter-sterilize and keep frozen. 12. LB medium: in 1 L* 10 g bacto-tryptone, 5 g yeast extract, 5 g NaCl. Adjust the pH to 7.4 with 1NNaOH and autoclave. LB plates: Add 15 g agar before autoclaving. LB/amp: Add ampicillin to a final concentration of 100 pg/mL. For LB/amp plates, let LB/agar cool after autoclaving and add 2 mL of the sterile ampicillin stock solution. Top agar: To a 100&L aliquot of LB add 0 7 g of bacto-agar and autoclave. 13. Plasmid DNA purification kit (any commercial kit is tine). 14. TBE: Make a 5X stock solution. For 1 L: 54 g Tris-base, 27.5 g boric acid, 20 mL 0.5M EDTA, pH 8.0. 15. Agarose and agarose gel electrophoresis apparatus (gels run in 1X TBE buffer). 16. DNA sequencing kit, radioactive isotope for sequencing, and a sequencing gel electrophoresis apparatus.

2.3. Production

of Recombinant

Protein

17. Phage mGPl-2 stock (see Note 1). 18. Suppressor containing E. coli host cells (i.e., JMlOl, JM109). 19. Isopropyl thio-P-n-galactopyranoside (IPTG): Make a fresh stock of 100 mM in water before use. 20. Rifampicin: Make a stock solution of 20 mg/mL in methanol. Keep in the dark at 4OC; it is stable for about 2 wk. 2 1. M9 salts: m 1 L; 6 g Na,HPO,, 3 g KI$PO,, 5 g NaCl, 1 g NH&l, pH 7.4. 22. [3s5’+Methionine (1000 Ci/mmol); EN3HANCE (DuPont); methanol; acetic acid. 23. X-ray film. 24. Lysozyme. 25. Phenylmethylsulfonylfluoride (PMSF, protease inhibitor).

2.4. Site-Directed Replacements This consistsof oligonucleotides containing the proposed substitutions ( 1O-20 bp long; see Section 3.4.1.). 2.5. Immunological

Assays

26. PBS: 10 mA4 sodium phosphate, pH 7.2, 150 mA4NaCl. 27. Radio-immune-precipitation-assay buffer (RIPA): 150 mMNaC1, 10 mMsodium phosphate, pH 7.2, 1% NP-40, 0.5% sodium desoxycholate, 0.1% SDS, 2% trasylol (protease inhibitor, add fresh before use). 28. 10% Bovine serum albumin (BSA) in PBS or RIPA.

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224 29. 30. 3 1. 32. 33. 34.

LiCl wash: 100 mA4Tris-HCl, pH 8.5,500 mMLiC1. Borate buffer: O.lM sodmrn borate, pH 9.0. Fixed Stu$zylococc~s aureu~ cells (PansorbinTM, Calbiochem). BLOTTO: 5% Nonfat dry milM0.02% (v/v) Antifoam A (Sigma) in PBS (8). ELISA plates and ELISA plate reader. Developer solution: 100 mM sodium phosphate, pH 6.0, 2% glucose, 8 pg/mL horseradish peroxidase, 0.15 mg/mL 2,2’-azinobis[3-ethylbenz-thiazoline sulfomc acid] (ABTS).

3. Method 3.7. Deriving the Nucleic Acid Sequence of the Gene and Design of OIigonucleotides 1. If the nucleic sequence of the protein is not available, derive the coding sequence from the protein sequence, using the codons most frequently used m E. colz (9) (see Note 3). 2. Introduce as many unique restriction sites as possible in order to permit rapid subsequent alterations in the gene. For that purpose, a few less-frequently used codons can be allowed. The design of the oligonucleotides is time- and patienceconsuming. It is useful to search for 4-bp palindromes or for pairs of complementary nucleotides, and then test the immediately adjacent bases on both stdes to see whether there is an allowed substitution of nucleotide that will still maintain the same amino acid, but will add a new restriction site to the sequence (see Note 4). 3. If the coding sequence does not contain a methionme as the first residue, add tatg at the 5’-end to allow cloning into an NdeI site in the multiple cloning site (MCS) region, as well as creating an ATG initiation codon. 4. If the coding sequence does not contain a stop codon, add a stop codon and a restriction site beyond it to allow cloning of the complete gene (see Note 5). 5. Divide the coding sequence into a series of short ohgonucleotides (30-40 bases long) that cover both strands of the DNA. Each double-stranded segment should end with a 4-6 nucleotide single-stranded overhang that can anneal to the complementary segment of the adjacent neighbor. Wherever possible, design the ends of the ohgonucleotides to match the ends of restriction sites in order to facilitate cloning into the MCS region of the vector, as well as for screening of transformants (see Note 6). At this point, the oligonucleotides are ready to be synthesized. The design of the MHr gene is shown in Fig. 1.

3.2. Construction of the Gene The gene is assembled in steps, as described: 1, Phosphorylate 200 pmol of each oligonucleotide, using T4-PNK in a total of 20 pL phosphorylation reaction. Incubate for 60 min at 37’C followed by 10 min at 65°C.

Oligonucleotide

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Replacement

N&I II II~GGTTGGGAAATCCCGGMCCGTACGTATGGGACGAGTCTTTCCGTGTTTTCTACGAGCAGCTGGATGAGG~CAC~TCTT90 31*lus

I

51

Pvu

71Bgl

II

CGTCGACCTACTCCTTGTGTTTTTCTAG .r MGWEIPEPYVWDESFRVFYEQLDEEHKKIF

91Nru I “IIm II CAAAGGTATCTTCGATTGCAT~CG~GATAACTCTCTGCGCCG~CCTGGCGACCCTGGTT~GTTACCACC~CCACTTCACCCACG~GAlSO GTTTCCATAGAAGCTAACGTAAGCGCT GTGGTTGGTGAAGTGGGTGCTTCT 104 KGIFDCIRDNSAPNLATLVKVTTNHFTHEE

I 17bo I ‘1AGCGATGATGGATGCGGCGAAATACTCTG 3GTGGTTCCGCACAAAAAAA TGCATAAAGATTTCCTcGwTCGGTGGTCTGTCTGC270 TCGCT TACCTACGCCGCTTTATGAGACTTCACCA GGCGTGTTTTTTTACGTATTTCTAAAGGGC CTTTTAGCCACCAGACAGACG N#i

1T

164

18r

AMMDAAKYSEVVPHKKMHKDFLEKIGGLSA MHr/79-84 xpn I 331 UP 1 '1 931 'Y GCCGGT~GACGCGAAAAACGTTGATTACTGCAAAGAATGGCTGGTT~CCACATC~GGTACCGATTTC~GTAC~GGT~CTG~360 CGGCCAEC GCGCTTTTTGCAACTAATGACGTTTCTTACCGA CAATTGGTGTAGTTTCCATGGCTAAAGTTf4 TGTTTCCATTTGACBI; 20T 4 t PVDAKNVDYCKEWLVNHIKGTDFKYKGKL.

Fig. 1. The derived nucleotide sequence of the MHr gene. The nucleic acid sequence of the MI-h gene was deduced from the amino acid sequence (4) using the E. coli codon preference (9). Restriction sites unique to the MHr gene are indicated. Synthetic oligonucleotides are marked with a vertical line at their S-end in the coding strand (even numbers) and on the 3’-end on the complementary strand (odd numbers). Individual variations from the codon usage table, as well as additional nucleotides that are not part of the MHr sequence, are underlined. The antigenic site MHr/79-84 is indicated.

2. Anneal the oligonucleotides in small groups of six oligonucleotides each (three complementary pairs): Mix 4 pL of each of six phosphorylated oligonucleotides, 3 uL of 1M NaCl, and 3 uL of Hz0 in an Eppendorf tube. Place the tube in a beaker, boil for 5 min, remove from heat, and let cool to room temperature (about 45 min) while still in the beaker. Move to ice (see Note 7). 3. Ligate 2 pL of annealed oligonucleotides (one group at a time) to l-2 pg of vector (cut with appropriate restriction enzymes to generate ends that will fit the single-strand overhangs of the oligonucleotides) using 1 pL T4 ligase and 2 uL 10X ligase buffer in a final volume of 20 pL. Incubate at 15°C overnight (see Note 7). 4. Transform: Mix 2-4 pL of ligation mixture with 20 PL competent E. coli host cells of your choice (see Note 2), and let sit for 40 min on ice. Heat-shock for 2 min at 42”C, add 500 uL LB, and shake for 1 h at 37°C. Plate on LB/amp plates.

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5. Isolate positive transformants by picking single colonies into 5 mL of LB, shaking overnight, and preparing plasmid DNA using a commercial plasmid purification kit or any other quick screen method. 6. Analyze transformants for the acquisition of sites unique to the synthetic gene (see Note 7). 7. It is recommended to sequence the entire gene (or gene segment), using the ohgonucleotides as primers and any commercial sequencing kit, to ensure a proper assembly of the gene. 8 If needed, transfer the complete construct to E. coli K38 cells (see Note 2)

3.3. Production of Recombinant Protein 3.3.1. Preparation of mGPI-2 Phage 1. Plate a dilution of the phage stock on a suitable host (such as JMlOl, JM103, JM109) on LB plates. Incubate overnight. 2. Grow JM109 cells at 37’C m 10 mL of 2X LB. While m log phase, As9s = 0.1, innoculate with a single plaque of mGPl-2 and continue to shake overnight. Pellet the cells and use the supernatant as phage stock (titer should be around 2.5 x lOi plaque forming units [PFU]/mL). 3 Dilute a 20-mL overnight culture of JM109 cells mto 2 L of 2X LB (500 ml/flask) and follow absorbance. When the culture reaches an Ass0= 0 1, infect with phage stock at a multiplicity of infection (MOI) of 10. Continue to shake overnight. 4. Add 60 g NaCl/L and dissolve. Centrifuge cells for 20 min at 10,OOOg(8000 rpm m JA- 10 rotor), and discard pellets. 5 Add 70 g polyethylene glycol (PEG) 8000/L, stir gently for 30 mm, and let stand overnight at 4°C. 6. Centrifuge at 10,OOOg for 30 min to collect the PEG-precipitated phage. Resuspend phage pellets in 20 mL M9 salts, add a drop of chloroform, and store at 4°C 7. Titer phage by mixing 10 yL of 1O-7-1&1o dilutions of phage stock and 0.2 mL JM109 cells with 2.5 mL top agar and pouring onto LB plates. Successful preparation will result in around 1014 total PFU.

3.3.2. Expression of Recombinant Protein 1. Grow E. coli K38 cells containing your construct in 10 mL LB to A5s0 = 0.4. 2. Infect culture with mGP l-2 (MO1 = 20) in the presence of 2 mM IPTG. 3. At 25 min after induction, pellet the cells and resuspend in 10 mL of M9 salts for 30 min. Add rifampicin to a final concentration of 200 ug/mL (dilute solution 1: 100) for an additional 25 min. 4. If desired, the recombinant protein can be labeled for 10 min by addition of 10 pCi of [35SJ-methionine (1000 Ci/mmol)/mL of culture Divide into I-mL aliquots, centrifuge, and freeze pellets at -20°C. For direct visualization of the recombinant gene product, proceed through steps 5 and 6. If you wish to contirm the reactivity of the recombmant protein with your antibodies, proceed through steps 7-9.

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5. Resuspend one tube of frozen labeled cells in 40 pL of water, mix with an equal volume of SDS/polyacrylamide sample buffer (sample buffer), boil for 3 mm, and apply directly to polyacrylamide gels. The percent of the running gel is determined by the size of your protein. 6. Fix the gels in 20% methanol/lO% acetic acid, prepare for fluorography using EN3HANCE, dry, and autoradiograph; or 7. Lyse one tube of labeled cells overnight in 250 pL of PBS, 0.2 mA4 PMSF, and 5 mg/mL lysozyme. 8. Remove cell debris by centrifugation in a microcentrifuge. 9. Analyze the reacttvity of the recombinant protein with existing antibodies by immuneprecipitation (IP) (see Section 3.5.1.).

3.3.3. Protein Purification and Verification of Protein Properties Large scale production of recombinant protein (see Note 8). 1. Grow E. coli K38 cells containing your construct at 37°C in LB/amp to an AJ9,,= 1.O. 2. Infect with mGPl-2 (MO1 = 20) in the presence of 2 mM IPTG. After 30 min, add rifampicin to a final concentration of 200 yg/mL (dilute stock solution 1: loo), and continue to grow the culture for additional 90 min. 3. Harvest the cells by centrifugation, rinse in M9 salts, and store the cell pellets at -20°C.

3.4. Site-Directed Replacement 3.4. I. Design of Oligonucleotides and Replacement of Gene Segments 1. Choose one or more regions of the protein that have been proposed as antigenic sites by any of the epitope mapping techniques (see Note 9). 2. Propose substitutions for critical amino acid residues within the site. Choose conserved as well as nonconserved substitutions (see Note 10). 3. Design oligonucleotides that will encode the newly introduced amino acids. Whenever possible, introduce an additional change to the nucleotide sequence, but not the coding sequence, to eliminate a restriction site within the oligonucleotide for the purpose of selection and screening (see Note 11).

3.4.2. Expression of Mutant Proteins and Verification of Protein Properties 1. Express the wild-type and mutant proteins containing the designed substitutions using the T7 polymerase/T7 promotor expression system as in Section 3.3.2. 2. Purify the recombinant proteins as needed. If your protein has an easily assayed activity, typical absorbance, or other physical or chemical characteristic, it is suggested that you verify that the substitutions did not destroy the biological activity of the protein (see Note 12). 3. Analyze the reactivity of your existing antibodies with the mutant proteins.

Alexander

3.5. Immunological

Analysis

3.5.1, IP with Antibodies 1. Add 1O-20 pL of preimmune serum to cell extracts and incubate for 90 min on ice. 2. Add extracts to a pellet from 100 pL of Pansorbin, resuspend the pellets gently with a Pasteur pipet, incubate for 30 min on ice, and centrifuge. Keep supernatant as precleared extract. 3. In an Eppendorf tube, add 10 pL of the specific immune serum to varying amounts of the precleared extracts in a total of 100 pL PBS or RIPA (see Note 13), and incubate for 90 min on ice. 4. During the incubation prepare Pansorbin: Pellet Pansorbin suspension (40 pL/IP tube); decant and resuspend the pellet in 1 mL of 10% BSA/RIPA or 10% BSA/ PBS and incubate for 20-30 min on we; centrifuge, decant, and resuspend pellet in the original volume with RIPA or PBS. 5. Collect antigen-antibody complexes by adding 40 pL of washed Pansorbin (from step 4) to each IP tube; incubate for 30 min on ice. 6. Collect Pansorbin cells with adsorbed immune complexes by centrifugation. 7. Wash Pansorbin pellets 1X in 1 mL PBS (or RIPA) and 2X in 1 mL LiCl wash. 8. Suspend pellets in 50 pL of sample buffer, boil for 3 mm to release antigen, centrifuge to remove the Pansorbin pellet and apply supernatants containing the mnnunoprecipitates onto gels as in Section 3.3.2.

3.5.2. ELISA Assay 1. Add l-5 pmol of antigen in 50 pL PBS to each well of a polystyrene microtiter plate and dry overnight at 37°C. 2. Fix by adding 50 yL methanol/well for 5 min, shake off and air dry for 5 min (see Note 14). 3. Block with 100 pL BLOTTO/well for 4 h. 4. Add 25 pL/well of specific antiserum (serially diluted in BLOTTO), and mcubate the plates overnight at room temperature. 5. Remove unbound antibodies and wash the plates 10X under gently running water. 6. Add 25 pL/well of 1:200-l :500 dilution of glucose oxidase-conjugated secondary antibody (affinity-purified goat antimouse or goat antirabbit) for 90 min at 37°C. 7. Wash off unbound secondary antibody 10X under running water. 8. Develop by adding 50 pL/well of developer solution for 45 min at room temperature. 9. Read the plate in an ELISA plate reader by measuring absorbance at 405 nm.

3.5.3. Competitive ELlSA (see Note 75) 1. Coat microtiter plates with 1 pmol antigen/well as described above. 2. Add 15 PL of twofold serial dilutions of competing antigen to each well. 3. Add 15 pL of the indicated antibody dilution (see Note 16), mix, and incubate for 90 min at 37’C. 4. Wash the plates and proceed with ELISA assay as described above. 5. Present values as percent of reactivity in the absence of competition.

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4. Notes 1. For overexpression, the vector pT7-7 (10) is recommended for the following reasons. Plasmid pT7-7 is a derivative of the ampicillin-resistant pT7- 1, which contains a multiple cloning site, a T7 promoter, a ribosome-binding site (RBS), and a translation initiation site at an efficient distance from the RBS. Phage mGP l-2 is a derivative of Ml3 phage, harboring the T7-specific RNA polymerase gene. pT7-7, mGPl-2, and E. coli K38 were generous gifts from S. Tabor, Harvard Medical School (10). Expression of the T7-specific RNA polymerase is under the control of the luc promoter and is induced by IPTG. The gene of interest is cloned downstream from the T7 promoter sequence in the plasmid pT7-7. Expression of the protein is achieved by infecting growing cultures of E. coli K38, containing your recombinant construct, with mGPl-2 phage and inducing the T7-specific RNA polymerase with IPTG in the presence of rifampicin, which inhibits cellular RNA synthesis. Thus, only genes that are under the T7 promoter will be expressed. In the example of MHr, these conditions resulted in virtually exclusive synthesis of recombinant MHr protein, which ultimately constituted over 5% of the total soluble protein in the cell. 2 In the original experiments performed in this lab, commercially available competent cells were used (DHSo, Bethesda Research Laboratories). However, chemical- or electrocompetent cells can be prepared according to standard procedures. For the expression of the recombinant protein, E. coli K38 was used, which is lad and produces a low level of repressor to facilitate induction. Thus, constructions that are made in any other strain should be transformed into K38 before induction. 3. Using the E. coli codon preference is recommended when the protein is to be expressed in this organism, because it greatly contributes to the stability of the expressed protein (11). 4. Using a computer program that will perform DNA manipulations, the coding sequence of the protein can be reverse translated to the nucleotide sequence and examined for ambiguous bases (usually appearing as letters other than A, C, G, T) for a choice that will generate a restriction site. In the MHr example, the restriction site could be generated for the enzymes BglII, NruI, BstEII, X’zoI, and &fl by using less frequently used codons to encode the same amino acids. The substituted nucleotides are underlined in Fig. 1. 5. tgagctctgca was added to the 3’-end to create the opal (tga) stop, and Sad and PstI restriction sites to allow cloning. 6. The gene was divided into 24 oligomers (12 pairs), as shown m Fig. 1. Watch out for restriction enzymes that give compatible overhangs. Avoid having two different oligonucleotides ending with the same overhang, especially if they are to be annealed in the same group. 7. In the case of MHr, the oligonucleotides were annealed in four groups of six oligomers each, and the gene was constructed in four steps as follows: a. Step 1: Oligonucleotides 19-24 were cloned into the S&I&t1 sites in the MCS region of the vector, creating pT7-7/MHr(l!k24). This was screened for the acquisition of a KpnI site.

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b Step 2: Oligonucleotides l-6 were cloned into the NdeI-BgZII sites of the vector. This segment was cloned in the opposite orientation, and was later extracted and ligated to the rest of the gene. This was screened for the acquisition of SnuBI and PvuII sites. c. Step 3: Annealed oligonucleotides 7-12 and 13-18 were ligated to pT7-7/ MHr(l9-24) as a BglII-Sal1 fragment, creating pT7-7/MHr(7-24). It was screened for the loss of EcoRI site and the acquisition of XhoI, NsiI, and BstAI. d. Step 4: The NdeI-BgnI fragment (from step 2) and the BglII-P&I (from pT77/MHr(7-24) were excised, annealed, ligated, and cloned as an NdeI-PstI fragment into pT7-7. (The plasmids containing the above fragments were cut with HueIII, which cuts the vector at eight sites, but does not cut the MHr gene; this prevented religation of the vectors involved and avoided purification of the fragments.) 8. Some of the immunological experrments can be performed using crude extracts containing the recombinant protein. However, in some cases, you will need to purify the recombinant protein to various degrees. This protocol will obviously be different for each protein studied. If antibodies are available, they can be used for affinity purification. (The procedure used was to disrupt the cells with glass beads in a Braun homogenizer in the presence of DNase [to lower viscosity] and PMSF. Cell debris was removed by centrifugation, and the crude extracts were subjected to a 50-100% ammonium sulfate fractionation followed by two consecutive size fractionations on Sephadex G-100 and G-50 columns, This resulted m essentially pure recombinant MHr protein). 9. For these studies, the antigenic site spanning amino acid residues 79-84 of the MHr molecule was chosen: Asp-Phe-Leu-Glu-Lys-Ile (see Fig. 1). This antigenic site represents an a-helix in MHr and contains a salt bridge. This site was strongly recognized by anti-MHr antibodies produced in rabbits, as well as guinea pigs, chickens, mice, and to some extent sheep (12). There are extensive data on the reactivity of rabbit (I, 13) and mouse (14) antibodies with peptides spanning this site. Furthermore, there are MAbs that react with the MHrY79-84 antigemc site and exhibit preferential sensitivity to substitutions within this epitope (14). 10. The following are examples of substitutions that were introduced into the MHr, and the reasons for choosing these replacements: Phe 80 and Leu 81 are both hydrophobic, buried residues. Peptide-based replacement studies found both residues to be essential, since they did not allow even highly conservative substitutions (14). Conservative substitutions of these residues, which were predicted to affect the antigenicity, were made to determine their anttgenic role m the context of the folded protein. Lys 83 was chosen for study because it is a hydrophilic residue exposed on the surface of the molecule, and was found in the peptide replacement studies to be a selectively replaceable residue (2,3). In this study, it was replaced with a conservative change to Arg. Based on previous peptide-based replacement studies, it was predicted that this change would not reduce stgnificantly the binding to the MAbs.

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11. The following are examples of the strategies used to introduce the above substitutions: Substitution of Phe 80 to Tyr was introduced by cloning an N&I-XhoI fragment containing TAC (Tyr) at position 241-243, and a C instead of T at position 235, to eliminate the NsiI site while still keeping His 77. Leu 81 to Ile was generated by cloning an NsiI-XhoI fragment containing A instead of C at position 244. This created a codon for Ile and abolished the XhoI site. Arg 83 was generated by cloning an NsiI-Sal1 fragment containing CGT instead of AAA at 250-252, and a C for T at position 234 to eliminate the NsiI site. (It is possible to use PCR-based mutagenesis to generate these mutations. The same criteria should be applied to the design of primers.) 12, In this case, we took advantage of the fact the MHr protein has a unique absorbance at A,,, owing to the iron center. Even though the possibility of localized conformational changes in the mutants cannot be excluded, it was shown that functional folding was maintained by the correlation between independent measurements of the protein concentration (Lowry assay) and the concentration of the intact di-iron site (A&, The UV/Vis absorbance spectra of the recombinant mutant proteins illustrated that the proteins were expressed at high levels, and that they folded properly around the iron center. 13. For nondenaturing conditions, we use PBS. The use of RIPA may result in some denaturation of the molecule, although it results in a lower background of nonspecific adsorption. 14. If you have reasons to suspect that the drying of the antigen onto the plate and the fixation with methanol will affect the antigen, it is possible to coat the plate in less harsh conditions by letting the antigen adsorb to the plate overnight without drying and avoid the fixation step: Add l-5 pmol of antigen in 50 yL of PBS to each well of a polystyrene microtiter plate, cover, and incubate overnight at 37°C. Pour off supernatant and do not fix. 15. The advantage of competitive ELISA is that it allows one to determine if the differences that are observed in reactivity of the antibodies with immobilized antigen on the plates reflect relative differences in the affinity of binding. In this assay, the target antigen is immobilized to the plates, and the various competing proteins are allowed to interact with the antibody in solution. 16. Competitive ELISA should be performed at a previously determined limiting concentration of antiserum.

Acknowledgment This work was supported by the US National Science Foundation. Thanks are given to Vince McGuire and Steve Alexander for comments on this chapter.

References 1. Tainer, J. A., Getzoff, E. D., Alexander, H., Houghten, R. A., Olson, A. J., Lerner, R. A., and Hendrickson, W. A. (1984) The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein. Nature 312, 127-133.

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2. Geysen, H. M., Tainer, J. A., Rodda, S. J., Mason, T. J., Alexander, H., Getzoff, E. D., and Lerner, R. A. (1987) Chemistry of antibody binding to a protein. Science 235,1184-l 190. 3. Getzoff, E. D., Geysen, H. M., Rodda, S. J., Alexander, H., Tainer, J. A., and Lerner, R. A. (1987) Mechanisms of antibody binding to a protein Science 235, 1191-l 196. 4. Hendrickson, W. A., Klippenstein, G. L., and Ward, K. B (1975) Tertiary structure of myohemerythrin at low resolution, Proc. N&l, Acad. Sci. USA 72,2 160-2 164. 5. Alexander, H., Alexander, S., Heeon, F., Fieser, T. M., Hay, B. N., Getzoff, E. D., Tainer, J. A., and Lemer, R. A. (1991) Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene. Gene 99,15 l-156. 6. Alexander, H., Alexander, S., Getzoff, E. D., Tainer, J. A., Geysen, H. M., and Lemer, R. A. (1992) Altering the antigenicity of proteins. Proc. Natl. Acad. Scz. USA 89,3352-3356.

7. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 8. Johnson, D. A., Gautch, W. J., Sportsman, J. R., and Elder, J. H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech. 1,3-8. 9. Konigsberg, W. and Godson, G. N. (1983) Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia cok Proc. Natl. Acad. Sci USA 80,687-691,

10. Tabor, S. (1990) Expression using T7 RNA polymerase/promoter system, m Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J, A., Seidman, J. G., and Stmhl, K., eds.), Green Press, New York, pp. 16.2.1-16.2.11. 11. Springer, B. A. and Sligar, S. G. (1987) High level expression of sperm whale myoglobin in Escherichia coli. Proc. Natl. Acad. Sci. USA 84,8961-8965. 12. Getzoff, E. D., Tamer, J. A., Lemer, R. A., and Geysen, H. M. (1988) The chemistry and mechanism of antibody bmding to protein antigens. Adv. Immunol. 43, l-98. 13. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102, 259-274.

14. Fieser, T. M., Tainer, J. A., Geysen, H. M., Houghten, R. A., and Lemer, R. A. (1987) Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti-peptide antibodies with a protein a-helix. Proc. Natl. Acad. SCI. USA 84,8568-8572.

21 Epitope Mapping by Region-Specified PCR Mutagenesis Takehiko Shibataand Masayuki lkeda 1. Introduction This technique was developed for mapping of epitopes of monoclonal antibodies (MAb) at the amino acid sequence level; i.e., the identification of amino acids of an antigenic protein involved in specific interactions with each antibody. The epitope mapping includes: 1. Construction of a random basesubstitution DNA library for the gene encoding the antigenic protein in the X-gtl 1 phageexpression-vector; 2. Testsfor the crossreactivitiesof proteins expressedin plaquesformed by phages in the library; and 3. An analysisof DNA sequencesof the mutantgenesencodingproteinswith altered crossreactivities. An efficient tool to generate a mutagenized DNA library is polymerase chain reaction (PCR) (I) under conditions that cause highly increased errors in DNA synthesis owing to base substitutions (resulting in amino acid substitution mutations; see Note 1). We added deoxyinosine 5’-triphosphate (dITP) to the reaction mixture of PCR to increase misincorporation of bases (i.e., base substitutions; see Notes 2 and 3). In addition, PCR is an excellent tool for specific mutagenesis within a defined DNA region; i.e., a DNA region to be mutagenized is specified by a pair of PCR primers flanking the region. One can amplify an entire gene for the antigenic protein or a subregion of the gene, if one has information about the approximate region of the epitopes (Fig. 1). The application of this technique requires: 1, A cloned geneencoding the antigenic protein; 2. Sequencedata of the geneand the flanking regions; and 3. At least two MAbs that crossreactwith the protein at different epitopes. From:

Methods In Molecular Biology, vol. 66: Epltope Mapping Protocols Edited by: 0. E. Morris Humana Press Inc., Totowa, NJ

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234 primer

1-

recA g-b

AT0

TAA primer ‘t

EJRI

PCR (dATP, dl-l-P, dGTP, dCTP, dlTP, ~aq DNA polymerase)

$

recA

ATG

2

EcoRl

ene

TAA

E&I

EcoRl

EcoRl cleavage 1

Isolation of EcoRI-fragments + t EcoRl

EcoRl

DNA-library formation on lambda-gtl l-vector

1

0 0

0

0

0

0

Plaque formation on E. coliY1090

0

Transfer onto membranes 4 Immuno-blotting experiment with ARM1 91 ARM193

Fig. 1. Outline of the mapping by region-specified PCR mutagenesis. This figure shows the outline of the procedure used for epitope mapping on RecA protein against anti-RecA protein monoclonal IgGs, ARh4 19 1, and ARM 193 (4). Open and closed circles in two big circles represent plaques expressing protein showing crossreaction and those showing no crossreaction with the indicated IgG, respectively.

One can use other techniques for random mutagenesis of a specified gene, such as in vitro mutagenesis by use of a mixture of oligonucleotides, including random replacements of bases or oligonucleotide cassettes(2,3). The current PCR mutagenesis has merits over other methods as follows:

Region-Specified PCR Mutagenesis

235

1, A region to which mutations are introduced is easily specified by use of a pair of DNA primers; 2. Restriction sites and a linker sequence required for the insertion of the amplified gene into an expression vector are easily introduced by designing the primer sequence; 3. Mutation rate is easily controlled by simple modifications of conditions for PCR that are high enough to obtain mutations to locate epitopes; 4. As far as we tested, all mutations obtained by PCR were base substitutions; and 5. Sufficient amounts of DNA for the construction of a library are obtained through the procedure.

We will describe a procedure applied to map epitopes on Escherichia coli RecA protein against anti-RecA protein monoclonal IgGs (4). In this procedure, we amplified and mutagenized the entire recA gene. Then, a DNA region encoding a C-terminal 94 amino acid region was cut out by EcoRI restriction enzyme and ligated into an EcoRI site of h-gtl 1 expression vector to construct a library (Fig. 1). Thus, the C-terminal region of RecA protein is expressed as a fusion protein with P-galactosidase. Protein in plaques obtained by phages derived from the library were transferred onto membranes, and the crossreactivities were tested by means of immunoblotting experiments against a couple of monoclonal IgGs, ARM1 9 1 and ARM1 93 (Fig. 2).

2. Materials 1. TE buffer: 10 mMTris-HCl, pH 7.5,1 nrjWethylene&amine tetraacetic acid (EDTA) 2. 1OX PCR buffer: 15 mM MgC12, 500 mA4 KCl, 0.0 1% gelatin, 100 mM Tris-HCl buffer (pH 8.3 at 25’C after diluted to 10 mA4). 3. DEAE membrane: NA45, Schleicher & Schuell (Dassel, Germany). 4 TBS buffer: 20 mMTris-HCl, pH 7 5, 150 mMNaC1. 5. TY plate: 1% (w/v) Difco (Detroit, MI) tryptone, 0.5% Difco yeast extract, 1% NaCl, 1.5% Dtfco agar 6. Plastic Petri dishes: Square-shaped dishes (10 x 14 cm) for screening of plaques of a DNA library. 7. Phage dilution buffer: 10 nnl4 Tris-HCl, pH 7.5, 10 mM MgSO,, 0.0 1% gelatin. 8. Nitrocellulose membranes for immunoblotting experiments: BA85 type (pore size 0.45 pm, 9 x 12.4 cm, Schleicher & Schuell) that are autoclaved at 121°C for 20 min, soaked in 50 mMisopropyl+n-thiogalactoside (IPTG), and dried before use. 9. 1% Bovine serum albumin (BSA): BSA (type 5, Sigma, St. Louis, MO) dissolved m TBS buffer. 10. Anti-(mouse IgG) antibody labeled with horseradish peroxidase: An affinitypurified preparation purchased from Kirkegaard & Perry Laboratories, Inc. (0.2 ug/mL, cat. no. KPL 14-18-02). 11. 4-chloro- 1-naphthol H202 solution, prepared just before the assay as follows: Dissolve 4-chloro-1-naphthol in methanol at 3 mg/mL, and dilute the solution sixfold by TBS buffer. Add 30% H202 (0.5 yL/mL).

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Library 1

Library 2

ARM191

ARM193

Anti-RecA-IgG Fig. 2. Examples of immunoblotting experiments to detect plaques expressing mutant protein for crossreaction.A plaqueindicatedby an arrowheadin eachpanel for Library 1 showed crossreaction with ARM193, but not with ARM191. That for Library 2 showedcrossreactionwith ARM19 1, but not with ARM1 93.

3. Method Generalmethodsfor restrictionenzymetreatment,gel electrophoresis,recovery of DNA from the gel, DNA ligation, phage experiments,cloning of DNA fi-agments into a sequencingvector, and DNA sequenceanalysis are described in detail in published laboratory manuals(5,6) and in other chaptersin this volume.

3.1. Design of Primers for PCR A pair of primers should be designed to have a cutting site for a suitable restriction enzyme and a linker sequenceto connect the amplified DNA in frame to an N-terminal portion of the La& gene at the unique EcoRI site on h-gtl 1 vector. In the mapping of epitopes of RecA protein, we amplified the entire RecA gene and ligated the C-terminal fragment generated by EcoRI

Region-Specified

PCR Mutagenesis

237

digestion into the EcoRI site of h-gtl 1 vector (Fig. 1). Thus, only one primer (primer 2) complementary to a region outside 3’-terminus of the gene was designed to have an EcoRI site. The primers used in this mapping were primer 1 (S-ATGGCTATCGACGAAACAA-3’) and primer 2 (S-GAATTCTGTCATGGCATATCCTT-3’).

3.2. PCR 1. Prepare a reaction mixture (100 pL) containing 1 pil4 each of primers flanking the sequence to be amplified, ca. 3 ng of the template DNA (linearized), 200 @4 each of dATP, dTTP, dGTP, and dCTP, 200 @4 deoxyinosine S-triphosphate (dITP), and 0.025 U of Tuq DNA polymerase/pL in 1X PCR buffer (lo-fold dilution of 10X PCR buffer). One can use “GeneAmp DNA Amplification Reagent Kit with AmpliTaq,” distributed from Perkin Elmer Cetus (Part No. N801-0055). In the mapping of epitopes of RecA protein, we used pBEU14 DNA (7) linearized by the treatment with BamHI as a template for PCR. 2. Load 30 pL liquid paraffin onto the reaction mixture to prevent evaporation during PCR process. 3. For each cycle of PCR, anneal primers onto the template DNA by mcubation at 37°C for 2 min, synthesize DNA at 72°C for 3 min, and denature the synthesized DNA by incubation at 94’C for 1 min. 4. After 25 cycles of PCR, treat the amplified DNA with phenol saturated with water, followed by chloroform extraction. 5. Add l/10 vol of 3Msodium acetate to the DNA solution and precipitate the DNA by the addition of 2.5 vol of ethanol at -80% for 2 h. 6. Recover the precipitated DNA by centrifugation at 15,OOOg for 20 min at 4’C, and dissolve the DNA in 20 PL TE buffer. 7. Treat the amplified DNA with EcoRI restriction enzyme and separate a fragment encoding the mutagenized gene by gel electrophoresis and trapping wtth a DEAE membrane.

3.3. Construction of a Library of Mutagenized DNA in A# 7 Vector 1, Mix the isolated DNA fragments with EcoRI fragments of h-gtl 1 vector DNA (ca. 1 pg) at 1: 1 molar ratio and ligate the fragments into the EcoRI site of the lucZ gene by incubation at 4°C overnight in a 5-pL reaction mixture. We used h-gt 11 cloning kit (h-gt 1 l/EcoRIICIAP-Treated Vector Kit cat. #2342 11, Stratagene) in which h-gt 11 DNA had been cut by EcoRI restriction enzyme and the cut sites had been treated with phosphatase to prevent self-ligation. 2. Package the ligated DNA into a h phage particle. We used a h in vitro packaging kit (code N.334Y, Amersham, Arlington Heights, IL) for this process. Since ligated DNA takes either of two orientations relative to the vector DNA, half of the phage particles contain the amplified DNA in frame with IacZ gene and will express a fusion protein with P-galactosidase, but the rest of the particles will not express the amplified DNA.

238 3.4. lmmunoblotting

Shibata and lkeda Experiments

1. Dilute the packaged phage suspension so that it will give approx 1O3plaques/100 cm*oftheTYplate(lOx 14cm). 2. Plate the phages with Eschenchia coli Y 1090 strain as host on TY plates by use of soft-agar (0.7%) overlay technique (described in detail m Chapter 23). 3. Transfer proteins in plaques on the plate onto two or more nitrocellulose membranes (9 x 12.4 cm) by placing the membrane on the plate at 37°C for 2 h. 4. Soak the membranes in 1% bovine serum albumin dissolved in TBS buffer for 2 h at room temperature for blocking. 5. Soak each membrane in a solution of a tested MAb (at an appropriate concentration depending on each antibody) dissolved in TBS buffer containing 0.1% bovine serum albumin for about 2 h at room temperature. 6. Wash the membranes with TBS buffer three times, and soak them m TBS buffer containing 0.1% bovine serum albumin and an antibody agamst the tested antlbodies labeled with horseradish peroxidase at room temperature for 2 h. In the mapping of epltopes of RecA protein against anti-RecA protein-mouse monoclonal IgG, we used an anti-(mouse IgG) antibody labeled with horseradish peroxidase. 7. Soak the washed membranes in a 4-chloro- 1-naphthol-H202 solution until appropnate expression of coloring reactions, wash the membranes with water, and dry them. 8. Compare the two or more membranes. Plaques that contam wild-type protein (with respect to the crossreaction with the tested antibodies) give a positive coloring signal on all membranes, and those that do not express the amplified gene give no positive signal on any membrane. 9. Mark plaques that give no positive signal on one (or some) of the membranes and give a positive signal on the other(s). Figure 2 shows examples m which the wildtype or mutant RecA protein expressed in plaques as fusion proteins reacted with an anti-RecA protein monoclonal IgG, ARM191, or ARM193, followed by a coloring reaction to detect the bound IgGs to the fusion proteins.

3.5. DNA Sequence Analysis 1. Pick up the phages in the marked plaques, and suspend them in a least volume of phage dilution buffer. 2. Purify these mutant phages by a series of single-plaque isolations, and amplify the isolated phages. 3. Prepare DNA samples from the phage particles. 4. Cut out DNA fragments encoding a mutated gene by use of appropriate restnction enzymes. 5. Reclone the DNA fragments into a sequencing vector (such as pUC19) for DNA sequence analysis. 6. Analyze the DNA-sequences of both strands by the dideoxyrlbonucleotide chain termination technique (8) to locate base substitutions. For these reactions, we followed a procedure described m a manual for Sequenase (United States Blochemical Co., Cleveland, OH) and used an automated DNA sequence analyzer. Figure 3 shows amino acid substitutions of RecA protein that prevent crossreaction with each anti-RecA protein IgG (4).

239

Region-Specified PCR Mutagenesis Anti-RecA protein-IgG

either or both

C-terminus

Mab156 ARM191

RecA protein (residue number) Fig. 3. A map of amino acid residues included in epitopes against anti-RecA protein-IgGs (4). Each box represents each amino acid residue. Filled boxes indicate amino acids of which replacement resulted in a loss of crossreactivity against the indicated IgG. Shadowed boxes indicate amino acids of which replacement resulted in a partial loss of crossreactivity

4. Notes 1. Thermus aquaticus (Tug) DNA polymerase is known to show a higher rate of errors in DNA synthesis, because of the absence of a proofreading exonuclease activity and the incubation at higher temperature (9). The higher frequencies of errors during DNA synthesis are a serious problem for gene amplification and DNA sequence analysis, but Taq DNA polymerase provides a simple technique for in vitro mutagenesis. 2. We added dITP to increase errors in the incorporation of nucleotides during PCR. We tested the concentration of dITP from 0.2 to 200 pA4, and found that 200 @4 dITP gave a lo-fold increase in the yield of mutant RecA protem that showed altered crossreactivities (4). By PCR in the presence of 200 pA4 dITP, we picked up 21 candidate plaques among 2000 plaques expressing the C-termmal94 amino acid region (approx 280 nucleotides) of RecA protein, and finally obtained 10 kinds of mutant recA genes for the crossreaction against anti-RecA protein IgGs. We detected 18 kinds among 25 mutations in 19 mutant recA genes obtained from several experiments, and found that all of them were base substitutions. Fifteen of the mutant recA genes had single base substitutions and the other four had two or three base substitutions (4). Under normal conditions for DNAsynthesis (in the absence of dITP), errors in synthesis by Taq DNA polymerase were reported to be caused by single base substitution mutations, and less frequently (about a quarter of the rate of base substitution) by frame-shift mutations (9). We have not tested concentrations of dITP higher than 200 pM, which might be worth testing.

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3. Other conditions that increase errors in DNA synthesis by Tuq DNA polymerase are available. These include an increase in the concentration of MgClz relative to the four dNTPs (dATP, dGTP, dTTP, and dCTP), a decrease in the dNTP concentrations, and higher pH (ZU). When the MgCl, concentration was increased from 4 to 20 mM in the presence of 250 w each of dNTP, the mutation rate was shown to increase by 71-fold, and an increase in pH from 5.1 to 8.2 increased the mutation rate by 56-fold (10). Variations in the relative concentrations among dNTPs and replacement of MgC12 by MnC12 were shown to reduce fidelity of DNA synthesis in PCR by Tuq DNA polymerase (IZ). These conditions would be useful to increase the mutation frequency during PCR.

Acknowledgments This study was supported partly by a grant for Biodesign Research Program from RIKEN

and partly by a grant from the Ministry

of Education,

Science,

and Culture.

References 1. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985) Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. 2. Kramer, K. and Fritz, H.-J. (1987) Oligonucleotide-directed constructton of mutations via gapped duplex DNA. Methods Enzymol. 154,350-367. 3. Reidhaar-Olson, J. F and Bowie, J U. (1991) Random mutagenests of protein sequences using oligonucleotide cassettes. Methods Enzymol. 208,564-587. 4. Ikeda, M., Hamano, K., and Shibata, T. (1992) Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis. J Biol. Chem. 267,629 l-6296. 5. Berger, S. L. and Kimmel, A R. (1987) Guide to molecular cloning techniques. Methods Enzymol. 152, 6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 7. Uhlin, B. E. and Clark, A. J. (1981) Overproduction of the Escherzchia coli recA protein without stimulation of its proteolytic activity. J. Bacterzol. 148,386-390. 8. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. SCL USA 74,5463-5467. 9. Tindall, K. R. and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27,6008-6013. 10. Eckert, K. and Kunkel, T. A. (1990) High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. l&3739-3744. 11. Leung, D. W., Chen, E., and Goeddel, D. V. (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1, 11-15.

22 Random Fragment Libraries Using Yeast Expression Plasmid Serge BBnichou and Genevibve lnchauspb 1. Introduction The natural immune response against pathogens can be extremely varied, ranging from a strong, broad humoral and cellular response capable of stopping disease progression to a weak, inadequate, or even deleterious response inefficient at controlling replication of the infectious agent. Epitope mapping provides one approach to the understanding of the interactions between the host immune system and the attacking pathogen. Current methods for epitope mapping rely on computer models, the use of chemically synthesized peptides (1,2), and/or the development of expression assays in prokaryotic or eukaryotic environments (3). The former approaches can be limited, since choice of sequences synthesized or of the chemistry involved may be inappropriate to allow for proper mimicry of native determinants. The latter usually involves an immunoscreening approach to clone isolation leading to the identification of the epitope sequences. We will describe here the basic principle and advantages of an epitope mapping approach based on the use of yeast-derived expression libraries. This approach is based on recombinant DNA that allows dissection of a protein of interest into its recognizable epitopes via the exploitation of an inexpensive and rapidly implemented technology. By this method, each continuous epitope is randomly produced as peptides of various length, 15-60 amino acid (aa) residues. The yeast expression system offers a number of advantages over the Escherichia c&‘-based expression system: 1. It has no toxin for mammals; 2. Many posttranslational

modifications

of the expressed antigens can take place

(seeNote 1); and From-

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3. Secretion of the protems or determmants into the culture flutd is typically achieved, simplifying purification procedures and assays for antibody-antigen recognition (#,5).

The basic principle of the approach is illustrated in Fig. 1 and can be outlined as followed: 1. An cl-factor-based expression vector, offering optimal use of the yeast machinery (including posttranslational modifications, increased secretion) is used for

expressionof peptide sequencesof interest. 2. The determinant domain to be analyzed (e.g., a viral sequence) is digested randomly and used for library construction in the expression vector. 3. The resulting library is screened with specific antibodies (mono- or polyclonals) and posmve clones isolated. 4. Further characterization of the reactive clones IS performed (e.g., by sequencing of the insert sequences, expression, and purificatton of the correspondmg

antigen) to correlate epitope reactivity with amino acid sequenceand chemical characteristtcs.

One interest of the approach is to combine both advantages offered by an epitope mapping strategy based on the use of synthetic peptides in terms of the capacity to identify sequential determinants and those offered by eukaryotic expression systems in terms of favoring a more complex conformation of the epitopes. The yeast approach is cost-effective compared to using synthetic peptides that can lead to a biased choice of sequences to be tested. This limitation is mostly overcome in the yeast system, since peptides are randomly generated from the larger sequence of interest, thus offering a wider combmation of sequences and a unique source of candidate peptides likely to encompass determinant sequences. Since the size of the digested inserts can be selected to range from 20-30 to 6040 aa or more (see Section 3.), it is possible, depending on the complexity of the antigenic structure studied, to select for a smaller or larger insert population to be cloned. Ultimately, the simultaneous construction of two libraries, expressing small or large cloned inserts, can be performed to favor an exhaustive screening of the determinant sequences. A typical size selection for digested inserts is 30-40 aa, although antigenic structures as large as 100 aa or more (e.g., granulocyte-macrophage colony-stimulating factors, mouse-112,human epidermal growth factor) have been properly processed and expressed using the yeast a-factor-directed synthesis (68). There are two basic requirements for a most efficient use of the approach: (1) the sequence (or clone) encompassing the determinant domain to be analyzed has to be available, and (2) knowledge on the titers and affinities of the antibodies under evaluation is useful to avoid nonspecific signals and/or background effects. At the stage of library construction, knowledge of the actual antigenic sequence per se is not required, although ultimately sequences of

243

Epitope Libraries in Yeast Immunological

screening of the epitope expressed in yeast

library

l-Transformation of the yeast strain W303-1B with the library plasmtd DNA and spreading on selective media. ZTransfer

of the yeast colonies

onto nitrocellulose

filters.

nitrocellulose filter petri dish with yeast colonies

3-Lysis of the cells. ,

* A * .

A .

.

yeastcoloniesonto nitrocellulose q 3MM paper soaked with lysls buffer

P-Treatment of the nitrocellulose filters polyclonal or monoclonal antibodies. 5-Rescue of library

plasmid

by immunoblot

from immunoreactive

with

yeast clones.

B-DNA sequencing of insert from library plasmid and deduction the amino acid sequence of the recombinant peptide expressed.

of

Fig. 1. Summary of the different steps involved in the construction and immunological screening of the epitope library expressed in yeast.

identified clones would have to be compared to the sequence of the protein studied for confirmation of specificity. An important criteria is the knowledge of, or at least some indication of the titers (or concentration) of antibodies that will be used for antibody probing of the library. Adequate antibody titers are essential when screening with polyclonal antibodies obtained from infected hosts, such as sera from infected patients or from immunized animals. A dilution of such antibodies is required (at least 1:100-l :300), since undiluted serum often crossreacts with antigens expressed from the yeast cells, thus resulting in uninterpretable background signals. Low-level antibody or low-affinity antibodies cannot be properly analyzed with the approach, unless strategies for concentration of the molecules (such as antigen-based affinity columns when purified antigens are available) can be applied. Mapping of monoclonal antibodies (MAb) using yeast expression libraries is usually very effective and can

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B6nichou and lnchausp&

be rapidly carried out. For example, a two-step procedure can be used, first using mixes of various antibodies followed by a second screening of the candidate clones using each antibody separately. Construction of the yeast expression/secretion vector pSE-X is shown in Fig. 2 (from ref. 9). The plasmid is based on a pUC 18 backbone, and is able to replicate in both E. coli and yeast. The pUC 18 polycloning region has been removed and five pieces of foreign DNA added: 1. The promoter and partial coding sequence(600 bp) of the a-mating factor gene (MFa- 1 gene, Fig. 1C); 2. A 35-bp fragment of plasmidpEX2 providing a stopcodon for eachopenreading frame of MFa-1 (10);

3. A 400-bp fragment of TRP5 yeastgenecarrying signalsfor genetermination (7); 4. The yeastselectablemarker UR43; and 5. The yeastorigin of replication (the 2 pm DNA). Figure 3 illustrates an example of the antigenicity of crude recombinant products directly tested from culture supernatants in ELISA tests (from ref. I I). In that case, a clone containing an identified determinant of the HIV Env. region (aa 591-642) is being used. As shown in Fig. 3A, 100% of the HIVpositive sera tested, but none of the HIV-negative human sera reacted with the crude pSE-env. 591-642 supernatant. When positive and negative sera were incubated under the same experimental conditions with culture supernatants from cells containing only pSE-X without an insert, no crossreactivity could be detected. When more dilute sera were incubated (from 1:250 to 1:1000; Fig. 4B), only a moderate decrease in the detection intensity could be monitored. Figure 4 illustrates the identification of both linear and conformational determinants using the yeast immunoscreening approach. Both types of determinants were identified in the glycoprotein E2 of hepatitis C virus (HCV) (12). Reactivity patterns obtained after migration of various recombinant peptides expressed from clones (Y l-Y42) that were found to be reactive with a monospecific antibody were analyzed using nondenaturing (Fig. 4A) and denaturing (Fig. 4B) conditions. The data suggest that at least one antigenic determinant that is nonlinear in nature and a linear determinant(s) might also be contained in Y 16 sequence. Further analysis of the determinant domains indicated that it was only possible to mimic some of them using linear synthetic peptides, thereby confirming the previous observation. Also shown in the figure is an indication of the high level of glycosylation that may result from the yeast expression machinery (Fig. 4C). The multiple bands present in each lane are probably the result of incomplete processing of the mata fusion peptide (8). Such analysis allows the determination of whether the carbohydrate portions of the antigens may be involved in the antigenic function of the molecule.

Epitope Libraries in Yeast

245 -I I a I I a I I a I 1 al

AT SIGNAL1

---ilREAEA~HWLQLKPGQPMV;KR--J Et locIor t KEK 2 “’

l3

KCXl

C

Fig. 2. Construction of the yeastexpression/secretion vector pSE-X. (A) Domainsof the o-factor precursor; below is an enlargementof one of the four hormonal repeats. KEX2 is a specific endopeptidase,whereasK.EXl and STE13 are specific exopeptidases(Fuller). (B) Map of pSE-X: yeastDNA (@Q), DNA functional in E. coli (BIB), DNA from pEX2 (4); Hind111is the cloning site,whereasPstI sitesareusedto recover someinserts.(C) Schematicrepresentationof the MFol-1 TRF? sequenceof pSE-X with a detailed enlargement(below) of the reading frame at the Hi&III cloning site (9).

Bhnichou and lnchauspk

246 A

2.0..

B

2.09

1.5 15E 5 l.OE E %

-

f

1.0 -

d 6

OS-

05 O1

is VI of crude

--

0t

supematant

‘/250 reciprocal

‘ho0 dilution

of

human

‘~1000 sera

Fig. 3. Reactivity of HIV-posmve (filled squares) and HIV-negative (open squares) human sera with pSE-env.591-642 m ELISA tests. (A) As a functton of the amount of antigen in crude culture supernatant (20, 10,5, 1 uL). Equal volumes of supernatant from cells carrying pSE-x without insert were employed as controls. Sera were used at a 1:250 dilution after preadsorption over yeast cell lysates. The background intensity was measured for all sera (mean A4s2 = 0.04 from 18 sera) m the absence (0 pL) of supernatant in the wells. The cutoff value (dashed line) corresponds to three ttmes the mean value (.4492= 0.05) obtained for negative sera. (B) As a hmctton of the rectprocal dilutions of the sera. Wells were coated with 20 l.tL of pSE-env591-642 supernatant. In the experiments shown in Fig. 4, the peroxtdase-detecting reaction was stopped after 15 min of incubation, Each point represents the mean duplicate values (I I).

2. Materials Materials consist of pSE-X plasmid and W303- 1B yeast strain (see Note 2).

2.1. Special Equipment 1. Refrigerated microcentrifuge

for 1S-mL tubes.

2. Incubators and shaker incubatorsfor bacteria at 37°C and yeastat 30°C. 3. Vortex mixer.

2.2. Special Reagents 4. Deoxyribonuclease

I: DNase I (Boehringer, Meylan, France).

Epitope Libraries in Yeast

247

Fig. 4. Westernblot andN-glycosidaseF digestionanalysisof yeast-expressedantigens. Samplesof YC, Yl, Y8, Y16, Y20, Y22, and Y42 were analyzed using either non denaturing(A) or SDS and P-ME-denaturing(B) conditions; 5-10 yg of proteins were loaded on the gels for each sampleanalyzed.N-glycosidase F digestion (+) or undigested(-) samplesof YC, Y8, and Y 16were analyzedon nondenaturinggels (C). Molecular-weight controls are representedfor denaturingconditions only (12). 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26.

Phenol/chloroform/isoamyl alcohol (PCI): 25/24/l. Ethanol 100%and ethanol 70% in water. BacteriophageT4 DNA ligase (Biolabs, Beverly, MA). Adenosinetiphosphate (ATP) stock solution: 20 mM in Hz0 (pH 7.0). Deoxyribonucleosidetriphosphates(dNTP) stock solution: 10&each of dATP, dCTP, dGTP, and dTTP in Hz0 (pH 7.0). Klenow fragment of E. coIi DNA polymeraseI (Biolabs). BacteriophageT4 DNA polymerase(Biolabs). Agarose,type II grade (Sigma, La Verpilliere, France). Hind11 restriction enzyme (Biolabs). Calf intestinal alkaline phosphatase(CIP) (Biolabs). Epicurian Coli SCSl supercompetentcells (Stratagene,Montigny-le-Bretonneux, France). P-mercaptoethanol:14.2it4stock solution (Bio-Rad). Nitrocellulose filters: 132- and 82-mm diameter(Amersham,Les Ulis, France). Salmon spermcarrier DNA: 10 pg/pL stock solution (Clontech, Palo Alto, CA). Whatman 3MM paper. Nonidet P-40 (Sigma). Horseradishperoxidase-labeledsecondaryantibody (Amersham). 3,3’-diaminobenzidine(DAB) (Sigma). Ni (NH&SO&, 6 HZ0 (Carlo Erba). Hydrogen peroxide (H202) 30% solution (Sigma). Glassbeads:425-600 ).t(Sigma). Sodium azide: NaN3 (Sigma).

248

Bhichou

and lnchausp6

2.3. Buffers 27. 500 mM EDTA. pH 7.5. 29. 10X DNase I buffer: 10 mM MnC&, 500 mM Tris-HCl, pH 7.5, and 1 mg/mL BSA (see Note 3). 30. 10X bacteriophage T4 DNA ligase buffer: 200 mM Tris-HCl, pH 7.5, 50 mM MgCl*, 50 mA4 dithiothreitol. 3 1. TE: 10 mMTris-HCl, pH 7.5,l mMEDTA. 32. 5M NaCI. 33. 3MNa acetate, pH 5.2. 34. 10X bacteriophage T4 DNA polymerase buffer: 0.33M Tris-acetate, pH 8.0, 0.66M K acetate, 0. 1M Mg acetate, 5 mM dithiothreitol, and 1 mg/mL BSA. 35. 10X CIP buffer: 10 mMZnC&, 10 mMMgC12, and 100 mMTris-HCl, pH 8.0 36. Li acetate/TE buffer for yeast transformation: 10 mM Tris-HCl, pH 7 5, 1 mM EDTA, and 100 mM Li acetate; autoclave. 37. Polyethylene glycol (PEG3350 from Sigma) at 40% (w/v) in Li acetate/TE buffer; filter-sterilize. 38. Yeast lysis buffer for immunological screening of the library (see Section 3.3.): 0.2M NaOH, 0.1% sodium dodecyl sulfate (SDS), and 0.5% of 14.2M 8mercaptoethanol solution. 39. Lysis solution for yeast DNA preparation (see Section 3.4.): 2% Triton X-100, 1% SDS, O.lMNaCl, 10 mMTris-HCl, pH 8.0, and 1 mMEDTA. 40. 10X phosphate-buffered saline (PBS): 1.SMNaCl, 100 mMNa phosphate; adjust the pH to 7.3. 41. Blocking solution: 5% nonfat dry milk in 1X PBS. 42. 5M potassium acetate. 28. IMTris-HCI,

2.4. Media 43. SOC medium: 20 g/L bacto-tryptone, 5 g/L yeast extract, 0.5 g/L NaCl; autoclave. Prior to use, add (per liter) 10 mL of 1MMgC12, 10 mL of lMMgSO,, and 10 mL of 2M glucose; filter-sterilized solutions. 44. LB-ampicillin medium: 16 giL bacto-tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 15 g/L of bacto-agar (if needed for plates); autoclave. When agar has cooled to 55V, add 0.5 mL/L of a 100 mg/mL filter-sterilized ampicillin stock solution (final concentration of 50 pg/mL of ampicillin). 45. YPDA medium: 20 g/L bacto-peptone, 10 g/L bacto-yeast extract, 20 g/L glucose, 40 mg/L adenine sulfate, and 15-20 g/L of bacto-agar (if needed for plates); adjust the pH to 6.8-7.0, and autoclave for 10 min at 121°C (see Note 4). 46. Synthetic dextrose minimal medium (SD) supplemented for URA3 plasmid selection in the W303-1B yeast strain (see Note 4): 6.7 g/L bacto-yeast nitrogen base without amino acids, 20 g/L glucose, 5 g/L casa-amino acids, 20 mg/mL final of each supplement (L-His, L-Leu, L-Tip, and adenine sulfate), and 15-20 g/L of bacto-agar (if needed for plates); adjust the pH to 6.8-7.0 and autoclave for 10 min at 121V.

Epitope Libraries in Yeast

249

3. Methods

3.1. Construction of the Epitope Library 3.1.1. Preparation of the Inserts 3.1 .1-l. DNASE DIGESTION (SEE NOTE 5 AND REF. 73) 1. Make up a stock solution of DNase I at a concentration of 1 mg/mL in 150 rrGl4 NaCl, 50% glycerol. Divide the enzyme solution into lo-pL aliquots, and store them at -2OOC. 2. Prepare freshly 1 mL of 10X DNase digestion buffer (see Note 3). 3. In three distinct microfuge tubes (to ensure complete digestion), prepare three separate reaction mixtures: Dilute 6 pg of the target cDNA into a final volume of 100 pL of 1X DNase digestion buffer (see Note 6). 4. Remove an aliquot of DNase I from storage at -2O”C, and dilute it 1: 1000 into 1X DNase digestion buffer. Add to each reaction mixture of 3 PL (3 ng) of diluted DNase I, mix gently, and incubate at 1YC for 15-30 min (previous digestions stopped at different time-points, e.g., every 5 min over a 30-min time period should have first been run to identify the optimal digestion time for the selected size of digested products wanted). 5. Stop the reaction by addition of 2.5 pL of 500 mMEDTA and 6 pL of 5MNaCl. 6. Pool the three digestions, and extract twice with an equal volume of PCI. Precipitate the DNA fragments with 2 vol of 100% ethanol (see Note 7), leave at -2O’C for 10 min, and recover the DNA by centrifugation at 12,000g for 15 min at 4°C. Wash with 70% ethanol, dry the pellet, and resuspend it in 30 pL of TE, 50 mMNaC1. 3.1 .1.2. SIZE SELECTION AND REPAIR OF DNA INSERTS 1. Purify the DNA fragments of the desired size, 50-150 bp, by electroelution through a 2% agarose gel (see Note 8). 2. Extract the eluted DNA with PCI, and precipitate it by addition of 0.1 vol of 3M Na acetate, pH 5.2, and 2 vol of 100% ethanol. Leave on ice for 10 min. Recover DNA by centrifugation at 12,000g for 15 min at 4°C. Wash with 70% ethanol, dry the pellet, and resuspend it in 30 pL of 1.5X T4 DNA ligase buffer. 3. Add 3 pL of 20 mMATP, 1.5 pL of dNTP stock solution, 7.5 pL HzO, and 0.6 U of T4 DNA ligase. 4. Incubate the reaction for 15 min at 4OC and then add 1 pL (5 U) of the Klenow fragment of E. coli DNA polymerase I. After incubation for 45 min at 4°C and 5 min at 15’C, stop the reaction by addition of 1.5 yL of 500 mM EDTA, 5. Extract with PCI, and precipitate DNA with l/l0 vol3MNa acetate, pH 5.2, and 100% ethanol. 6. Resuspend the DNA in 35 pL of HzO, and add 5 pL of 10X T4 DNA polymerase buffer, 1 pL of dNTP stock solution, and 3 PL (3 U) of T4 DNA polymerase. Incubate at 37°C for 10 min, and then stop the reaction with 1 pL of 500 mMEDTA.

250

BBnichou and lnchauspe

7. Extract the DNA inserts twice with PC& and precipitate DNA with Na acetate, pH 5.2, and 100% ethanol. Redissolve the pellet m 12 pL of TE, pH 7.5. 8. Check the amount and quality of the inserts by analyzing an aliquot (l-2 l.tL) on a 2% agarose gel.

The DNA inserts are now ready for blunt-end cloning m the pSE-X expression vector. 3.1.2. Preparation of the Vector The pSE-X plasmid must be prepared for blunt-end cloning of inserts at its unique Hind111 restriction site (see Fig. 2). 1. In a total volume of 20 l.tL, digest 5 pg of the pSE-X plasmid with HzndIII enzyme for l-2 h at 37’C. 2. When digestion is complete, add 1 pL of dNTP stock solution and 1 yL (5 U) of the Klenow fragment of E coli DNA polymerase I and incubate the reaction for 15 min at room temperature. 3. Extract the reaction mixture with PC1 and precipitate DNA with l/10 vol3MNa acetate, pH 5.2, and 100% ethanol. Recover the DNA by centrifugation for 15 mm at 4°C. After washing with 70% ethanol, resuspend the pellet in 20 pL of HzO. 4. Dephosphorylation of the vector: To the 20 I.IL of digested plasmid DNA, add 10 ltL of 10X CIP dephosphorylation buffer, 70 l.tL of HzO, and 5 U of CIP. Incubate at 37OC for 30-60 mm 5. Extract with PC1 and ethanol-precipitate DNA. Wash with 70% ethanol, dry the pellet, and resuspend it in 11 PL of HzO. 6. Check the recovery and integrity of the prepared vector by electrophoresis on agarose gel (1%).

3.1.3. Ligation of Inserts to Vector DNA Perform the ligation of the dephosphorylated pSE-X vector with the bluntended inserts as follows (see Note 9): 1. Transfer 100 ng of the dephosphorylated vector to a microfuge tube and mix with a lo-fold excess of inserts (10 ng of 50-150 bp sized inserts). 2. Add 1 PL of 10X T4 DNA ligase buffer, 0.5 ltL of 10 mMATP (and HZ0 to 9 pL if necessary), and 1 I,~L (5 U) of T4 DNA ligase. 3. Incubate the ligation overnight at 16’C, and then inactivate the T4 DNA ligase for 10 min at 70°C.

3.1.4. Transformation of E. coli with the Ligation Mixture For library transformation of E. coli, we used commercial Epicurian Coli SCS 1 supercompetent cells from Stratagene. The transformation is performed in duplicate with 5 pL each of the ligation mixture and 125 pL of competent bacteria as recommended by the manufacturer.

Epitope Libraries in Yeast

257

1. Transfer 125 uL of thawing competent cells in a 15-mL polypropylene tube. Add 2.2 pL of a fresh 1: 10 dilution of 14.2MP-mercaptoethanol giving a 25-mMfina1 concentration. Mix gently and leave on ice for 10 min. 2. Add to the bacteria 5 uL of the ligation mixture, mix gently, and leave on ice for 30 min. 3. Heat-shock for 45 s in a 42°C water bath, and then leave on ice for 2 min. 4. Add 0.9 mL of SOC medium and incubate at 37’C for 1 h with shaking. 5. After the l-h regeneration, spread two aliquots of 10 and 50 uL onto LB-ampi plates to estimate the transformation efficiency and the number of independent clones of the library (see Notes 10 and 11). 6. Transfer the other 960 uL of the transformation into 200 mL of LB-ampi medium and grow overnight at 37V with shaking. 7. After overnight growth, centrifuge the 200~mL culture and recover the library plasmid DNA using a classical procedure for plasmid maxiprep.

3.2. Spreading

in Yeast of the Epitope Library

For each screening

of the library, the yeast host strain W303-1B (Mata,

Zeu2, his3, uru3, trpl, ade2) is transformed

with 10 yg of library plasmid

DNA

and spread on selective media for URA3 plasmid selection. The procedure describedbelow givesanefficiency of aboutlo4 transformantsfor 1 pg of plasmid. 1. Inoculate 20 mL of YPDA complete medium with a single colony from a freshly grown YPDA plate of yeast strain W303-1B. Incubate overnight at 30°C with shaking (220 r-pm). 2. Dilute the 20-mL preculture into 100 mL of final YPDA medium, and incubate with shaking for 3-4 more hours. The culture should have an OD at 600 nm of about 0.6. 3. Pellet the cells by centrifugation at 25OOg for 5 mm at room temperature. 4. Discard the supernatant, and resuspend the cells in 50 mL of Li acetate/TE buffer. Centrifuge at 2500g for 5 min. 5. Discard the supernatant, and resuspend the cells in 2 mL of Li acetate/TE buffer. Incubate at 30°C for 1 h with shaking. 6. In a sterile micromge tube, mix 10 ug of library plasmid DNA together with 4 pL of a 10 pg/pL stock solution of salmon sperm carrier DNA. 7. Add 200 pL of yeast cells, mix well, and incubate for 10 min at room temperature. 8. Add 800 uL of a 40% PEG3350 solution. Mix gently and incubate at 30°C for 1 h. 9. Heat-shock for 25 min in a 42°C water bath. 10. Pellet the cells by centrimgation for 5 s only. Discard the supernatant and resuspend the cells in 1 mL of YPDA medium. Centrifuge again for 5 s, remove the supernatant, and resuspend the cells in 1 mL of YPDA. 11. Centrifuge for 5 s, remove the supematant, and resuspend the cells in 3 mL of SD selective medium. 12. Spread 0.2 mL of the cell suspension/l40mm diameter plate on SD selective medium for URA3 plasmid selection. Incubate plates, colony side down, at 30°C for 2-3 d. Spread all the transformatron mixture on 24 plates.

BtMchou and lnchauspb

252 3.3. Immunological

Screening

of the Library

1. Nitrocellulose transfer of yeast colonies: Afier 2-3 d of growth, lift the colonies by gently placing a 135~mm diameter nitrocellulose filter, numbered side down, on the surface of each plate in contact with colonies, until it is completely wet. Mark the filter in three asymetric locations by stabbing through it and into the agar with a sterile needle. 2. Yeast lysis: Place a piece of Whatman 3MM paper (140~mm diameter) in the top of a 140~mm Petri dish and soak it with 10 mL of yeast lysis buffer. Using forceps, remove the filter from the plate and place it, colonies side up, onto the soaked 3MM paper. Cover the filter with the bottom of the dish and incubate for 30 min at room temperature. 3, Store the plates at 4°C until the results of the immunological screening are available. 4 Remove the filter from the Whatman paper with forceps and eliminate the excess of lysed yeast colonies by washing the membrane in a stream of deionized water. 5. Transfer the nitrocellulose filter into a bath containing the blocking solution. Use a minimum of 15 mL of blocking solution for each 135~mm filter. Incubate with shaking for 2 h at room temperature or overnight at 4’C (see Note 12). 6. Remove the filter from the blocking solution and place it m a heat-sealed bag. Add 5 mL of the primary antibody diluted in the blocking solution and incubate overnight at 4°C (or at least 2 h at room temperature) with shaking (see Notes 13 and 14). 7. Discard the primary antibody (see Note 15) and wash the filters, with gently shaking, twice in 1X PBS for 5 min, once m 1X PBS-0.5% NP40 for 10 min, and three times in 1X PBS for 5 min. Use a minimum of 15 mL of washing solutions for each 135~mm filter. 8. In a heat-sealed bag, incubate each filter for 2 h at room temperature with 5 mL of horseradish peroxidase-labeled secondary antibody diluted in the blocking solution. 9. Discard the secondary antibody (see Note 15) and wash the filters three times in 1X PBS for 5 min, twice in 1X PBS-0.5% NP40 for 10 min, and four times in 1X PBS for 5 min. The filters are now ready for detection. 10. Preparation of the detection solution (see Note 16): Dissolve 20 mg of DAB and 0.6 g of Ni(NH&(SO&, 6Hz0 in 100 mL of 100 mMTris, pH 7.5. Just prior to developing the filters, add 20 pL of Hz02 to the DABMi solution. 11. Transfer the 100 mL developing solution into a 200~mm glass Petri dish, and place the washed filter in this solution. Incubate at room temperature, gently shaking until the background of negative colonies becomes slightly visible. Use the same 100 mL of detection solution for the development of the 24 filters. 12. To stop the reaction, transfer the filters into a bath of 0.5 g/L NaNs in HzO, and then rinse the filters several times with H20. The immunoreactive colonies appear slate black in color. 13. Identify the location of the potential positive colonies on the yeast plates stored at 4OC. Spread these colonies again on SD-selective media, and repeat the process of screening to confirm the immunoreactivity of the peptides expressed by these colonies. Use as a negative control the yeast colonies transformed with the pSE-X vector without insert.

Epitope libraries in Yeast 3.4. Rescue of the DNA Plasmids from lmmunoreactive

253

Clones

After positive colonies have been confirmed and isolated, the library plasmid should be recovered for DNA sequencing of the insert. Since preparation

of plasmid DNA from yeast is not useful for restriction and sequence analysis, the plasmid are introduced

into E. coli. A rapid procedure for yeast DNA prepa-

ration is given below. 1. From a fresh Petri dish of the positive yeast clone, transfer with a toothpick cells from four to five colonies to 100 pL of TE, pH 7.5, into a microfuge tube. 2. Add 0.2 mL of yeast lysis solution, 0.2 mL of PCI, and 0.3 g of glass beads. 3. Vortex at high speed for 2 min, and centrifuge at 12,000g for 5 min at room temperature. 4. Transfer the aqueous phase to a new microfuge tube, and precipitate the DNA by addition of 0.1 vol of 5M K acetate and 2 vol of 100% ethanol. Leave at room temperature for 2 min. 5. Recover the DNA by centrifugation at 12,000g for 5 min. Wash the pellet with 70% ethanol, dry under vacuum, and resuspend it in 25 pL of HZ0 6. Use 1-2 pL of yeast DNA to transform electrocompetent DH5o E coli strain (see Note 7). 7. Prepare plasmid DNA from the E. coli transformants by standard methods. 8. Determine the DNA sequence of the insert using the specific 16-mer oligonucleotide TGCCAGCATTGCTGCT priming within the MF-al sequence of the pSE-X vector. 9. Deduce the amino acid primary structure of the immunoreactive recombinant peptide expressed by yeast clone.

4. Notes 1. The a-factor-based expression vector can tolerate the production of large amounts of foretgn gene product that can be hyperglycosylated on the outer chains with an additional 30-50 kDa/chain (4). This might explain the high-mol-wt recombinant peptides sometimes seen that can have an altered antigenicity. 2. The pSE-X expression vector and yeast strain W303-1B are available from the authors. 3. MnClz is very unstable in solution and should be stored at -2O’C as small aliquots of 1M stock solution. 4. It is convenient to prepare sterile stock solutions for each yeast growth supplement: 2 g/L for adenine sulfate stock solution, and 10 g/L for L-Tip, L-His, and L-Leu stock solutions. These solutions are sterilized by filtration and can be stored for extensive periods at 4°C. The media should be prepared by adding the appropriate volumes of these stock solutions to the ingredients of YPDA or SD media. 5. Before performing the DNase digestion protocol of the target cDNA, it should be tested to determine the amount of enzyme to use and the time of digestion required to generate DNA fragment size ranging from 50 to 150 bp.

Nnichou

and lnchauspb

6. A cDNA fragment encoding the protein of interest or a plasmid carrying this cDNA should be used as target DNA to generate the inserts. 7. It is not necessary to add salts to precipitate DNA, since you have already added NaCl to stop the DNase reactions. 8. See other chapters in this volume and ref. 14 for electroelution procedure of DNA fragments, electrotransformation of E. coli, and DNA sequencing. 9. Set up a control ligation that contains the plasmid vector alone without addition of inserts. 10. From these plates, recover plasmids from 10 random colonies to estimate the ratio of plasmids containing inserts (recombinant plasmids) and the average size of these inserts. 11. Uniform representation of random short fragments of the library should be verified. One approach to do so is to transform a portion of the library in E. coli. Duplicate colony blots can then be mdlvidually hybridized to 32P-labeled synthetic oligonucleotides representing a well-spaced region of the sequence of interest. 12. At this stage, nitrocellulose filters can be stored at -2O’C for several months, dried, and sandwiched between 3MM paper. 13. Polyclonal sera or MAb (purified or ascites fluids) can be used at a final concentration ranging between 1 and 50 pg of immunoglobulins/mL. If the antibody concentration is unknown, try several antibody dilutions to determine the correct range that gives acceptable background, yet still allows detection of the denaturated antigen on Western blot. 14. To prevent crossimmunoreactivity with yeast components, all animal polyclonal sera should be preimmunoadsorbed over yeast lysates as follows. Disperse Baker’s yeast in culture supernatant from yeast cells harboring pSE-X (without insert) at a concentration of 1 g/mL, and boil the suspension for 20 mm. After cooling, adjust to pH 7.3 with NaOH, and add 0.05 vol of 10X PBS. Dilute the sera at the desired concentration in the yeast suspension, and incubate at 4OC overnight with gentle shaking. Centrifuge the mixture at 3000g for 10 min at 4’C, and use the supernatant directly for incubation with the nitrocellulose filters. 15. The diluted antibody solutions can be stored at 4°C for more than 1 mo in the blocking buffer (with 0.02% final of NaNs) and reused several times. 16. The sensitivity of detection is enhanced by adding nickel or cobalt ions to the DAB substrate solution; in the presence of these ions, the product is slate black in color (IS).

References 1. Modrow, S., Hahn, B. H., Shaw, G. M., Gallo, R. C., Wong-Staal, F., and Wolf, H. (1987) Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes m conserved and variable regions. J Viral. 61,570-578. 2. Westhof, E., Altschuh, D., Moras, D., Bloomer, A. C., Mondragon, A., Klug, A., and Van Regenmortel, M. H. V. (1984) Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature 311, 123-126.

Epitope Libraries in Yeast

255

3. Scott, J. K. and Smith, G. P. (1990) Searching for peptide hgands with an epitope library. Science 249,386390 4, Ballou, C. E. (1982) Yeast cell wall and cell surface, in Molecular Biology ofthe Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 335-360. 5. Miyajima, A., Bond, M. W., Otsu, K., Ken-Ichi, A., and Arai, N. (1985) Secretion of mature mouse interleukin-2 by Succharomyces cerevisiae. use of a general secretion vector containing promoter and leader sequences of the mating pheromone a-factor. Gene 37, 155-161. 6. Ernst, J. F., Mermod, J. J., Delamarter, R. J., Mattaliano, R. J., and Moonen, P. (1987) O-glycosylation and novel processing events during secretion of a-factor/ GM-CSF fusion in Saccharomyces cerevistae. Biotechnology 5,83 l-834. 7. Miyajima, A., Bond, W. M., Otsu, K., Arai, K.-I., and Arai, N. (1987) Secretion of mature interleukin-2 by Succharomyces cerevtsiae: use of a general secretion vector containing promoter and leader sequences of the mating pheromone a-factor. Gene 37,155-161. 8. Brake, A. J., Merryweather, J. P., Coit, D. G., Heberlein, U. A., Masiarz, F. R., Mullenbach, G. T., Urdea, M. S., Valenzuela, P., and Barr, P. J. (1984) a-Factor directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Nat1 Acad. Sci. USA. 81,4642-4646.

9. Madaule, P., Gairin, J. E., Benichou, S., and Rosier, J. (1991) A peptide library expressed in yeast reveals new major epitopes from human immunodeticiency virus type 1. FEMS Microbial. Immunol. 76,99-108. 10. Stanley, K. K. and Luzio, J. P. (1984) Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO .I. 3, 1429-1434. 11. Gairin, J. E., Madaule, P., Traincard, F., Barr&, E., and Rossier, J. (1991) Expression in yeast of a cDNA clone encoding a transmembrane glycoprotein gp4 1 fragment (aa. 591-642) bearing the major immunodominant domain of human immunodeficiency virus. FEMSMicrobiol. Immunol. 76, 109-120. 12. Mink, M. A., Benichou, S., Madaule, P., Tiollais, P., Prince, A. M., and Inchauspt, G. (1994) Characterization and mapping of a B-cell immunogenic domain in hepatitis C virus E2 glycoprotein using a yeast peptide library. Virology 200,246255. 13. Anderson, S. (1981) Shotgun DNA sequencing using cloned DNAseI-generated fragments. Nucleic Acids Res. 9,3015-3026. 14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laborutory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 15. DeBlas, A. L. and Cherwinski, A. M. (1983) Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. Anal. Biochem. 133,2 14-219.

23 Epitope Mapping Using Random Fragment Expression Libraries in h Phages Hartmut Porzig and Kenneth D. Phillpson 1. Introduction The efficient detection of antigenic epitopes in a protein by screening a recombinant h. phage expression sublibrary of the antigen cDNA was first described by Mehra et al, (I). Subsequently, the method has found multiple applications. We have used it to identify the epitopes for a panel of monoclonal antibodies (MAbs) raised against the canine myocardial Na+- Ca2+exchanger (2), which was first cloned by Nicoll et al. (3). Once the cDNA of a protein is cloned, it provides an efficient tool to create partial polypeptide sequences as targets that may be recognized by MAbs directed against an unknown portion of the intact protein. This allows precise localization of the antibody-binding site. The information can then be used to analyze the topology of the native protein, or even functional organization, if antibody-binding has functional effects. The main drawback is the fact that the strategy will only work with antibodies that interact with more or less continuous peptide sequences. Amino acid sequences forming a topographic binding site created by folding of the native protein, but localized far apart in the primary sequence, will not be recognized. Moreover, even antibodies that strongly react with the denatured antigen on Western blots may not bind with sufficient strength to the folded forms of the fragments produced as fusion proteins. In the case of the Nat-Ca2+ exchanger, only about one-quarter of the strongly immunoblot-positive MAbs showed high affinity to bacterially expressed polypeptides. Successin localizing an epitope will also depend on the characteristics of the protein sequence and/or on the mean fragment size in the sublibrary, and may be higher than in our study (1). The fact that this method works in spite of the complex structure of most epitopes in native proteins (4) From: Methods m Molecular Biology, vol 66 Epltope Mapplng Protocols Ed&d by G. E. Morris Humana Press Inc , Totowa, NJ

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Porzig and Philipson indicates that relatively few amino acid residues within a linear sequence may often determine to a large extent the overall antibody-binding affinity. Basically, the method includes the following steps: Digestion of the fulllength cDNA clone of the antigenic protein with DNase to yield random DNA fragments that are then inserted into the hZAP phage vector (5). The ensemble of inserts, each coding for a small piece of the protein, is referred to as a sublibrary. On induction with isopropylthio-P-D-galactoside (IPTG), the corresponding polypeptide fragments are expressed as fusion proteins with P-galactosidase in Escherichia coli and blotted to nitrocellulose filters. These filters are then probed with the various antibodies. Specific binding of MAbs is detected by peroxidase- or alkaline phosphatase-based indicator systems.From positive phage clones, the plasmid insert is excised and amplified. The plasmid inserts are end sequenced to determine their position relative to the full-length protein. Since an antibody usually reacts with several overlapping polypeptides, the minimal length of a linear epitope can be derived from the region of overlap. Figure 1 summarizes the result of our studies with the Na+-Ca2+exchanger protein. Epitopes could be localized for four MAbs. Three of the MAbs recognized the same domain in the hydrophilic part of this membrane protein. Not too surprisingly, a sequence of highly charged amino acid residues sits in the center of this irmnunodominant epitope. From the methodological point of view, three interesting conclusions can be drawn from these results: 1. A short linear sequenceof 15amino acid residueswithin a highly immunogenic region is sufficient to form a high-affinity MAb-binding site. 2. One antibody (R3Fl) recognized two different neighboring epitopes that may reflect foldmg of the polypeptide chain in this particular area of the native protein. 3. One antibody (C2C 12) required an unusually long sequence of 154 amino acid residues (corresponding to 462 bp in the expressed cDNA fragment) for high-

affinity binding. This observation suggeststhat identification of some epitopes may require large fragments of DNA (500 bp or higher).

2. Materials 2.1. Construction of the cDNA Sublibrary of the Antigenic Protein in ZAP 1. GeneCleanTM (Bio 10 1, La Jolla, CA) for isolatingDNA fragmentsfrom agarosegels. 2. DNase I (Sigma [St. Louis, MO] DN-EP) is stored at -20°C as a stock solutron of 1 mg/mL in O.OlN HCl.

3. 10X DNA digestion buffer: 500 mMTris-HCl, 10 mMMnC12, 1 mg/mL BSA, pH 7.5. 4. ChromaSpinTM100 columns(Clontech Laboratories, Palo Alto, CA). 5. T4 DNA polymerase(Stratagene[La Jolla, CA], 100U/mL). 6. 5X T4 DNA polymerasebuffer: 250 mM Tris-HCl, 50 mA4[NH&S04, 25 mA4 MgC12, 50 timercaptoethanol,

pH 8.0.

259

il Phages EXTRACELLULAR

H2N

a

e

k

g

1

h

i

bNi

700

600

4iO INTRACELLULAR

Fig. 1. A model of the Na+-Ca2+ exchanger with the location of MAb epitopes as determined by the technique described in this chapter. The model is a modified version of the one first proposed by Nicoll and Philipson (6). Hydrophilic loops connecting the putative transmembrane domains l-l 1 are labeled a-k. The numbers attached to the large intracellular loop f between transmembrane domains 5 and 6 indicate the approximate position of amino acid residues as given in ref. 6. The bars correspond to the relative location and extension of identified antibody epitopes. R3Fla and R3Flb designate the two parts of the split epitope for this MAb (reproduced with permission from ref. 2).

7. TimeSaverTM cDNA Synthesis kit (Pharmacia, Piscataway, NJ). 8. h~~PII/EcoRIIGlgapackTM Clonmg Kit (Stratagene).

2.2. Immunological

Screening of the Expression Sublibrary

9. Bacteria: The XL,l- blue strain of E. coli (Stratagene) is used in conjunction with the hZAP phage system (5). 10. Disposable sterile plastic labware: Polystyrene Petri dishes (not tissue-culturetreated) with diameters of 10 and 14 cm; conical centrifuge tubes (screw-cap) for 15- and 50-mL volumes (NUNC [Roskilde, Denmark], Corning [Corning, NY], Sterilin [Stone, UK], and many others); pipets (lo-, 5-, and l-mL sizes), pipet tips for Eppendorf or Gilson variable volume pipets. 11. Nitrocellulose filter disks with 90- and 132~mm diameters fitting into the above Petri dishes (Schleicher & Schuell, Keene, NH).

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12. IPTG stock solution: 1.1915 g IPTG (mol wt 238.3) made up to 10 mL with distilled or deionized water (dH,O) (final concentration 500 m/U). Store frozen at -20°C. 13. Maltose stock solution: 20 g maltose made up to 100 mL with dH,O. Sterilize by filtration through 0.2~pm membrane filters. Stable at room temperature. 14. MgCl, stock solution: 20.33 g MgClz x 6 Hz0 made up to 100 mL with dH,O. Sterilize by filtration. Stable at room temperature. 15. Ampicillin stock solution: 100 mg/mL m dH,O. Sterilize by filtration. Divide into 50-pL portions and store at -2OV. 16. NZYM medium: 10 g NZ amine (Type-A casein hydrolysate), 5 g NaCl, 1 g casamino acids, 5 g yeast extract, and 2 g MgSO, x 7 H,O, are dissolved in 900 mL dH,O, adjusted to pH 7.5 wtth NaOH, and made up to 1000 mL. NZYM agar (agarose): Weigh 3.75 g agar (or 1.75 g agarose) into 1-L glass bottle, and add 250 mL of the NZYM medium. Then autoclave. After autoclaving and coolmg down to 50-6O”C, swirl the bottles to distribute the agar evenly in the medium. Stable for several weeks at room temperature. 17. Luria-Bertani (LB) medium: 10 g bacto-tryptone, 5 g bacto-yeast extract, and 10 g NaCl are dissolved in 900 mL dl&O. Adjust pH to 7.4 with NaOH and make up to 1000 mL with dH*O. Autoclave. It is stable for several weeks at room temperature. 18. SM Medium (sterile, for storage and dilution of phages): 5.8 g NaCl, 2 g MgSO, x 7H,O,50 mL of 1M Tris-HCl buffer, pH 7.5, are made up to 1000 mL with dH,O. Autoclave. It is stable at room temperature. 19. MTBST (washing and blocking buffer) 10X concentrated: For 1 L, dissolve 81.8 g NaCl(l.4M), 41.8 g morpholinopropane sulfonic acid (Mops) (0.2A4), 16.6 g Tris-HCl (O.l4M), 20 mL Tween-20 (2%). The pH of this solutton will be -7.2. Dilute 1:10 prior to usage. It is stable at room temperature but discard if bacterial or algal growth is detected (turbidities). Prior to use as a blocking buffer, add 10 g/L nonfat dried milk. 20. Mops-Tris (MT) buffer: Dissolve 10.465 g Mops (final concentration 500 mM) and 3.634 g Tris-HCl (final concentration 300 mM) and make up to 100 mL with dH,O. pH will be -7.2. Stable at room temperature after sterile filtration. 2 1. NiC12 stock solution: Dissolve 4 g NiCl in dHzO, and make up to 50 mL. Final concentration is 80 mg/mL. Stable at room temperature. 22. Diaminobenzidine indicator solution to detect protein-bound antibodies (make fresh for each experiment): For 100 mL, weigh out 50 mg 3,3’-diaminobenztdine (DAB) tetrahydrochloride, add 2 mL NiClz stock solution, 20 mL MT buffer, and 78 mL dHzO. Immediately prior to use, add 30 pL of a 30% hydrogen peroxide (HzOz) solution. 23. Secondary antibody: Horseradish peroxidase (HRP)-conjugated goat antimouse IgG (whole molecule), e.g., Sigma A-4416. Stable for up to a year at 4°C.

2.3. Maferlals R8qUir8d for Amplifying and Sequencing DNA Fragmenfs Coding for Antibody-Blndlng Fusion Proteins 24. XL-1 blue cells, R408 helper phage (Stratagene). 25. TYE broth: For 1 L: 5 g NaCl, 5 g bacto-yeast extract, 10 g bacto-tryptone. Sterilize by autoclaving. Stable for several weeks at room temperature.

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26. TE: 10 mA4 Tris-HCl, 1 mMEDTA, pH 8.0. Stable at room temperature. 27. 2X YT broth: For 1 L: 10 g NaCl, 10 g bacto-yeast extract and 16 g bactotryptone. Sterilize by autoclaving. Stable for several weeks at room temperature 28. TBE buffer (5X concentrated): For 1 L dissolve 54 g Tris-HCl, 27.5 g boric acid, and 10 mMEGTA in 900 mL dH,O, adjust to pH 8.0, and make up to 1000 mL with dH,O. Stable at room temperature. 29. Agarose 0.8% for flat-bed gel electrophoresis: Dissolve 1.2 g agarose (DNAgrade) in 150 mL TBE, and autoclave. Stable for months at room temperature. 30. DNA mol-wt markers: Gibco-BRL (Gaithersburg, MD) Cat. #561 l-015. Use a 1: 10 dilution. Store at -20°C. 31. Primers: Universal primer and Ml3 (reverse) primer (USB/Amersham, Arlington Heights, IL). Store at -2O’C. 32. Restriction enzyme: EcoRI (20 U/uL). Store at -20°C. 33. Restriction buffer: Combine in an Eppendorf tube 60 yL dH,O, 10 uL 10X concentrated buffer (supplied together with the restriction enzyme), and 1 uL EcoRI enzyme. Make fresh for each experiment. 34. Sample buffer for electrophoretic separation of DNA probes: Combine 5 mL 50% glycerol, 20 uL of 0.5M EDTA stock solution, pH 8.0,40 mg bromphenol blue, and 40 mg of xylene cyanol, and make up to 10 mL with dHzO. Store at -20°C. 35. Stock solutions for alkaline denaturatlon of DNA: a. 2M NaOH, 2 mM EDTA. b. 2M ammonium acetate, pH 4.6. Both are stable at room temperature. 36. DNA sequencing: SequenaseTM DNA sequencing kit V.2.0 marketed by USB/ Amersham. Store at -2OOC. 37. [o,-35&‘+dATP (store at -8O”C, usable for at least 6 wk) 38. DNA sequencing gel apparatus (e.g., Gibco/BRL model S2). 39. Polyacrylamide gel materials. 40. Materials for autoradiography: Autoradiographic film: Kodak X-OMATTM AR or BioMaxTM MR. Exposure cassettes: soft vinyl-covered cassette with velcro closure from Sigma. 41. Equipment for photographic documentation of electrophoresis gels: Polaroid DS-34 direct screen camera equipped with a red filter (Wratten 2A and Wratten 22) and a flat face hood.

3. Methods

3.1. Preparatlon of the Expression Sublibrary in LZAP 3.1.1. Isolation of cDNA Insert The procedure assumes that the protein of interest has been cloned into a plasmid vector, such as pBluescript. 1. Excise the coding region of the cDNA (-30 ug) by digestion with appropriate restriction enzymes.

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2. Separate the piece of cDNA coding for the protein from other DNA fragments on an agarose gel. 3. Extract the DNA insert using GeneClean. Depending on the size of the insert and the efficiency of DNA recovery, the yield of cDNA will be about 5 pg. 4. Determine the purity and approximate concentration by agarose gel electrophoresis.

3.1.2. Digestion of cDNA with DNase The objective is to digest the cDNA to produce random fragments from about 100-600 nucleotides in length.

ranging

1. In a typical digestion, mix 20 PL cDNA insert (-6 pg), 3 I.& of 1OX DNA digestion buffer, and 7 pL of DNase I (freshly diluted lO,OOO-fold) at room temperature for 45 min. 2. Stop the reaction by the addition of EDTA (5 mM final concentration). 3, Check the efficacy of digestton by running an aliquot (1.5 yL) of the final sample on an agarose gel. A visible smear of DNA should be observed to span the appropriate size range. Conditions (DNase I dilution; time of digestion) may need to be adjusted to ensure proper extent of digestion. 4 To remove small DNA fragments (
3.1.3. Subcloning DNA Fragments into ;tZA P The ends of the cDNA fragments are first made blunt with T4 DNA polymerase. 1. Add to the DNA fragments (12 pL, -1 pg) 6 uL 5X DNA polymerase buffer, 1.5 uL 2 mM dNTP, 9.5 uL H20, and 1.4 uL T4 DNA polymerase. Allow the reaction to proceed for 1 h at 15’C. 2. Dilute to 100 pL with H,O and extract with chloroform/phenol. 3. Ligate EcoRIINotI adapters on the ends of the cDNA and insert m hZAP arms following the instructions in a Pharmacia Timesaver cDNA synthesis kit (180 yL final volume). 4. Package the h DNA (150 pL) using Gigapack Gold following the instructions of the manufacturer. 5. Determine the size of the library using standard techniques. The library should contain a few hundred thousand plaque forming units (PFU). 6. Determine the fraction of phage-containing inserts by excision of phagemids (see Section 3.3.). Isolate phagemids from individual colonies and analyze by agarose gel electrophoresis after EcoRI digestion (see Note 1). 7. Amplify the hZAP sublibrary following standard procedures and store at 4OC.

3.2. /mmunologica/

Screening

of the Sub/i&my

This part of the method includes five sequential

steps:

1. The preparation of bacterial cell stock expressing h phage receptors; 2. Infection of these cells with phage containing the sublibrary;

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3, Blotting of the expressed fusion proteins onto nitrocellulose 4. Screening the blots with MAbs; and 5. Recovery of positive phage clones.

filters;

3.2.1. Preparation of Bacterial Cell Stock E. coli from the purchasedXL-1 blue stock aregrown on NZYM agarplates. 1. Melt agar in a microwave oven. Pour approx 30 mL m a sterile 50-mL centrrfuge tube. 2. After cooling to -50°C (if you cannot keep it in your hand for 10 s, rt is still too hot!) add 30 l.tL ampicillin stock and 300 nL maltose (required to induce the gene that codes for the h phage receptor), mix gently by inversion, and pour into a 90-mm Petri dish. 3. After hardening (keep the hd partially open during this time), invert the plates and store at 4°C. 4. On the next day, remove bacteria from stock wrth a sterile platinum maculation loop and streak immediately on the agar plate (prewarmed at 37°C for 30 min). 5. Incubate the plate overnight at 37°C 6. Remove a well-Isolated single colony from the plate and inoculate into 4 mL of LB medium (supplemented with 40 lrL maltose stock and 40 pL MgCI, stock solutron) m a 15-mL conical centrrfuge tube. 7. Seal the agar plate with the remaining colonies with ParafilmTM around the lid and store at 4°C. Additional cultures can be removed from this “mother plate” for several weeks provided sterihty is maintained. 8. Incubate cells in the LB medium overnight at 37°C m a trlting shaker. 9. Sediment the cells (-4000g for 10 min) and discard the supernatant. Resuspend the cells in 7-8 mL of sterile 10 miWMgC12 solution. Read optical density (OD) in a photometer at h = 600 nm. 10. Dilute with sterile 10 mM MgC!l* solution to OD w 1, corresponding to a cell density of -2 x 108/mL. Cells can be stored at 4°C and used for -1 wk

3.2.2. Phage Infection of Bacteria and Preparation of Filter Blots with Expressed Fusion Proteins

For a library screen,useat leasttwo 140~mmNZYM agarplateswith about 5 x 1 O4 PFU/plate

for each antibody.

Eight plates (16 filters) for the srmulta-

neous screenof four MAbs can easily be handled in a single assay. 1. Pour 60 mL of hot NZYM agar (without additions) mto each of the 140~mm plates (use 25 mL for 90-mm plates), let cool down, and store overnight at 4OC. 2. On the next day, prepare as many sterile glass vials (10 mL) as there are plates. In each vial, mix 270 nL of the bacterial stock suspension of OD 1 with 1 ltL diluted phage stock from the library to produce 5 x 1O4PFU. Dilute phage as required in SM medium. 3. Incubate the cell suspension for 15 min at 37°C.

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4. During that time, melt NZYM agarose in the microwave oven and keep it in a 5O“C water bath. 5. Mix gently 8 mL of the warm agarose with the infected cell suspension in one vial and pour immediately onto one of the prewarmed agar plates (use 3 mL for a go-mm plate). The agarose hardens very rapidly. Therefore, handle only one vial and one agar plate at a time. Distribute the agarose evenly on top of the agar. 6. Transfer all plates into an incubator set to 42%. Incubate for 3.5-4 h. By that time, a lawn of bacteria will have grown, containing tiny lytic halos where a phage infection has occurred. 7. In the meantime, soak nitrocellulose filters in IPTG solution (10 mA4, prepared from the 500 mM stock by dilution in dH,O; no sterility required). Handle the filters with gloves. Prepare twice as many filters as you have plates. After a few minutes, remove the filters from the solution and air-dry them on a clean surface. IPTG 1srequired to induce the expresslon of the recombinant polypeptldes, which are fused to the bacterial /3-galactosidase protein. Twenty milliliters of IPTG solution will be sufficient to soak five filters simultaneously. 8. Label the dry filters with a soft lead pencil on the side that will face the bacteria 9. When the plates are ready, place filters quickly but carefully on top of the cultures. Avoid air bubbles by bending the filter such that first contact is made in a line across the middle of the plate, and then allow the filter to be drawn onto the plate by capillary force. Do not move the filter once it has made contact with the culture surface. 10. Mark the position of the filter on the culture by piercing it (and the underlymg agar) at several places with a thick needle to ensure that positive plaques can be unequivocally localized. 11. Incubate for -3 h at 42’C. 12. Lift the first set of filters from the cultures and replace by a fresh set of IPTG-soaked filters. Make sure that the labeling of the position of the second filter matches that of the first (see Note 2). Transfer used filters into new Petri dishes containing blocking buffer (MTBST supplemented with milk powder) and store at 4% 13. Continue the incubation for another 3 h. 14. Remove the second set of filters and transfer into blocking buffer as above. Seal the phage-infected cultures with Paratilm and store at 4°C.

3.2.3. Screening of the Protein Transblots with MAbs 1. Prepare a working dilution of the appropriate antibodies in MTBST supplemented with 0.02% NaN3. Use the same antibody concentration that gives a maximum response in a conventional immunoblot (see Note 3). 2. Wash the filters (4X) in MTBST and incubate pairwise for 1 h on a shaker at room temperature with the specific antibodies. Recover the antibody solution for repetitive use (three to five times). 3. Prepare working dilution of the second, HRP-conjugated antibody m MTBST (dilute as recommended by the supplier, usually 1: 1000-l :5000). 4. Wash filters 4X in MTBST and incubate for 1 h with the second antibody.

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5. Wash filters 4X in MT. Add the DAB indicator solution (seeNote 4). The color develops within -20 min. Longer incubation periods do not enhance the signal, but will increase the background. The indicator solution is rinsed away with dH,O (use gloves when working with DAB; the compound is carcinogenic). Positive clones will appear in both filters as small, dark, doughnut-shaped rings, slightly larger on the second filter than on the first one.

3.2.4. Recovery of Positive Phage Clones The position of positive clones is identified on the original agar plate using the immunolabeled filter as a template. 1. Search the inverted plate on a light box and label the position of the clone on the bottom of the plate. 2. Punch out the area of the clone with a sterile inverted Pasteur pipet or with a disposable polyethylene transfer pipet. Prewet the pipet with SM medium to avoid sticking of the agar plug to the inner surface of the pipet. If more than one clone on a plate has been labeled by a given antibody, takeeachof them individually. 3. Transfer each plug into 1 mL SM medium containing 40 pL chloroform, vortex, and elute overnight at 4’C. Typically, the yield per plug will be about lo7 PFU/mL. 4. Eliminate contaminating negative clones by a secondary screening using much lower PFU densities. Prepare 1: 10, 1: 100, and 1: 1000 dilution of the positive clones picked in the first screen. 5. Mix 1 PL phage dilution with 90 pL of ODl XL-1 blue cells, plate on 90-mm agar plates, and prepare immunolabeled filters as described above. 6. Pick well-isolated uncontaminated positive clones. After elution in 1 mL SM/ chloroform solution, about lo6 PFU are obtained from a single plaque. This phage stock is stable for about 1 yr at 4°C.

3.3. Excision of Plasmid DNA For the in vivo excision of the pBluescript plasmid from the hZAP II vector, follow the protocol suppliedby Stratagene.However, we reducedthe reaction volumes to one-tenth of the suggestedvalues such that everything could be done in sterile I-mL Eppendorf tubes, rather than using 50-mL conical tubes.

After working successfully through this protocol, bacterial colonies should be available growing on LB/ampicillin agar, which contain the pBluescript phagemid, including the cloned DNA insert. 3.4. Plasmid Amplification

and DNA Preparation

1, Transfer at least two colonies from each plate into conical lo-mL tubes containing 4 mL TYE broth supplemented with 100 pg/mL ampicillin. 2. Incubate overnight at 37’C in a shaking device. 3. Use the cultures for a plasmid miniprep according to the Promega WizardTM Miniprep protocol. The overall yield from a 4-mL culture is about 15-20 pg DNA

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suspendedin 50 yL TE buffer. Five microliters of thts DNA preparation are diluted to 500 yL with dHZOandusedfor the photometric determination of DNA purity and concentration. 3.5. Digestion and Size Fractionation of Plasmid DNA 1. To checkfor the sizeof the insertwithin the plasmid DNA, digest a sampleof the above DNA preparatton with EcoRI Add 3 pL DNA stock from each sample to 7 pL restrictton buffer and mcubatefor 1h at 37°C. 2. Prepare the agarose minigel from 25 mL hot 0.8% agarose in TBE buffer. Prior to gel casting,add2.5 pL ethidium bromide solution (10 mg/mL), andmix carefully. 3 Mix each of the digested samples with 2.5 pL of sample buffer For size stan-

dards, 9 pL of the 1:10 diluted sizemarker solution are combmed with 1 pL of the 10X concentrated enzyme buffer and 2.5 pL of sample buffer. 4. Centrtfuge and then heat the samples briefly to 90°C for 5 mm. 5. Apply 2 pL/lane of each sample to the gel. Set voltage to -60 V and contmue the separation until the dye front has migrated about one-third the length of the gel. 6. Visualize the separation on a UV transilluminator and document it with a Polaroid Direct Screen camera (see Note 5).

3.6. DNA Sequencing After alkalme denaturation of double-stranded plasmid DNA accordmg to standard procedures, the sequencing procedure follows the protocol given in the Sequenase 2.0 instructions (see Note 6). 3.7. Analysis of Sequence Data and Identification of Epitope Location The sequence for both ends of the isolated, antibody-binding inserts are read for 30-70 bases starting from the primer sequence. Using the program “bestfit” m the GCG software package (7), these sequencesare compared to the known sequence of the cDNA coding for the antigenic protein (retrtevable from GenBankTM). Since sequences from both ends of the insert are available, its position with respect to the full-length clone can be precisely determined (see Note 7). Partially overlapping sequences from different inserts reacting with the same antibody can then be used to identify the minimal sequence common to all antibody-reactive fragments within the sublibrary. This core sequence IS assumed to code for the epitope of this particular antibody. A typical result m epitope localization is shown in Fig. 1. 4. Notes 1, Since all inserts in the sublibrary are derived from a single piece of cDNA, the likelihood of finding an epitope in an expressed fusion protein is relatively high when large numbers of phage are screened. Therefore, it IS acceptable if only a fraction of phage in the sublibrary contains inserts. This fraction, however, should be over 20%.

A Phages 2. Screening two filters from the same plate facilitates the discrimination between truly positive clones (positive on both filters) and false positives (usually present on only one of the filters). 3. Only those monoclonals should be used for sublibrary screening that give strong signals with the antigen in immunoblots. Although, in theory, an antibody that binds weakly to a blotted protein band might show high-affinity binding to a bacterially expressed fusion protein, we never observed such a case. On the other hand, even with MAbs reactmg strongly in the tmmunoblot it may be impossible to Identify an epitope with this technique. 4. Popular detection systems in addition to the DAB method, which is described here, include alkaline phosphatase and chemiluminescence-based methods (e.g., the ECL-system marketed by Amersham). We have used all three techmques, but prefer the DAB method because of low background levels and the low frequency of false positives. It is true that DAB is somewhat less sensitive than the other two indicators. However, the higher sensitivity is paid for by a considerable amount of additional work required to eliminate false posmves. Moreover, the ECL system is also much more expensive. 5. It is a distinct advantage if different clones reactmg with the same antibody yield variable Insert sizes. Such a tindmg suggests that the eprtope can be fairly accurately determined using the information from overlapping sequences Sometimes the insert appears m two bands rather than one. Thts may occur if an EcoRl site is within the epitope of your antibody. 6. The followmg changes to the Sequenase 2.0 protocol are recommended: a Use two primers (universal and reverse) m parallel assays for each DNA sample to obtain sequences from both ends of the insert. b. The incubation should be extended to at least 10 min at room temperature. Approximate timing is sufficient. After adding stop solution, the samples can be stored at -20°C until the sequencing gel is ready for loading. 7. Even though the final analysis is a rather straightforward procedure, in our experience, there was one major problem that repeatedly prohibited the successful localization of an antibody-reactive insert: In several cases, the sequence obtained with one of the two primers, as expected, could be localized within the primary sequence of the protein, whereas there were problems with the sequence obtained from the second primer. It either showed no homology at all with the cloned cDNA, or else it showed homology with a fraction of the DNA that corresponded to a locus so far apart on the primary sequence that it could not be part of the same sublibrary fragment. This phenomenon can be explained by tandem formation durmg sublibrary construction. In our case, such tandem inserts did form between fragments originating from disparate parts of the primary sequence or between fragments of the protein cDNA and fragments of the pBluescript vector. Since the frequency of tandem formation within a sublibrary is unknown, it is absolutely essential to isolate as many positive clones as possible to increase the probability of finding a sufficient number of useful ones.

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Acknowledgment The work of the authors was supported by Swiss National Science Foundation Grant 31-9473.88, by National Heart, Lung, and Blood Institute Grant HL-2782 1, by the Laubisch Foundation, and by the American Heart Association, Greater Los Angeles Affiliate.

References 1. Mehra, V., Sweetser, D., and Young, R. A. (1986) Efficient mapping of protein anttgenic determinants. Proc. Natl. Acad. Sci. USA 83,7013-7017. 2. Porzig, H., Li, Z., Nicoll, D. A., and Philipson, K. D. (1993) Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies. Am. J. Physiol. (Cell Physiol.) 265, C748-C756. 3. Nicoll, D. A., Longoni, S., and Philipson, K. D. (1990) Molecular cloning and tinctional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger. Science 250,562-565. 4.

Laver, W. G., Air, G. M., Webster,R. G., and Smith-Gill, S.J. (1990) Epitopeson

protein antigens: misconceptions and realities. Cell 61,553-556. 5. Short, J. M., Femandez, J. M., Serge,J. A., andHuse,W. D. (1988) Lambda ZAP: a bacteriophage lambda expression vector with m vivo excision properties. Nucleic Acids Res. I&7583-7600. 6. Nicoll, D. A. and Philipson, K. D. (1991) Molecular studies of the cardiac sarcolemmal sodium-calcium exchanger. Ann. NY Acad. Scz. 639, 18 l-l 88. 7. Devereux, J., Haeberli, P., and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12,387-395.

24 Random Fragment Libraries Displayed on Fllamentous Phage Lin-Fa Wang and Meng Yu 1, Introduction Phage-display peptide libraries (1-4) have become powerful tools for identification and characterization of peptide mimicries that bind to specific selector molecules, such as antibodies (‘5-7). The technology depends on random peptide sequences, displayed on the surface of filamentous bacteriophages, being allowed to interact with antibodies or other ligates. Ligates are usually immobilized on a solid support, such as a Petri dish, microplate, or microbeads, and binding phages are specifically enriched by several cycles of affinity selection (1,8,9). The displayed peptide responsible for binding to the antibody can be identified by directly sequencing the encoding insert in the genome of the recombinant phage. This random epitope library strategy has the potential advantage of being able to identify critical residues within an epitope (10) and of providing mimotopes (II), which can mimic discontinuous epitope structures (12,13). (See Chapters 17 and 18 of this volume for more details.) In this chapter, we will describe a slightly different random expression strategy for epitope mapping using phage-display technology. Rather than expressing totally random synthetic peptide sequences, our approach relies on construction of a random fragment expression library using small DNA fragments generated by partial digestion of target gene fragment(s) using DNase I. Although such a gene-targeted fragment library does not have the diversity exhibited by random synthetic peptide libraries, its limited complexity and presentation of the authentic antigen sequence, rather than a mimotope, makes it an effective method for mapping epitopes (14). Depending on the size of DNase I fragments selected, it is possible to construct recombinant phages disFrom: Methods in Molecular Biology, vol 66’ Epltope Mapplng Protocols Edited by. G. E. Morris Humana Press Inc., Totowa, NJ

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playing relatively large peptide fragments, which may be useful in other applications, such as development of diagnostic reagents and phage-based recombinant vaccines. Although there have been reports that describe the mapping of epitopes using polyclonal antibodies and phage-display peptide libraries, it is a technically difficult task owing to a high level of nonspecific binding. In contrast, we find that gene-targeted fragment libraries are able to provide much more conclusive results when polyclonal antibodies are used. One example is given in Fig. 1. The target antigen under investigation was the 646-aa major capsid protein p72 of African swine fever vnus (ASFV) (Z-5). From this particular mapping experiment using DNase I fragments in the range of 150-300 bp (coding for peptides of 50-l 00 amino acid residues), there are several interesting observations: 1. It seemsthat the N-terminal region of p72 is more immunogenicthan the remaining part of the molecule It will be interestingto determine if this is related to the topographical location of the p72 protein in assembledvirions. 2. Pig polyclonal antibodiesreacted with the epitope fragment displayed in phage clone A-9 in ELBA test,but not in Westernblotting, indicating that this epltope is conformation-dependent. 3. The antigenic region defined by phageclonesA- 10and A- 11 also overlaps with a 7-aa epitope defined by a mouse monoclonal antibody (MAb) raised against p72 (see Fig. 1C). This indicates that the region is immunogenic

not only m the

target animals (pigs), but also in mice. 4. Clones A- 11 and A- 12 actually contained a hybrid peptide msert that was derived from two different parts of the p72 protein, randomly joined together in-frame before the recombmed fragment was inserted, m-frame again, into the vector (see Fig. 1B for more details). At present, it is not clear whether both of the original peptide fragments from the hybrid insert are antigenic, but it is conceivable that,

if the library sizeis large enough,it is possibleto isolatehybrid epitopesthat may contain different determinantsof a discontinuousepltope If high-resolution mapping of antibody-binding sites is desired, one may use smaller DNase I fragments for library construction. Figure 1C illustrates one of such mapping experiments that we have carried out to map an MAbdefined epitope for ASFV ~72. In thuscase, smaller DNase I fragments in the range of 50-l 00 bp were used for library construction. Affinity selection using MAb 6F4, an MAb known to react with p72 in Western blotting, led to the isolation of four different classesof positive phage clones containing overlapping peptides as shown in the figure. From this, a 7-aa antibody-binding site was determined. This same antigenic region was also detected by pig antisera as described in phage clones A- 10 and A- 11. It should be emphasized that, although the given examples were based on relatively simple gene-targeted random fragment libraries, the same prmciple

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can be applied to much more complex genome-targeted random epitope libraries, since the phage-display systemhas a capacity to generate a library of 107-1 O9 independent clones (5,9,13). This has recently been demonstrated by Jacobsson and Frykberg (16) in isolation of IgG- and fibronectin-binding domains using a genome-targeted phage-display library constructed from the total genomic DNA of Staphylococcus aureus. 2. Materials All reagents should be of AR grade. All solutions and buffers should be autoclaved or filter-sterilized where appropriate. Sterile tubes and tips should be used. Unless otherwise stated, all molecular biology reagents are obtained from Promega (Madison, WI). 2.1. General 1 Antibodies: MAb(s) or polyclonal anttbodies of interest, rabbit ant+Ml3 antibodies, and horseradish peroxidase (HRP)-conjugated secondary antibodies. 2. Bacterial strains and plasmids. Escherzchta colz MC1061 and K91Kan, phage expressionvector fUSE 1, all obtained from Smith (I, 9). 3. Equipment from Bio-Rad (Hercules, CA): power supply, horizontal agarose gel electrophoresis tank, Bio-Dot Microfiltration unit, sequencing gel apparatus, gel dryer, and Gene Pulser electroporator. 4. Darkroom facthties for Polaroid photography and for X-ray film developing. 5. Rocker and orbital shaker.

2.2. Library

Construction

6. Recombinant gene: usually a pUC clone containing the gene coding for the protem recognized by the antibodies of interest. 7. Enzymes: RNase-free DNase I, T4 DNA polymerase, restriction enzyme PvuII, calf intestinal alkaline phosphatase (CIP), T4 DNA hgase. 8. DNase I buffer 50 mM Tris-HCl, pH 7.6, 10 mM MnCl*, kept frozen at -2O’C. Stable for at least 1 yr. 9. EDTA: 0.5Mat pH 8.0 10. TE buffer: 10 mM Tris-HCl, pH 7.6, 1 nit4 EDTA. 11. Phenol:chloroform: 1: 1 mixture of TE-saturated phenol and chloroform. 12. Sodium acetate:3M at pH 5.2. 13. Ethanol: 100 and 70%, kept at -2O“C. 14. 50X TAE buffer (per liter): 242 g Tris base, 57 mL glacial acetic acid, 100 mL 0.5MEDTA, pH 8.0. Make 1X solution every month. 15. End repairing buffer: 40 mMTris-HCl, pH 8.5,lO mM(NH4)2S04, 5 mMMgC12, 5 rnA4 DTT, 0.5 mM EDTA, 150 ug/mL BSA, and 100 uil4 dNTPs. Each of the components were made separately as a 10X stock and kept at -20°C. Before use, make the 1X solution by diluting in water, and use on the same day

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A

400

200

600

I

p72 (646 aa)

4

A-IO A-l

I A-lla

B

A-llb

C

684-l

241 270 I I LLCNVQDMH.KPHQSKPILTDENDTQZWC!TIi (3)

634-2(2) 634-3

(3)

684-4

(4)

DMHKPHQSKPILTDEND ILTDENDT SKPILTDENDT PILTDENDTQR

Fig. 1. Summary of epitope mapping results for the major capsid protein p72 of ASFV. (A) The numbersgiven on top of the figure representthe amino acid residue numbersof ~72. The bars underneaththe p72 protein (shown as an elongatedarrow) indicate epitope fragments selectedusing several different ASFV-infected pig sera. The slanted region of clone A-9 indicates it contained a conformational epitope that reactedwith the pig antibodies in ELISA, but not in Westernblotting. Clones A-l 1

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16. GeneClean DNA Purification Kit from BIO- 101 (San Diego, CA). 17. Gene Pulser cuvet (0.2 cm) from Bio-Rad. 18. Antibiotics stock solution: tetracycline (Tet) at 20 mg/mL in absolute ethanol and kanamycin (Kan) at 50 mg/mL in water are kept at -20°C. Unless otherwise stated, the working concentration of antibiotics is at a 1: 1000 dilution, i.e ,20 &mL for Tet and 50 pg/mL for Kan (e.g., LB/Tet/Kan medium represents LB containing Tet at 20 pg/mL and Kan at 50 pg/mL). 19. LB medium: 1% bacto-tryptone, 0.5% yeast extract, 1% NaCl. 20. Terrific broth (TB) medium: dissolve 12 g bacto-tryptone, 24 g yeast extract, and 4 mL (5.04 g) glycerol in 900 mL water. Autoclave 90-mL portions m 125-mL bottles. When cooled, add 10 mL of separately autoclaved potassium phosphate solutions (O.l7MKH,PO,, 0,72MK,HPO,) to each bottle. 2 1, PEG solution: Dissolve 100 g PEG 8000 and 116.9 g NaCl in 475 mL water (may need heating to 65’(Z), autoclave to sterilize, and keep at 4’C. 22. TBS buffer: Dilute 10X TBS (per liter: 90 g NaCl, 60 g Tris base, adjust pH to 7.9 with HCl) to 1X with distilled water. Store at room temperature for up to 6 mo. 23. NaN, solution: make a 20% (w/v) stock solution and keep at 4’C. Use at 1:lOOO dilution to a final concentration of 0.02%. Caution: this is a toxic chemical, Handle with gloves, and label the tube with an appropriate warning sign. 24. Dimethyl sulfoxide (DMSO) from Sigma (St. Louis, MO). 25. TBS/gelatin: Dissolve 0.1 g gelatin m 100 mL TBS by autoclaving, and store at room temperature for up to 6 mo.

2.3. Library Screening 26. TBST buffer: TBS containing 0.5% (v/v) Tween-20. 27. Blocker solution: TBST containing 5% (w/v) skim milk powder and 1% of Ml 3 phage solution (approx 1013 phage particles/ml). Make fresh before use. 28. Streptavidin magnetic beads (SMB): 1 mg/mL suspension, Promega 29. Magnetic separation stand (two-hole), Promega.

and A- 12 are composed of two peptides as further detailed below. (B) Schematic representation of the two recombined epitope fragments obtained for clones A-l 1 and A-12. The arrows indicate the direction of gene expression, In each case, the signal peptide region (at the N-terminus of gene III) is first fused to peptide-a, then to peptide-b, and finally to the coding region for the mature pII1 protein. Clone A- 11 contained two peptides from different regions of p72, whereas clone A- 12 contained a tandem repeat structure with peptide 12a being much smaller than 12b. (C) Sequence alignment for four different epitope fragments isolated by affinity selection using MAb 64F. The numbers given in parentheses on the left indicate the total number of independent clones isolated within each class of insert. Shown on top is the amino acid sequence from the region (aa 241-270) covering the 7-aa consensus sequence (underlined) shared by all of the clones.

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30. Biotinylated sheep antimouse antibodies, Amersham (Buckinghamshire, UK) 3 1. Elution buffer: O.lNHCl (pH adjusted to 2.2 with glycine), 1 mg/mL BSA. Store at 4°C up to 6 mo. (Optional: Add 0.1 mg/mL phenol red to momtor the pH of the solution.) 32. 1M Tris-HCl, pH 9.5. 33. Nitrocellulose membrane: 0.45 pm from Schleicher & Schuell (Dassel, Germany). 34. Blotto solution: TBST containing 5% (w/v) skim milk powder. Make fresh before use. 35. Plastic bag and heat sealer. 36. Container with flat bottom (e.g., square Petri dishes). 37. Chemiluminescent Substrate (POD) Kit from Boehringer (Mannheim, Germany) 38. X-ray film. for example, Kodak X-Omat AR5 39. Autoradiography cassettes. 40. Light box.

2.4. Characterization of Epitope-Displaying

Phage Clones

4 1. Multichannel pipet, ELISA plates, and microplate shaker: all from Titertek Flow Laboratories (McLean, VA). 42. Microplate reader: Multiskan MS, from Labsystems (Helsinki, Finland) 43. Coating buffer: 50 mMTris-HCl, 150 mMNaC1, pH 9.0. 44. PBST. Dilute 10X PBS (10.7 g/L Na,HPO,, 3 9 g/L NaHzP04, 80 g/L NaCl, pH 7.2) to 1X with distilled water, and add Tween-20 to a final concentration of 0.05% (v/v). Store at room temperature for up to 6 mo. 45. Blocking solution: PBST containing 1% skim milk powder; make fresh before use. 46. Citrate acetate buffer: Make 100 mL of 1M sodium acetate and 10 mL of 1M citric acid. Adjust the sodium acetate solution to pH 5.9 with approx 1.5 mL of the citric acid. 47. TMB substrate: Dissolve 100 mg of 3,3,5,5,-tetramethylbenzidine (Sigma) in 10 mL DMSO to make a 42-mA4 solution. Store at 4°C in small ahquots (0.5 mL) for up to 12 mo. Prewarm at 37°C for 10 min before use. 48. Substrate solution: Make fresh by mixing 18 mL of distilled water with 2 mL of citrate acetate buffer and 0.2 mL of the TMB substrate. Add 2.5 pL 30% H,02 just before use. 49. Stopping solution: 1M H,SO+ 50. Tuq polymerase and PCR reagents, use as recommended by supplier. 5 1. Oligo primers: III-5 (5’-GGT TGG TGC CTT CGT AGT-3’), III-3 (5’CCA TGT ACC GTA ACA CTG-3’), and 35S (S-CCC TCA TAG TTA GCG TAA CG-3’). 52. PCR machine: Hybaid OmniGene or any similar thermal cycler capable of carry-

ing out microplate PCR. 53. PCR Sequencing Kit from USB (Cleveland, OH).

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3. Methods

3.1. Library Construction 3.1.1. Generation of Random Fragments by DNase I Partial Digestion The procedures given below can be used with plasmid DNA containing the target gene insert, PCR-amplified gene fragment, or chromosomal DNA. If more DNA is required, we recommend setting up multiple tubes rather than increasing the volume of each reaction. See Note 1. 1. Resuspend DNA sample in water at a concentration of 200 pg/rnL, divide into four 50-pL aliquots, and keep on ice. 2. Dilute DNase I in ice-cold DNase I buffer at final concentrations of 4, 2, 1, and 0.5 U/n& respectively. 3. Start the DNase I reaction by transferring 17.5 pL of diluted DNase I solution from each of the above four concentrations into a tube containing 50 pL of the DNA sample prepared in step 1. 4. Incubate at 15°C for 10 min. 5. Stop the digestion by adding in 2.5 pL of O.SMEDTA solution, and keep the tubes on ice. 6. Take 2 pL of digested mtxture from each of the four tubes, and analyze the digestion patterns on a 2% agarose-TAE gel. 7. Combine the two tubes that give optimal digestion patterns (i.e., most DNA fragments are distributed in the range of 50-300 bp), and bring the total volume to 500 yL with TE buffer. Extract once with an equal volume of phenol:chloroform, followed by precipitation with 2 vol of absolute ethanol in the presence of 0.3M sodium acetate. Leave the tube at -20°C for 30-60 mm. 8. Pellet the DNA by centrifugation in an Eppendorf centrifuge at 4°C for 10 min, wash the pellet twice with 70% cold ethanol and dry under vacuum for 15 min. 9. Resuspend the pellet in 50 pL of end-repairing buffer, add 10 U of T4 DNA polymerase, and incubate at 15°C for 60 min. 10. Separate DNA fragments by electrophoresis in a 2% agarose-TAE preparative gel, and cut out the gel slice containing DNA fragments in the range of 100-300 bp. 11. Purify DNA fragments from the gel slice using GeneClean Kit following supplied instructions. Elute the DNA in a final volume of 20 pL water (see Note 2).

3.1.2. Vector Preparation 1. Digest 2 ug of fUSE1 vector DNA with 10 U of PvuII enzyme in a total volume of 20 pL. Incubate at 37°C for 60 min. 2. Dilute the digestion mixture by adding 24 pL of water and 5 pL of 10X CIP buffer. Add 1 pL of CIP enzyme (1 UIyL) and incubate at 37°C for 30 min. Add another 1 pL of CIP enzyme, followed by 30-min incubation. 3. Bring the total volume to 100 pL by adding 50 pL water, and extract this diluted mixture with an equal volume of phenol:chloroform, followed by ethanol precipitation in the presence of 0.3M sodium acetate. 4. Resuspend the DNA m 5 pL water.

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3.1.3. Ligation 1. Mix 5 pL vector DNA with 20 pL end-repaired DNase I fragments, followed by addition of 3 pL 1OX ligase buffer containing ATP. 2. Start the ligation reaction by adding 1 PL of T4 DNA ligase (3 U/pL), followed by incubation at 14OC for 2-4 h. 3. Add an additional 1 pL ligase, followed by overnight incubation at 14’C. 4. Inactivate ligase by heating at 65°C for 15 min. 5. Bring the total volume to 100 pL with water, followed by phenol:chloroform extraction and ethanol precipitation. 6. Resuspend the DNA in 5 pL and keep on ice until use.

3. I. 4. Electropora tion 1. Prepare electro competent cells of E: coli stram MC1061 using protocols provided with the Gene Pulser electroporator. Quickly freeze 120~pL aliquots m liquid nitrogen and keep at -80°C until use. 2. Thaw three tubes of competent cells on ice, combine the cell suspension, and transfer to the tube containing the 5 pL ligated DNA mixture prepared in Section 3.1.3. 3. Conduct six separate electroporations, each with approx 60 @. of cell-DNA mixture in a 0.2-cm cuvet, using settings at 2.4 kV, 25 pF, and 200 n (see Note 3). 4. After each electroporation, immediately transfer the mixture into a lOO-mL flask containing 10 mL prewarmed LB medium with tetracycline at 0.2 pg/mL. After the completion of the last electroporation, incubate the flask at 37OC for 60 min with gentle shaking (at 150 rpm). 5. Plate 25-, 50-, and 100~pL aliquots onto LB/Tet plates for colony counting, and incubate the plates at 37°C overnight. 6. Transfer the rest of the culture to a 1-L flask containing 190 mL prewarmed LB/Tet medium, and incubate for 12-16 h at 37°C with vigorous shaking (at 300 rpm). 7. Transfer the 200-n&. culture to a centrifuge bottle and spin for 15 min at 10,OOOg. Transfer the supernatant to a clean centrifuge bottle and repeat the spin. 8. Collect the supernatant from the second spin in a clean bottle and add 0.15 vol of PEG solution (i.e., 30 mL for 200 mL supernatant). Invert the bottle several times and incubate on ice for at least 2 h (see Note 4). 9. Centrifuge at 12,000g for 30 min at 4°C. Completely remove the supernatant. 10. Resuspend the phage pellet in 10 mL of TBS buffer by pipeting, followed by incubation at room temperature for approx 30 min to dissolve the pellet completely (see Note 5). 11. Transfer the phage solution to a SO-n& centrifuge tube and spin for 10 min at 10,OOOgto remove insoluble materials. 12. Repeat PEG precipitation by adding in 1.5 mL PEG solution, followed by incubation on ice and centrifugation as in steps 8 and 9. 13. Completely dissolve the phage pellet in 1.6 mL TBS as in step 10, and transfer the phage solution to a 2-mL Eppendorf tube.

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14. Spin for 5 min to remove insoluble materials and transfer the supernatant to a clean 2-mL tube. 15. Repeat PEG precipitation as above by adding in 240 l.rL PEG solution, followed by incubation on ice and centrifugation. 16. Finally dissolve the phage pellet in 1 mL of TBS containing 0.02% NaNs. 17. Spin again to remove insoluble materials, and split the supernatant into two amber-colored Eppendorf tubes, one with 0.1 mL and the other with 0.9 mL. Keep the 0. l-n& tube at 4°C as working stock, and freeze the other at -80°C in the presence of 7% (v/v) DMSO for long-term storage (see Note 6).

3.1.5. Titering Phage Transducing Units (TU) 1. On the night before, maculate 1 mL LBKan with E. coli strain K91Kan and shake overnight at 37’C. 2. Use 100 pL of the overnight culture to inoculate 10 mL TB/Kan medium in a lOO-mL flask. Shake vrgorously at 37°C until mid- to late-log phase (see Note 7). 3. Slow the shaking down to around 100 rpm to allow sheared F-pili to regenerate. Use the cells within approx 60 min. 4. During the slow shaking of the bacterial culture, make a serial dilution of phage solution in TBS/gelatin covering the range 1:10-7,1:1~,1:1~9, and l:lO-lo. 5. Mix 10 yL K9 IKan cells prepared in step 3 in a 1.5-n& Eppendorf tube with 10 ltL each of the diluted phage solutions, and incubate at room temperature for 10 min for phage infection. 6. Add 1 mL LB medium containing 0.2 yg/mL tetracycline and incubate at 37’C for 30 min (with gentle shaking if convenient). 7. Plate 50- and 100~pL aliquots onto LB/Tet/Kan plates, followed by overnight incubation at 37’C. Count the colony numbers to determine phage titer.

3.2. Library Screening 3.2.1. Affinity Selection Using Streptavidin Magnetic Beads (SMB) The protocol given below is to be used for mouse monoclonal antibodies (MAb). The same protocol can be used for antibodies from other species with appropriate biotinylated bridging antibodies in step 4. 1. Prepare K91Kan cells as in Section 3.1.5, steps l-3. 2. Set up a phage-antibody incubation solution in a 2-mL flat-bottom Eppendorf tube by mixing 180 yL blocker, 10 pL antibody, and 10 ltL phage. Incubate at room temperature for 45-60 min (see Note 8). 3. Meanwhile, place 100 pL of SMB suspension (1 mg/mL) in a 2-mL flat-bottom Eppendorf tube, and wash the beads three times with 0.5 mL blocker solution. 4. Resuspend the washed SMB in 190 l.tL blocker solution, followed by the addition of 10 PL biotinylated antimouse IgG antibodies. Incubate the solution at room temperature for 30 min (see Note 9). 5. Wash the beads three times (2 min each) with 1 mL TBST.

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6. Transfer the 200~pL phagsantibody incubation mixture from step 1 to the tube containing the washed beads, followed by a further incubation at room temperature for 30 min. 7. Wash the beads five times as in step 5. 8. Resuspend the beads in 200 pL TBS, and transfer to a 0.5-mL cone-shaped Eppendorf tube. Position the tube bottom against the magnetic separation stand, so that the magnetic beads will “swim” toward the bottom of the tube rather than toward the side of the tube, as in a normal operation. Completely remove the TBS buffer using a thin plpet tip, so that a small compact pellet IS formed at the bottom of the tube. 9. Elute bound phages by incubating the beads with 40 pL elution buffer at room temperature for 10 min. 10. Place the tube bottom against the magnetic separation stand as in step 8, remove the 40 pL supematant using a thin plpet tip, and immediately transfer to a 2-mL tube containing 16 yL 1M Tris-HCI, pH 9.5, for neutralization of the eluted phage solution. 11. Add 100 PL K91Kan cells prepared as in Section 3.1.4., and incubate the tube at room temperature for 10 mm for phage mfectlon. 12. Add 1 mL LB contaming 0.2 pg/mL tetracyclme and incubate at 37°C for 30 mm with shaking. 13. Take 100 pL for making dilutions of 1: 10, 1: 100, and 1: 1000 m prewarmed LB medium. 14. Onto LB/Tet/Kan plates, plate m duplicate 200~pL ahquots of the undiluted culture as well as the three diluted cultures made above in step 13. Incubate the plates (eight total) at 37°C overnight 15. Transfer the remaining culture from step 11 into a 250~mL flask containing 30 mL prewarmed LB/Tet/Kan medium, and shake vigorously at 37°C overnight. 16. Purify phages from the supematant of this 30-mL culture by three times PEG precipitation as described in Section 3.1.4. This can be used as an enriched library for a second cycle of affinity selection, if required (see Note 10).

3.2.2. Colony Lift lmmunoblotting All incubation

steps are carried out at room temperature.

1. Select two to four plates, from step 14 of Section 3.2. l., which have a colony density in the range of 100-500 colonies/plate (see Note 11). 2. Place a piece of precut circular nitrocellulose membrane onto the surface of the plate. Make sure that the membrane makes an even contact with the place so that it will be completely wet by the moisture from the plate within l-2 min. Mark the orientation of the membrane in relation to the plate so that it can later be correctly superimposed (see Note 12). 3. Lift the membrane and immediately transfer to a container (e.g , a square Petri dish) contaming 30-50 mL of blotto. Gently rock for 15 min. 4. Change the blotto solution and rock for an additional 15 mm.

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5. Seal the membrane inside a plastic bag with one side open, add 2 mL of MAb solution diluted in blotto at 1:5 for MAb tissue-culture supernatant or 1: 100 for ascitic fluid, and seal the remaining side. Incubate the bag for 30 min with gentle rocking (see Note 13). 6. Wash three times (5 min each) with approx 50 mL TBST. 7. Proceed as in step 5, except that an HRP-conjugated sheep antrmouse antibody is used at 1: 1000 dilution. 8. Wash as in step 6. 9. Develop the blot using the Chemiluminescent Substrate (POD) kit from Boehringer Mannheim and X-ray films following the instructions given by the supplier (see Note 14). 10. After drying the film, place it on a light box, so that the plate containing the corresponding bacterial colonies can be aligned with the signals (black dots) on the film. Pick up the positive clones using individual toothpicks and patch onto a fresh LB/Tet/Kan plate, followed by overnight incubation at 37°C. Keep the plate at 4°C as the master plate for positive phage clones.

3.3. Characterization

of Epifope-Displaying

Phage Clones

3.3.1, Phage Minipreparation 1. Pick up a single colony from the above master plate, and innoculate 2 mL LB/Tet medium in a lo-mL culture tube. Vigorously shake the tubes overnight at 37’C (see Note 15). 2 Remove cells by centnfugation in a 2-mL tube for 5 min at room temperature. 3. Transfer the supernatant to a clean tube and repeat the centrifugation as above. 4. Carefully take 1.6 mL supernatant from the final spin and transfer to a 2-mL tube containing 240 l.tL PEG solution. Invert the tube several times and either incubate the tube on ice for at least 2 h or leave the tubes at 4°C overnight 5. Collect phage precipitate by centrifugation at 4°C for 15 min. 6. Dissolve the phage pellet m 30 p.L TBS by pipeting, followed by 30-mm incubation. 7. Spin for 5 min to remove insoluble materials. (The phage solution 1sready to be used in ELISA, dot-blotting, or Western blotting analysis.)

3.3.2. ELISA All incubations, except for substrate development, are carried out at 37OC with gentle shaking on a microplate shaker at setting 6. 1. Make a 1:5000 dilution of rabbit anti-Ml3 antiserum in coating buffer, and use 50 l.tL/well to coat an ELISA plate (see Note 16). Incubate the plate for 60 min. 2. Wash the plate three trmes (5 min each) with PBST. 3. Add 100 nL blocking solution to each of the wells and incubate for 30 min. Discard after incubation. 4. Add 50 pL phage solution, serially diluted in blocking solution starting from 1: 100 (see Note 17), followed by incubation for 60 min. 5. Wash as m step 2.

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6. Add 50 pL MAb, diluted in blocking solution at 1: 10 for tissue-culture supernatant or 1: 100 to 1: 1000 for ascitic fluid, followed by incubation for 60 min. 7. Wash as in step 2. 8. Add 50 pL HRP-conjugated sheep antimouse IgG diluted in blocking solution at 1:2000, followed by incubation for 60 min. 9. Wash as in step 2. 10. Add 50 pL substrate solution, and incubate at room temperature for 10 min. 11. Stop the reaction by adding 50 pL stopping solution. 12. Read the absorbance at 450 mn.

3.3.3. Dot Blotting All incubations are carried out at room temperature. 1, Wet a precut nitrocellulose membrane (10 x 14 cm) in TBS buffer, and assemble the membrane into the Bio-Dot Microfiltration unit following given instructions. 2. Serially dilute phage solutions in PBS starting at I:100 (see Note 17). The dilution can be conveniently carried out in an ELISA plate, since the Bio-Dot unit has the same 96-dot/well format. 3, Under vacuum, apply each 20 pL of diluted phage solution into a corresponding well. Wait for l-2 min after all samples are applied. Slowly release the vacuum and disassemble the unit. 4. Remove the membrane from the unit and immediately transfer to a container with 50 mL blotto. Gently rock for 30 min. 5-10: Follow steps 5-10 described in Section 3.2.2. (see Note 18).

3.3.4. Colony PCR and DNA Sequencing We find that .it is most convenient to carry out PCR and sequencing reactions in microplates. However, the following procedures are equally applicable for using tubes as long as minor adjustments are made accordingly for PCR conditions. 1. Place 14 pL water into each well of a microplate that is suitable for plate PCR application. 2. Use a toothpick with a sharp tip to transfer cells from a colony to a corresponding well by gently touching, rather than digging, the colony, and then mixing in water. 3. Heat the plate at 1OO’C for 2 min, followed by immediate cooling on ice. Keep on ice until the next step. 4. Set up a PCR reaction cocktail as follows (the volumes given are for one reaction): 2.5 pL 10X PCR buffer 2.5 pL 25 mMMgClz 4 p-L dNTPs, 1.25 mM 1 pL forward primer 111-5, 10 pmol/pL 1 pL reverseprimer 111-3, 10 pmol/pL 0.25 U of Tag polymerase

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5. 6. 7. 8. 9. 10. 11.

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Add 11 pL of this cocktail mixture into eachwell, andmix by pipeting. Seal the top with one drop of oil. Carry out a PCR amplification for 25 cyclesat 94OCfor 1 min, 50°C for 2 min, and 7YC for 2 min (seeNote 19). After amplification, take 5 pL reaction mixture for analysison a 1% agaroseTAE gel to checkfor insert sizeand the quality of PCR products. Transfer 7 pL from the remaining PCR product into a 0.5-n& Eppendorftube, add 1 pL exonucleaseI (supplied with the PCR Sequencingkit), and incubateat 37°C for 15 min. Inactivate the enzymeby heating at 80°C for 15 min. Briefly spin liquid to the bottom of the tube. Add 1 pL shrimp alkaline phosphatase(also supplied with the PCR Sequencing kit), and incubate at 37OCfor 15min. Inactivate the enzymeasin step 8. The PCR fragment (approx 9 pL total) is ready to bedirectly sequencedusing the PCR Sequencingkit and the internal primer 35S,following detailed procedures provided with the kit (seeNote 20).

4. Notes

4.1. L/bray Construction 1. It should be noted that there are other ways of generating random DNA fiagmentsother than DNaseI partial digestion. The other commonly usedmethod is by sonication(e.g., in ref. 16). A detailed protocol for generation of random fragmentsby sonicationcan be found in Chapter 7, vol. 23 of this series.The remaining procedures, from step 9 of Section 3.1.1, will be equally applicable to sonicatedDNA fragments. 2. To achieve the best yield from purification using the GeneCleankit, an effort should be madeto reduce the samplevolume and, hence,reduce the size of the agarosegel sliceafter electrophoresis.For purification of smaller fragments (50100bp), it is better to usethe Mermaid Clean kit (also from BIO-lOl), specially formulated for this purpose. 3. We recommend the use of new cuvets for this. The samecuvet can be used for multiple electroporationsof the samesample.For optimal performance, cool the cuvet on ice for 1 min betweeneachusage.Under theseconditions, we normally get a pulse of 4.0-4.6 ms. 4. Although 2-h of incubation on ice is usually enough for PEG precipitation of phages,overnight incubation at 4°C can give a slightly better yield. It is also a convenient break point in the protocol. This is true for all other PEGprecipitation stepsdescribed in this protocol. 5. Owing to the filamentous shape,phage particles in PEG precipitatesare hard to dissolve. The 30-min incubation is essentialfor phageparticles to diffuse completely. If convenient, the phagesolution may alsobe kept at 4OCovernight in the presenceof 0.02% NaNs.

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6. The phage solution purified by 3x PEG precipitation is usually good enough for the application described here. However, further purification by CsCl, gradient centrifugation (see ref. 9 for details) is recommended If the phage library is to be kept for a long period and for multiple applications. Although the phage particles purified by 3x PEG precipitation are stable at 4’C, we find that the antigenicity of certain recombinant phages displaying foreign epitopes decreases with time, probably because of degradation by trace amounts of contaminating proteases. 7. The growth of the bacterial culture IS best monitored by a spectrophotometer at 600 nm. However, we find that a 5-h vigorous shaking at 300 rpm, followed by 30-min incubation at 100 rpm, usually gives satisfactory results.

4.2. Library Screening 8. The optimal ratlo of antibody vs phage is hard to determine because both the phage titer and antibody titer vary from one experiment to another. Our common practice is to use the phage solution at approx lOI TU/mL, and the antibody solution at approx 0.1 pg/mL for purified antibodies or 10-100 pg/mL for crude antibodies. 9. The use of a biotinylated antispecies antibody as a bridge is not absolutely necessary. It IS possible to blotinylate the antibody of interest and bind it directly to the SMB. However, we recommend the use of a biotinylated bridging antibody for two reasons: a. It is convenient to operate since there is no need to biotmylate individual antibody molecules. b. This may also help the later elutlon process, since the binding between the bridging antibody and the primary antibody provides an additional break point during elution, and 1s more homogeneous m bmdmg affinity than the interaction between the primary antibody and individual phage-displayed epitope fragments. 10. Although multiple pannings can usually enrich the population of binding phages, there is an associated danger of losing relatively weak binders or slow-growth phage clones. When polyclonal antibodies are used for affinity selection, we especially recommend carrying out a single panning, followed by colony lift immunoblotting. 11. If possible, the plates should be used within a day to two for colony lift. However, old plates, kept at 4’C up to a week, have also been used in our laboratory with satisfactory results. 12. There are several methods one can use to mark the membrane for correct superimposition. The easiest method we use is as follows: a. Cut three small triangles off the edge of a circular membrane at positions approx correspondmg to 12, 1, and 4 o’clock. b. After placing the membrane onto the plate surface, mark the corresponding positions on the place using a permanent marking pen.

Phage- Display Fragment Libraries c. Overexpose one film in step 9 of Section 3.2.2. to reveal the three triangles on the edge of the membrane. d. Superimpose the overexposed film to the film with the best exposure and mark the triangles. Then superimpose this second film onto the plate to identify the positive colonies. 13. A convenient way of keeping the membrane flat is to put the bag in the middle of a thick heavy book (e.g., a telephone directory readily available in every laboratory), which is in turn placed on top of a rocker. The 30-min incubation time is the minimum time required, but can be extended to incubation at 4°C overnight to fit in with other ongoing experiments. This is also true for the incubation with conjugated antibody in step 7 of Section 3.2.2. 14. The exposure time required may vary from one experiment to another. We normally carry out three exposures at 10, 30, and 60 s, and then a 5-min exposure while the first three films are being developed. Less-sensitive methods can also be used if the expected signals are strong (e.g., 4-chloro- 1naphthol as substrate in the ProtoBlot Western Blot HRP System from Promega). However, it is better to avoid the use of alkaline phosphatase (AP)-conjugated antibodies, since the endogenous AP activity from E. coli can cause a high background.

4.3. Characterization

of Epitope-Displaying

Phage Clones

15. The phage production yield can be increased by using TB medium, and incubating the culture for an extend period of 36-48 h. 16. The use of anti-Ml3 antibodies to capture the phage particles is optional. Phage particles can also be directly coated in an ELISA plate. However, we found the capture ELISA gives more consistent results than direct coating. 17. It is essential to include a control phage in the binding assays. Since the cloning vector fUSE1 is a nonproductive phage, which contains a nonfunctional gene III, we normally use a closely related fLJSE2 vector (see Ref. 9) for production of control phages. For initial screening studies, a single point at each dilution is acceptable. Duplicate or triplicate points should be used in more detailed binding analysis later on. 18. Owing to space limitation, we presented only two of the most convenient binding assays here. There are other assays that can be used in confirming antibody binding. One frequently used method is Western blotting (e.g., see refs. 14 and 16). More recently, Dyson et al. (17) have reported a method for direct measurement of phage-ligate binding via phage titering, which can be used for comparison of different binding affinities of recombinant fusion phages. 19. If primers other than those described here are used, the PCR conditions may have to be optimized accordingly. 20. The direct DNA sequencing of PCR products following enzymatic degradation of primers and dNTPs in the PCR buffer (18) is the most convenient methods that we have come across. One can, of course, follow more conventional ssDNA sequencing methods for characterization of phage insert (see ref. 9 for detailed procedures on preparation and sequencing of ssDNA phage DNA).

Wang and Yu Acknowledgments We thank G. P. Smith of University of Missouri, Columbia for providing the fUSE expression system and a set of comprehensive protocols.

References 1. Scott, J. K. and Smith, G. P. (1990) Searching for peptide ligands with an epitope library. Science 249,38&390. 2. Cwirla, S. E., Peters, E. A., Barrett, R. W., and Dower, W. J. (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87,6378-6382.

3. Devlin, J. J., Panganiban, L. C., and Devlin, P. E. (1990) Randompeptide libraries: a source of specific protein binding molecules. Science 249,404+06. 4. Felici, F., Castagnoli, I., Musacchio, A., Jappelli, R., and Cesareni, G. (1991) Selection of antibody ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. Mol. Biol. 22, 30 l-3 10. 5. Cortese, R., Felici, F., Galfre, G., Luzzago, A., Monaci, P., and Nlcosia, A. (1994) Epitope discovery using peptide libratles displayed on phage. Trends Biotechnol. 12,262-267.

6. Cortese, R., Monaci, P., Nicosia, A., et al. (1995) Identification of biologically active peptides using random libraries displayed on phage. Curr. Opin. Biotechnol 6, 73-80. 7. Scott, J. K. and Craig, L. (1994) Random peptide libraries. Curr Opin Biotechnol 5,40-48. 8. Parmley, S. F. and Smith, G. P. (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73,305-3 18.

9. Smith, G. P. and Scott, J. K. (1993) Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 217,228-257. 10. Du Plessis, D. H., Wang, L.-F., Jordaan, F. A., and Eaton, B. T. (1994) Fine mapping of a continuous epitope on VP7 of Bluetongue Virus using overlapping synthetic peptides and a random epitope library. Virology 198,346-349. 11. Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986) A priori delineation of a

peptide which mimics a discontinuousantigenic determinant.Mol. Immunol. 23, 709-715. 12. Balass, M., Heldman, Y., Cabilly, S., Givol, D., Katchalski-Katzir, E., and Fuchs, S. (1993) Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library. Proc. Natl. Acad. Sci. USA 90, 10,638-10,642. 13. Lane, D. P. and Stephen, C. W. (1993) Epirope mapping using bacteriophage peptide libraries. Curr. Opin. Immunol. 5,268-271. 14. Wang, L.-F., Du Plessis, D. H., White, J. R., Hyatt, A. R., and Eaton, B. T. (1995) Use of a gene-targeted phage display random epitope library to map an antlgenic determinant on the bluetongue virus outer capsid protein VP5. J. Immunol. Methods 178, 1-12.

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15. Lopez-Otin, C., Freije, J. M. P., Parra, F., Mendez, E., and Vinuela, E. (1990) Mapping and sequence of the gene coding for protein ~72, the major capsid protem of African swine fever virus. Virology 175,477-484. 16. Jacobsson, K. and Frykberg, L. (1995) Cloning of ligand-binding domains of bacterial receptors by phage display. BioTechniques 18,878--885. 17. Dyson, M. R., Germaschewski, V., and Murray, K. (1995) Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes. Nucleic Acids Res. 23, 1531-1535. 18. Hanke, M. and Wink, M. (1994) Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. BioTechniques 17,858-860.

25 Epitope Mapping by Expression of Restriction Enzyme or PCR Fragments in Bacterial Plasmids Johannes A. Lenstra and Arnoud H. M. Van Vliet 1. Introduction Antibodies induced by native antigens often crossreact with denatured antigen or with peptides that contain a segment of the antigen polypeptide chain. The epitopes recognized by these antibodies are linear or sequential, i.e., specified by the linear sequence of the amino acid residues rather than by the protein conformation. Presumably, linear epitopes correspond to flexible parts of the surface of the antigen (1). These epitopes can be localized by synthesis of peptides and testing their antigenicity, i.e., the binding by antibodies. However, this is not practical for antigens of several hundreds of residues. In this chapter, we describe a convenient alternative, the prokaryotic expression of fragments of the coding sequence. In most systems,these fragments are expressed as part of a fusion protein, i.e., coupled to a bacterial polypeptide, which serves as a convenient carrier or provides a tag for affinity purification. A literature review (2) showed that almost without exception antibodies that recognize a prokaryotic expression product of a viral gene segment also recognized the viral antigen on a Western blot, an operational criterion for the recognition of a linear epitope. Therefore, conceptually, these expression products are as antigens equivalent to denatured protein fragments (2). The localization of an epitope follows directly from the location of the gene fragment that on expression yields an antigenic recombinant product (3,4). The starting material for epitope localization via prokaryotm expression is the gene encoding the antigen. For a eukaryotic gene, a cDNA clone rather than a genomic clone is preferred. The many prokaryotic expression systems that are available can be combined with several methods to generate fragments From

Methods m Molecular Biology, vol. 66’ Epitope Mappmg Protocols Edited by G E Morris Humana Press Inc , Totowa, NJ

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from the gene of interest. Table 1 summarizes the advantages and disadvantages of the plasmid expression systems pEX (5,6), the pGEX (78) and pQE (9), respectively, which are among the most widely used systems and are described in this chapter. pEX seem to be the most reliable workhorse for localizing the epitope, whereas soluble products from the pGEX and pQE systems are more suitable for ELISA or immunization. For the latter purpose, expression of the same insert in two systemswith different fuston proteins may be useful: After the first immunization with the product from one system, the product from the other can be used for boosting or for detection of the immune response specific for the insert. Table 2 lists the relative merits of the methods used to generate gene fragments. Restriction enzyme fragments and PCR products yield a rapid localization of the antigenic regions. Expression of oligonucleotides (10) can be useful for an accurate localization as an alternative for synthesizing peptides. The construction of DNase-generated fragment libraries (I I) and the sequencing of the clones may be accurate, but are relatively time-consuming. An interesting option is the screening of random expression libraries (12) to obtain mimotopes, linear sequences that mimic linear or conformational epitopes. However, because of the number of clones that have to be screened,this can be better combined with phage-display systems (see other chapters of this volume). This chapter describes the use of the pEX, pGEX, and pQE systems.Figure 1 shows a flow scheme that leads from gene to epitope. Cloning and characterization of clones can be done by adapting standard procedures (13) with the appropriate vectors and hosts. The immunoscreening of colonies (14,15) is common for all three systems.Rapid preparation procedures yield pEX, pGEX, or pQE expression products suitable for Western blotting, which reveals the localization of the epitope. As an example, Fig. 2 shows a Western blot of pQE proteins, which reveals the difference in specificity of two antisera. Affinitypurification methods are specrtic for the pGEX and pQE systems,respectively. 2. Materials 2.7. Host Cells and Vectors 1. pEX: Escherichia coli pop 2 136,recA+ andcarrying the Cl857 allele coding for the temperature-sensitive repressor of the h P, promoter is a suitable host strain. pEXl1, pEX12, and pEXI3 can be obtained via the American Type Culture Collection. 2. pGEX: commonhostsstrains,suchasDH5a or JM109, for the pUC plasmid family canbe used.Plasmidsaremarketedby PharmaciaBiotech (Uppsala,Sweden). 3. pQE: plasmidsandMIS[pRBP4] hostcells,carrying the pRBP4 plasmid with the 1acIq gene coding for the lac repressor and a kanamycin resistancegene, are marketed by Qiagen Inc (Hilden, Germany).

Table 1 Prokaryotic Vector PEX

pGEX

Expression Induction

Vectors Expression format

Temperature inactivates ts repressor ofh Pa promoter IPTGon tuc promoter

C-termlnal insertion in cro+galactosidase fusion protein

Reliable expression of virtually all inserts of up to 1500 bp

C-terminal insertion in glutathione S-transferase

IPTG on tat promoter

C- or N-terminal I-Ike tag

Expression product often soluble AfIinity purification of soluble fusion protein Optional cleavage of fusion protein Expression product often soluble Only short sequence contributed by vector Affiuity purification of soluble and insoluble fusion proteins with Ni-agarose No crossreaction of antisera with affinity tag

Rs 8

PQE

Advantages

of

Disadvantages

Ref.

Fusion protein insoluble Slow growth of clones at 30°C Reaction of many antisera with P-galactosidase part of fusion proteins Expression and solubility variable

2-6

Expression and solubility variable Often degradation of products of small inserts

7,8

Table 2 Methods of Generating

DNA Fragments

Method Restriction enzyme digestion (3,4.10, I I) PCR (4)

Synthetic oligonucleotides (ZO) DNase (I 1) or sonication fragments; exonuclease digestion of positive clones

Random-sequence fragments (12)

Encoding

Antigenic

Expression

Advantages Fast Fast

Accurate locahzatron Potentially accurate

One library for all antibodies Yields antigenically allowable sequence variants May yield mimotopes that mimic conformatronal eprtopes

Products Disadvantages Accuracy of epitope mapping limited by available restriction sites Cloning of PCR products may be difficult; possible sequence errors introduced by thermostable polymerase Syntheses of many oligonucleotides expensive Construction of library and sequencing positrve clones time-consuming Results unpredictable Possible cross-reaction with out-of-frame expression products Screening and sequencing of positive clones time-consuming Only for short epitopes

Prokatyotic Expression

291

Recombinant antigens by expression in bacteria DNA containing ORF of antigen gene fragments GE, PCR, DNase) ligation in expression plasmid cloning, DNA analysis I I protein minipreps * immunoscreening WesteXblotting DNA Galysis -3 e localization of epitope large-scale purification of fusion protein -m immunization EGA u 5 immunogenicity serology, etc. Fig. 1. Flow schemeof the constructionandanalysisof clonesproducing recombinant antigens.

2.2. Primers 4. pEX upstreamPCR or sequencingprimer: S-GATTGGTGGCGACGACTCCTGG (3156-3 177 in pEX2 with the EcoRI site at 31963201). 5. pEX downstreamPCRor sequencingprimer: 5’ TGAATTAATTCTAGAGCCGGAT (3274-3253). 6. pGEX upstreamsequencingprimer: 5’-GGTGGCGACCATCCTCC. 7. pGEX downstreamsequencingprimer: 5’CCGTCATCACCGAAACGCGC. 8. pQE upstreamsequencingprimer: 5’-GGCGTATCACGAGGCCCTTTCG (promoter region) or 5’-GAATTCATTAAAGAGGAGAAA (ribosome binding site). 9. pQE downstream sequencingprimer: 5’-CATTACTGGATCTATCAACAGG.

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Fig. 2. Localization of epitopesby Westernblotting of pQE expressionproduct. An antiserumfrom sheepinfectedwith the intracellularbacteriaCowdria ruminantium recognizes the major antigenic protein (MAP-l) from a cell lysate and epitopes within residues 1-152 and 47-152. A crossreactingantiserum againstthe related Bhrlichia recognized the product containing residues 1-152, but not the product containing 47-152 (16).

2.3. lmmunoscreening 10. LB/amp: Luria-Bertani (LB) medium (13) supplemented with 100 pg/mL ampicillin, addedjust before use. 11. For pQE in M15[pREP4]: LB/amp/kana, LB/amp with 50 ug/rnL kanamycin; add the antibiotics just before use. 12, LB/amp plates: 1.6%agarosein LB/amp (for the pQE clonesin MlS[pREP4] in LB/amp/kana), autoclavewithout antibiotics, let cool to ca. 50°C add the antibiotics, pour the plates, store at 4’C, and use within a few days (seeNote 4). Prepare one set of plates for growing and the samenumber of plates for expression. For library screening,chooselarge plates of the samesize as a filter that fits the electroblot apparatus. 13. For pGEX and pQE: 1 n&f isopropyl-S-n-thiogalactopyranoside (IPTG, 0.24 mg/mL) in HzO, and store at -2OV. 14. Nitrocellulose (NC) filter, sterilized and fitting the LB plates.Never touch with barefingers. pGEX andpQE: saturatewith 1mMIPTG, dry betweenfilter papers, and use within 3 d. 15. China ink. 16. Whatman 3MM sheets,2/filter andjust larger than the filter. 17. Whatman 3MM sheets,fitting the electroblot apparatus. 18. 5% SDS in H20. 19. Flat-bottomedplastic boxesthat can contain the filters.

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Prokaryotic Expression

20. Transfer buffer: 192 mM glycine, 25 mM Tris, 20% methanol, pH 8.3 (not adjusted); store at room temperature. 21. Gelatm wash buffer: 0.5% gelatin in PBS (13), autoclave, add 0.1% Triton X-100, mix, and store at room temperature. Beware of microbial infection. Optionally, add just before use phenylmethylsulfonylfluoride (PMSF) to 37 pg/mL from a stock of 25 mg/mL in 2-propanol. If stored at 4°C the gelatin has to be redissolved at 37°C prior to use. 22 Polyclonal antisera or monoclonal antibodies (MAbs): Dilute fresh in gelatin wash buffer (see Note 9). 23. Sucrose/Tris/EDTA: 15% sucrose, 50 mMTris-HCI, pH 8.0,50 rnA4EDTA, sterilize by filtration. 24. 10 mg/mL lysozyme; store at -20°C. 25. Triton/Tris-HCl: Add 0.1% Triton X-100 to 50 mMTris-HCl, pH 8.0, autoclave, and store at 4’C. 26. E. coli protein extract: Grow the host strain in 2 mL LB at 37°C overnight; transfer to 800 mL LB in a large flask, and shake at 37’C overnight; spm down (4000g for 10 min) and resuspend in 8 mL sucrose/Tris/EDTA; add 2 mL (20 mg) lysozyme, mix thoroughly (but do not vortex), and leave at 0°C for 40 min; add 12 mL 0.1% Triton/Tris-HCl, and leave for 15 min at O’C; spin for 1 h at 50,OOOg and at 4’C, aliquot the supernatant in 1-mL portions, and store at -20°C The protein concentration should be IO-20 mg/mL. 27. Second antibody, alkaline-phosphatase conjugate directed against the species of the first antibody; store at 4°C. 28. Alkaline-phosphatase buffer: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl*; autoclave. 29. NBT (chromogenic agent): 50 mg/mL nitroblue tetrazolium in 70% dimethylformamrde; store at 4’C. 30. BCIP substrate: 50 mg/mL 5-bromo-4-dichloro-3-mdolyl phosphate in dtmethylformamide; store at 4°C. 3 1. 0.6% Ponceau S in 3% trichloroacetic acid.

2.4. Protein Minipreps 32. ForpEX products: SDS lysis buffer: 5% SDS, 50 mMTris-HCl pH 8.0, 15 mM2mercaptoethanol. 33. For pGEX and pQE products: 100 mMIPTG, 24 mg/mL in HZO, store at -20°C. 34. For pGEX and pQE products: PBS + 1% (v/v) Triton X-100. 35. For pGEX and pQE products: bath sonicator. 36. 3X concentrated Laemmli loading buffer: 0.188M Trrs-HCI, pH 6.8, 6% SDS, 30% glycerol, 15% 2-mercaptoethanol, 0.03% bromophenol blue; store at-20°C.

2.5. Large-Scale Purification 2.5.7. Purification of p EX Fusion Proteins 37. Sucrose/Tris/EDTA: ilize by filtration.

15% sucrose, 50 mMTris-HCl,

pH 8.0,50 WEDTA;

ster-

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38. Triton/TE: add 0.2% Triton X-100 to sterile TE (10 mMTris-HCl, EDTA) and mix. 39. For pGEX and pQE products: bath sonicator.

pH 8.0, 1 mM

2.5.2. Affinity Purification of pGEX Fusion Proteins 40. Triton/PBS: Add 1% Triton X-100 to the autoclaved PBS and mix. 41. Glutathione-Sepharose 4B beads (Pharmacia), wash just before use (see Section 3.4.2.). 42. Elution buffer (fresh): 50 n&f Tris-HCl, pH 8.0, 15 mM glutathione.

2.5.3. Affinity Purification of pQE Fusion Proteins 43. Ni2+-NTA agarose (Qiagen Inc.): Equilibrate before use (see Section 3.4.3.2., step 2). 44. Sonication buffer: 50 mMNaH2P04, 0.3MNaCl; adjust to pH 7.8 with NaOH. 45. Wash buffer: 50 mMNaH2P04, 300 mMNaC1, 10% glycerol; adjust to pH 7.8 with NaOH. 46. 0.5M imidazole in wash buffer. 47. 10% TCA. 48. Buffer A: 6A4 guanidiniumHC1, 0.01MNaH2P04, O.OlMTris; adjust to pH 8.0 with NaOH. 49. Buffers B-E: 8Murea, 0.01MNaHzP04, 0 OlMTris, adjust to pH 8.0 (buffer B, use 2MNaOH); to pH 6.3 (buffer C, use half-concentrated HCI); topH 5.9 (buffer D); or to pH 4.5 (buffer E); do not autoclave; check and adjust pH of buffers just before use. 50. Buffer F: 6M guamdinium-HCl, 0.2M acetic acid.

3. Method

3.1. Cloning and Analysis of Clones 1. Digest cDNA and plasmid wtth the appropriate restriction enzyme(s) by standard methods (13). Figure 3 shows circular maps of pEX, pGEX, and pQE plasmids with the restriction sites for characterization of clones. Figure 4 shows the polylinker regions of representatives of the pEX, pGEX and pQE systems, respectively. Choose the vector in which the insert DNA will be expressed in the correct reading frame. 2. PCR products with 3’-A overhangs can be cloned by any of the reported procedures (17): by using linearized vectors with complementary 3’-T overhangs, by including restriction sites in the primers, or by polishing the PCR product with a DNA polymerase. We treated the PCR product with E. co11DNA polymerase I, ligated it into a blunt-ended linearized vector (e.g., by digestion with EcoRV or SmaI), and selected the desired clone by colony hybridization, 3. Ligate restriction enzyme or PCR fragment and the linearized plasmid DNA as described (13, see Note 1).

Prokatyotic Expression

295 HmdIII,llO

EaoRVJ303

4ooo 5808bps cro-1acZ

XboIJ

C

EooRI,30

polylinker &is \

/

3000 amp _ 2500

256fBglI

500

pQE9 3440 bps

1000

*

Fig. 3. Circular restriction maps of (A) pEX12, (B) pGEX-2T, representatives of the corresponding expression systems.

and (C) pQE9 as

Lenstra and Van Wet

296

A pEX1

~mal JJ&

& a

Pstl rlilndlll) GCCCGGGGATCCGTCGACCTGCAGCCAAGCllGCTGATlGATlGA ARGSVDLPPSLLID*

pEX2

m && EcoRI

a J@g

Pst1

~Hlndlll)

GAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTTGCTGATTGA EFPGfRRPAAKLAD*

pEX3

JIJ& J&g&

*

m Pstl rHlcdlI1) GAATTAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTTGCTGA ELIPGDPSTCSQAC* pEXl1

Smsl -iiT EtPRI 2!dL J&& SamHI sell Pstl (Hlndlll) AC!---GCCCGGGGTACCGAATTCACTAGTGCATGCGGATCCGTCGACCTGCAGCCAAGCTTGCTGATTGATTGA ARGTEFTSACGSVDLPPSLLlD*

pEX12

Smal Xmel -&&!

&mJj&gSa(l (WI ) LEfL L?FdL GAATTTTTCCCGGGGTACCGAATTCGCATGCGGATCCGTCGACCTGCAGCCAAGCTTGCTGATTGATTG EFFPGYRIRHRtRRPAAKLAD*

pEXl3

Smal Xmal -EcoRl

J&Jal!L && Jp& &g& GAATTAATTCCCGGGGTACCGAAT7CACTAGTGCA7GCGGGCT7GCTGA ELlPGVPNSLVHADPSTCSQAC*

Pstl

(HlndlIl)

Fig. 4A

B pGEX*lN

-sJ& J@&

ECoRl

CCAAAATCGGATCCCCGGGAATTCATCGTGACTGACTGACGA PKSDPREFIVTD'

pGEX.lIT m

&gQ

CTGGTTCCGCGTGGATCCCCGCAATTCATCGTGACTGACTGACGA LVPRGSPEFIVTD*

A lhranbln pGEX-2T

sne_l &&I

J&g

CTGGTTCCGCGTGGATCCCCGGGAATTCATCGTGACTGAC7GAC7G LVPRGSPGIHRO* A Thronbin

pGEX.JX

x m

&oJ

CTGATCGAAGGTCG7GGGATCCCCGGGAATTCATCGlGACTG LIEGRGIPGNSS' A Factor Xa

Fig. 4B

Prokatyotic Expression

297

C

pQEl0 BernHI-PstI Hmdlll ATGAGAGGATCTCACCATCACCATCACCATACCCATCCOTCGACCTGCAGCCAAGCTTAA MRGSHHHHHHTDPSTCSPA* NE11

m M Jg!&l&g ATGAGAGGATCTCACCATCACCATCACCATCCOTCGACCTGCAGCCAAGCTTAATTAG MRGSHHHHHHGIRRPAAKLN"

Fig. 4. Multiple cloning sites of (A) pEX, (B) pGEX, and (C) pQE vectors. Other pGEX derivatives: pGEX-2TK with a kinase recognition site for labeling of expression proteins; the pGEX-4T series with more restriction sites and thrombin cleavage, and the pGEX-5X series with more restriction sites and factor-X cleavage (8). For other pQE vectors, see the QIAexpress manual (9).

4. Transform by electroporation as prescribed by the manufacturer (e.g., Bio-Rad) rather than by heat-shock of competent cells (13). (See Note 2 on the host cells for pQE constructs.) Heat-shocks of the pEX host (5 min at 34°C followed by 2 min on ice; recovery 1 h at 30°C) cannot be carried at 42’C because of the temperature-sensitive repression system. This reduces the efficiency of transformation via the classical procedures to 2-5 x 10s transformants1p.g. 5. Grow pEX transformants always at 30°C (Instead of 37°C) with 100 pg/mL ampicillin (instead of the normal 50 pg/mL). Grow pGEX and pQE transformants at 37’C, but also with 100 pg/mL ampicillin; for pQE in MlS[pREP4], supplement with 50 pg/mL kanamycin. 6. The size of the insert of recombinants can be estimated by restriction analysis of miniprep DNA (Z-1) or by PCR. The pEX PCR primers listed in Section 2.2. tolerate an annealing temperature of 60°C. Sequencing of clones can be done via standard methodology with the primers listed in Section 2.2. (see Note 3).

3.2. Immunoscreening Immunoscreening of colonies is a convenient procedure to select antigenic clones. This step is essential with libraries of DNase or random-sequence fragments. It may also be usefiA to select clones with the correct orientation and reading frame after insertion of restriction enzyme or PCR fragments that encode antigenic

regions. We found that the procedure

of Stanley originally

developed for pEX (1415; see Fig. 5) works equally well for pGEX and pQE. 1. Plate out the transformed cells on LB/amp plates (LB/amp/kana plates for pQE in MlS[pREP4]). If necessary, plate at different densities to obtam 50-200 colo-

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298 Growth

3OYx 20h t--

-Expression 42°Cx 2h

\-

5%SDS 9% x 2mIn

Master plate

Electrodution 50~ x 30 mtn

Ponceau 5 start7 .

. l

l

Anhbady stain

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.

.

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.*

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.

.

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Fig. 5. Schematic overview of the immunoscreening

2.

3. 4. 5.

(14,15).

nies/9-cm plate for subcloning and 104-lo5 c&/300-cm* plate for library screening. Spread well; for large plates, this is most efficiently achieved with the end of a sterile lo-mL glass pipet raked across the surface at intervals of a few millimeters; repeat this perpendicularly to the first direction. Grow 20-24 h at 30°C (pEX) or 16 h at 37“C (pGEX, pQE). Let the plate dry for 15 min with the lids from the plates. Prewarm fresh LB/amp plates (LBlampkana plates for pQE in M 15 [pREP4]) for the expression. Mark the plates by touchmg the agar on three places with a toothpick dipped in China ink. Use blunt-ended forceps or clean gloves, and place the NC filter (pGEX, pQE: saturated with IPTG and dried) on the plate. Let the middle part touch first, and gradually lower to both sides while the filter gets wet. Avoid the trapping of air bubbles. Do not move the filter once it is in contact with the colonies.

Prokaryotic Expression 6. Use forceps to peel off the NC filter carefully and transfer, colonies up, to the prewarmed plate. Check if the Iif? has been complete 7. Expression in pEX: Incubate for 2 h at 42OC (see Note 6); expression m pGEX or pQE: Incubate for 4 h at 37°C. 8. Put the original plates back at 30°C (pEX) or 37’C @GEX and pQE) for a few hours until colonies reappear; then store at 4’C. 9. For each filter, soak two Whatman sheets (just larger than the filter) in 5% SDS and place on a glass disk. Avoid air bubbles. 10. After completion of the expression, peel the NC filter from the plate with a forceps and transfer to the 5% SDS Whatman papers, colonies up and without air bubbles. Put a lid on the disk and incubate in a 95’C oven for 15 min. This solubilizes the fusion protein. The colonies turn transparent and colorless. 11. Wet the Whatman paper that tits the electroblot apparatus in transfer buffer and drain well. Set up a sandwrch of two of these sheets, NC filters with the lysed colonies upward (as many as can be accommodated without overlap between the filters), then agam two Whatman sheets, filters, and so on, per sandwich up to six layers of filters. Carefully layer the papers and filters on top of each other and remove air bubbles by rolling lightly with a pipet over the Whatman sheets 12. Assemble the sandwrch in the electroblot apparatus, colonies facing the negative electrode. Apply 50 V for 30 min. The proteins bmd nreversibly to the NC filter, whereas the SDS is removed. 13. Remove the NC filter from the blotting apparatus and put in a tray with gelatin wash buffer. Remove remaining bacterial debris by carefully rubbing the filter, wearing a glove. Carry out the following incubations on a rocking platform. 14. Incubate twice for 5 min in gelatin wash buffer. If colonies are still visible as glassy lumps, repeat steps 13 and 14. 15. Make an appropriate dilution of the antibody in 10 mL of gelatin wash buffer (see Note 7). UseJust enough to cover the filters. With polyclonal antisera, add l/10 vol of E. cob extract to the Qlunon. Incubate the filters for 30-60 mm in the diluted antiserum. 16. Pour off the diluted antiserum in a tube (see Note 8). 17. Incubate for 5 min in gelatin wash buffer; discard the buffer. Repeat this twice. 18. Incubate for 30-60 mm m conjugated second antibody, diluted in gelatin wash buffer. Dilute as recommended by the manufacturer (see Note 9) 19. Incubate for 5 mm in gelatin wash buffer. 20. Incubate for 5 min in PBS and repeat this once. 2 1. Incubate for 5 min in AP buffer. 22. Just before use, prepare the substrate solution: 5 mL AP buffer with 35 PL NBT and 17.5 pL BCIP substrate. Keep in the dark. 23. With only a few filters simultaneously: Drain the filters well and transfer to a Petri dish with the substrate solution. Take care that the whole filter is in contact with the substrate solution. The staining depends on diffusion, so too many filters simultaneously may not stain equally. 24. Incubate l-20 min under continuous movement until a purple staining becomes visible. Incubating too long will stain all colonies.

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25. Stop the color development by rinsing thoroughly with TE and blot dry on filter paper. Repeat steps 22-25 with the remaining filters. 26. Using a ballpoint pen, indicate positive colonies by arrows. 27. Counterstain the colonies by incubating the filters for a few seconds in Ponceau S. Save the Ponceau S for reuse, and destain by rinsing the filters several times with H,O until the colonies are visible. Filters can be destained completely (see Notes 10-12). 28. Localize the positive colonies on the master plate by aligning the China ink markers on the filter and the plate on a lightbox. 29. To purify a positive clone, suspend the colony in 10 mL PBS, spread 100 yL of loo, 10-r, 10-2, and 10d3 dilutions on fresh LB plates with the appropriate antibiotics, and repeat the immunoscreening from step 2 onward. 30. Inoculate l-2 mL LB/amp (LB/amp/kana for MlS[pREP4]) with the purified positive colony and grow overnight at 30°C (pEX) or 37°C @GEX, pQE). 3 1. For long-term storage, add to the overnight culture 15-50% glycerol and put at -80°C.

3.3. Small-Scale Purification 3.3.7. pEX Protein Minipreps 1. 2. 3. 4. 5. 6. 7. 8.

Grow a small overnight culture of the pEX clones in LB/amp at 30°C (see Note 13). Dilute 50 pL in 2.5 mL LB/amp and shake for 2 h at 3O’C. Incubate for 1.5 h at 42°C in a shaking bath, Transfer 1.5 mL to an Eppendorftube and centrifuge for 2 min at 5000g. Discard the supernatant. At this stage, the pellet can be stored at -20°C. Resuspend the pellet completely in 100 PL SDS lysis buffer (see Note 14). Pierce the lid of the tube with a needle and boil for 5 mm. Add 50 pL 3X cont. Laemmli gel loading buffer, vortex vigorously, continue heating for 2 min, and vortex again. 9. Run 15 yL on a 7.5% SDS-polyacrylamide gel, The expression products are 115 kDa or larger. Optionally, analyze by Western blotting (see Note 15).

3.3.2. pGEX or pQE Protein Minipreps 1. Grow a small overnight culture of the clone in LB/amp. For pQE expression, use LB/amp/kana (see Note 13). 2. Prewarm 1 mL of fresh LB/amp (LB/amp/kana for MlS[pREP4]) at 37°C and then add 10 pL of the culture. 3. Grow for 2 h at 37°C under vigorous shaking. 4. Add IPTG to 1 mMfina1 concentration and shake for 3-4 h at 37OC 5. Centrifuge for 1 min at 10,OOOgin a minifbge. 6. Resuspend the cells in 100 PL PBS/Triton. 7. Sonicate for 1 min in a bath sonicator. 8. Centrifuge for 5 min at 10,OOOg at 4°C and transfer the supematant (soluble proteins).

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9. Resuspend the pellet in 100 pL PBS-Triton (insoluble proteins). 10. Analyze 10 pL of the soluble and the insoluble proteins on a 12% (pGEX) or 15% (pQE) SDS-polyacrylamide gel. The pGEX product of clones without insert (27 kDa) is predominant, but the amount of product depends on the insert (see Notes 15 and 16).

3.4. Large-Scale

Purification

3.4.1. pEX Fusion Proteins I. Grow an overnight culture of the pEX clone in LB/amp at 3O’C. 2. Dilute 200 pL in 10 mL LB/amp and grow for 2 h at 30°C. 3. Place in a preheated shaking incubator bath at 42°C and incubate for 90 min under continuous shaking. 4. Centrifuge for 10 min at 5OOOg. 5. Discard the supematant and resuspend the pellet completely in 200 pL sucrose/ Tris/EDTA by pipeting up and down; transfer to an Eppendorf tube (see Note 17). 6. Add 20 JJL (200 pg) lysozyme, mix, and leave at room temperature for 10 min (see Note 18). 7. Add 300 pL Triton/TE, mix, and somcate in a bath somcator for 15 mm at room temperature to shear the DNA. 8. Spin at 10,OOOg for 5 min; remove the viscous DNA-containing supernatant as completely as possible. 9. Resuspend completely in 500 pL Triton/TE by pipeting up and down through a yellow pipet tip and sonicate again. 10. Spin down for 2 min and remove the supematant. 11. Repeat steps 9 and 10 until the pellet is no longer viscous. 12. Resuspend the pellet completely in 250 pL PBS. 13. Store at -20°C (see Note 19). 14. Analyze the samples on a 7.5% SDS-polyacrylamide gel. The amount needed depends on the clone. Try 5 pL for Coomassie blue staining or Western blotting. After adding 3X Laemmli buffer, the samples can be loaded without boiling. Do not store in Laemmli buffer (see Note 19).

3.4.2. A Hnity Purification of pG EX Fusion Proteins Most, but not all, pGEX fusion proteins are soluble. This has to be tested by purification. Soluble fusion proteins can be purified by affinity selection. Insoluble fusion

a protein miniprep (see Section 3.3.1.) prior to a large-scale proteins can only be partially purified. 1. 2. 3. 4.

Grow 5 mL of overnight culture in LB/amp (100 pg/mL at 37°C). Transfer to 500 mL of LB/amp and shake for 2 h at 37°C. Add IPTG to 1 mM and shake for 4 h at 37°C. Centrifuge the cells for 10 mm at SOOOgat 4’C. Resuspend in 10 mL Triton/PBS and transfer to a 50-mL tube

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5. Sonicate for 10 min on ice. The color of the suspensron turns dull gray-brown (see Note 20). 6. Centrifuge for 10 min at 5000g at 4°C 3.4.2.1. SOLUBLE FUSION PROTEINS 1. Add to 400 pL glutathione-Sepharose beads stock 1 mL Triton/PBS, centrifuge for 10 min at 5OOg, remove the supernatant, wash three more times with Triton/ PBS, and resuspend in 400 uL T&on/PBS. 2. Transfer the supernatant from step 6 (Section 3.4.2.) to a new tube and add 400 pL washed glutathione-Sepharose beads. Mix and incubate on a rocking platform overnight at 4°C. 3. Collect the beads by centrifirgation for 10 min at 500g. Spmning at >3000 rpm will destroy the beads! 4. Wash the beads twice with PBS. 5. Resuspend the beads in 400 PL elution buffer and incubate for 15 min at room temperature or for 1 h at 4’C on a rocking platform. 6. Centrifuge for 10 min at 500g and transfer the supernatant to a new tube. 7. Repeat the elution twice with fresh elution buffer and pool the supematants (see Note 21). 8. Analyze 10 pL on a 12% SDS-polyacrylamide gel (see Note 22). 3.4.2.2. INSOLUBLE FUSION PROTEINS 1. Resuspend the pellet from step 6 (Section 3.4.2.) in 10 mL Triton/PBS, and centrifuge for 10 min at 5000g. Repeat this twice. 2. Resuspend the pellet m 20 mL PBS. 3. Analyze 1,3, and 10 pL on a 12% SDS-polyacrylamide gel.

sonicate,

3.4.3. Affinity Purification of pQE Fusion Proteins First test the solubility tion 3.3.2.). 1. 2. 3. 4.

of the fusion protein by protein mmipreps

(see Sec-

Grow 5 mL overnight culture of the pQE-M15[pREP4] clone m LB/amp/kana. Transfer to 500 mL LB/amp/kana and shake for 2 h at 37°C. Add IPTG to 1 mA4and shake for 4 h at 37°C. Centrifuge the cells for 10 min at 5OOOg at 4°C and discard the supematant. At this stage, the pellet can be stored at -20°C.

3.4.3.1. SOLUBLE FUSION PROTEINS 1. Resuspend the pellet of step 4 (Section 3.4.3.) in sonication buffer at 5 mL/g wet wt (ca. 25 mL for a 500~mL culture). 2. Add lysozyme to 1 mg/mL and Incubate on ice for 30 min. 3. Sonicate on ice for 1 min and then leave for 1 min on ice. Monitor the cell breakage by measurmg the decrease of A600nm of a I:200 dilutron. 4. Repeat step 3 until the ..&e does not decrease further (ca. three times). 5. Mix 2 mL vol Ni2+-NTA agarose with 2 mL sonication buffer mix, distribute over Eppendorf tubes, and centrtfuge for 5 s at 10,OOOg. Remove the supernatant,

Prokatyotic Expression

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9. 10.

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wash twice with 2 mL sonication buffer, and resuspend in 2 mL sonication buffer. Centrifuge the lysed culture for 20 min at 10,OOOg at 4°C. Transfer the supernatant to a new tube. Mix the supernatant with the equilibrated Ni-NTA agarose and incubate on a rocking platform for at least 1 h at 4°C. Remove the plunger from a 5 mL syringe and plug the bottom of the syringe with sterile glass wool. Transfer the Ni-NTA/expression culture mix to the syrmge and let the Ni-NTA agarose settle on the glass wool. Wash the resin with 40 mL wash buffer. Elute with a 30-mL gradient of O-O.5M imidazole in wash buffer. Collect 1-mL fractions on a 15% SDS-PAGE (see Notes 23 and 24). Do not boil the samples, but heat for 10 min at 37”C, since acid-labile protein bonds are hydrolyzed by boiling in imidazole (see Note 22).

3.4.3.2. INSOLUBLE

FUSION PROTEINS

1. Resuspend the pellet of step 4 (Section 3.4.3.) in buffer A at 5 mL/g wet wt (ca. 25 mL for a 500-mL culture), and incubate on a rocking platform for 1 h at room temperature. 2. Mix 2 mL Ni2+-NTA agarose with 2 mL buffer A, distribute over Eppendorf tubes, and centrifuge for 5 s at 10,OOOg. Remove the supernatant, wash twice with 2 mL buffer A, and resuspend in 2 mL buffer A. 3. Centrifuge the mixture of step I for 15 min at 4°C at 10,OOOg and collect the supernatant in a fresh tube. 4. Mix the supematant with the equilibrated Ni2+-NTA agarose and mcubate on a rocking platform for at least 1 h at room temperature or overnight at 4°C. 5. Remove the plunger from a 5-mL syringe and plug the bottom of the syringe with sterile glass wool. Transfer the Ni-NTA/expression culture mix to the syringe and let the Ni-NTA agarose settle on the glass wool. 6. Wash the Ni-NTA agarose with 15 mL buffer A. Save the flowthrough. 7. Wash with 15 mL buffer B. Save the flowthrough. 8. Elute with 15 mL buffer C. Save the flowthrough. 9. Elute with 15 mL buffer D. Save the flowthrough. 10. Elute with 15 mL buffer E. Save the flowthrough. 11. Empty the column by washing Ni-NTA agarose with 15 mL buffer F. Save the flowthrough. 12. Store the fractions at 4°C. Freezing will precipitate the urea. 13. Test 10 pL of the fractions on an SDS-polyacrylamide gel. To prevent precipitation of guanidinium hydrochloride in fractions A and F, carry out a TCA precipitation: Add to 10 pL of sample 90 uL PBS and 100 uL 10% trichloroacetic acid, leave for 20 min on ice, centrifuge for 15 min at 10,OOOgand at 4”C, remove the supematant, wash the pellet with 100 yL ethanol (-20’(Z), dry the pellet, and suspend in Laemmli gel loading buffer (see Note 25). Figure 6 shows the elution of a pQE fusion protein by buffer C.

Lenstra and Van Wet

Fig. 6. Affinity purification of a pQE fusion protein containing residues47-152 from MAP-l of C. ruminantium (16).

4. Notes 1. Colonies carrying inserts cannotbe identified via blue-white screening.Always include a control in which the ligation has been carried out without insert; this should give fewer colonies than the ligations with insert. 2. E. coli MlS[pREP4] is preferred for cloning of pQE constructsif the positive recombinants can be selectedvia expression,e.g., by immunoscreeningor by protein minipreps. However, if the pQE constructshaveto be verified at the DNA level, e.g., by restriction-enzyme cleavageor sequencing,the pREP4 plasmid may interfere. In this case,first clone in a normal host like DH5a or JM109, and transfer to MlS[pREP4] after verifying the construct. 3. Occasionally transformantsthat look perfect at the DNA level fail to produce a fusion protein of the correct size. Therefore, we prefer to screen the initial transformantsby expression,i.e., by immunoscreeningof colonies (see Section 3.2.) or by protein minipreps. 4. If the platesaretoo wet or too dry, the colonieswill not be completely transferred to the filter. 5. Throughoutthe whole procedure,usea blunt-endedforcepsandwear cleangloves to handle the NC filters. 6. For screeningpEX libraries, an oven with a fan is recommendedto warm up the platesquickly andevenly,resultingin the samelevel of expressionfor all colonies. 7. The optimal dilution of the antibody dependson the antibody titer. Typical dilutions are the same as for Western blotting: 1:500 to 1:10,000 with polyclonal sera;with MAbs 1:500 for ascitesfluid and 150 for culture supernatants.There is no needto preabsorbMAbs with the E. coli extract.

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8. The diluted antiserum can be stored for a few days at 4°C and used for another screening. 9. This procedure may also be combined with peroxidase conjugates and other stainmg methods (15). However, the alkaline-phosphatase staining has a low background and is relatively stable. 10. The same filters may be screened a second time after removing all Ponceau stain. However, the positives of the first screening keep their color. If filters are to be screened more than one time, use the most pure antiserum first. 11. For Western blots, the same incubation and staining procedure can be used. 12. As positive control of the immunoscreening, pEX colonies can be screened with monoclonal anti@-galactosidase) (Promega, Madison, WI). pGEX colonies can be screened with rabbit anti(glutathione S-transferase) (we raised an antiserum against the recombinant glutathione-S-transferase in rabbits). pQE colonies can be screened with an Ni-NTA-alkaline phosphatase conjugate (Qiagen) or with an MAb against the MRGSH6 sequence (Fig. 4C, Qiagen) and a conjugated second antibody. 13. Always include a clone without insert to check the increase in size by the expression of the insert. 14. A high concentration of 5% SDS is needed to solubilize the pEX fusion protein. 15. We never failed to obtain expression in pEX, but occasionally pGEX and pQE products were degraded intracellularly. 16. For the detection of low amounts of pGEX or pQE fusion proteins, use the specitic antisera or conjugates (see Note 12) on Western blots. 17. Pellets of pEX products are difficult to resuspend, especially if the procedure has been scaled up or if the bacteria have been spun down too hard (10,OOOg). Optionally, include a DNase treatment. 18. The sucrose buffer as well as the lysozyme digestion contribute to the purity of the pEX fusion protein. 19. pEX samples are degraded in Laemmli buffer and should be stored as pellet resuspended in PBS. 20. Excessive sonication of pGEX clones may lead to a contamination of the pGEX product with other proteins. Alternatively, monitor the cell lysis via the A,,, (see Section 3.4.3.1.) step 3). 21. Glutathione-Sepharose beads can be reused: Incubate the beads with PBS supplemented with 3M NaCl for 10 min at room temperature; wash three times with PBS and store in PBS with 20% EtOH. However, only reuse with the same antigen. 22. ELISA plates may be coated with soluble pQE or pGEX proteins dissolved in 15 mA4Na2C03, 35 mMNaHCOs, pH 9.6, at 0.5-l yg/mL by incubation for 1 h at 37’C and overnight at 4“C (17). 23. The elution of pQE fusion proteins depends on the pH and on the insert. Therefore, check all flowthroughs on an SDS-polyacrylamide gel. 24. pQE fusion proteins can show an aberrant molecular weight (ca. 5 kDa or larger) on SDS-polyacrylamide gel. 25. Urea used for the elution of insoluble pQE proteins reacts at >37’C with amino groups on the protein. This may affect the antigenicity.

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Acknowledgments The pEX procedures as well as Fig. 5 are partly from K. K. Stanley. The pQE procedures (9) have been adapted from the text of J. Crow and K. Hence (Qiagen Inc, Diagen GmbH). We thank C. E. d’oliveira (Utrecht), A. J. A. M. van Asten (Utrecht), and J. Ribbe (Qiagen) for useful discussions. References 1. Van Regenmortel, M. H. V. (1989) Antigenic cross-reactivity between proteins and peptides: new insights and applications. Trends Biochem. Sci. 12,237-240. 2. Lenstra, J. A., Kusters, J. G., and Van der Zeijst, B. A. M. (1990) Mapping of viral epitopes with prokaryotic expression systems (review). Arch. Viral 110, l-24 3. Nuijten, P. J., van der Zeijst, B. A. M , and Newell, D. G. (1991) Localization of tmmunogenic regions on the flagellin proteins of Cumpylobacter jejuni 8 1116 Injkt. Immun. 59, 1100-l 105. 4. Van Vliet, A. H. M., JongeJan, F., van Kleef, M., and van der Zeijst, B. A. M (1994) Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium. Infect Immun. 62, 1451-1456. 5. Stanley, K. K. and Luzio, J. P. (1984) Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding from human liver proteins. EMBO J. 3, 1429-1434. 6. Kusters, J. G., Jager, E. J., and Van der Zeijst, B. A. M. (1989) Improvement of the cloning linker of the bacterial expression vector pEX Nucleic Aczds Res 17, 8007.

7. Smith, D. B. and Johnson, K S. (1988) Single-step purification of polypeptides expressed in Escherlchia coli. Gene 29,263-269. 8. The world of Pharmacia Biotech ‘95-96, catalogue of Pharmacia Biotech. 9. Qtaexpress booklet (1992) of Diagen GmbH, Qiagen Inc. 10. Kusters, J. G., Jager, E. J., Lenstra, J. A., Koch., G., Posthumus, W. P. A., Meloen, R. H., and Van der Zeijst, B. A. M. (1989) Analysis of an mnnunodommant region in the peplomer protein of avian coronavirus infectious bronchitis virus. J. Immunol. 143,2692-2698. 11. Lenstra, J. A., Kusters, J. G., Koch, G., and Van der Zeijst, B. A. M. (1988) Antigenicity of the peplomer protein of infectious bronchitis virus. Mel Immunol. 26,7-l 5. 12. Lenstra, J. A., Erkens, J. H. F., Langeveld, J. P. M., Posthumus, W. P. A., Meloen, R. H., Gebauer, F., Correa, I., Enjuanes, L., and Stanley, K. K. (1992) Isolation of sequences from a random-sequence expression library that mimic viral epitopes. J. Immunol. Methods 152, 149-157. 13. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spnng Harbor Laboratory, Cold Spring Harbor, NY. 14. Stanley, K. K. (1983) Solubilization and immune-detection of /3-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Res 11, 4077-4092.

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15. Stanley, K. K. (1988). Expression screemng of cDNA libraries in pEX. Methods Mol. Blol. 4,329-342.

16. Van Vliet, A. H. M., Van der Zeijst, B. A. M., Camus, E., Mahan, S. M., Martinez, D., and Jongejan, F. (1995) Use of a specific immunogenic region on the Cow&% ruminantium MAP1 protein in a serological assay. J. CIzn Mxrobiol. 33, 2405-2410.

17. Costa, G. L., Grafsky, A., and Weiner, M. P. (1994) Cloning and analysis of PCRgenerated DNA fragments. PCR Methods Applic. 3,338-345.

26 Epitope Mapping of Proteh Antigens by Expression-PCR (E-PCR) David E. Lanar, Kevin C. Kain, and Henry B. Burch 1. Introduction It is often the casethat one has a cloned gene for a protein and a monoclonal antibody (MAb) that recognizes an epitope within that protein. Determination of the amino acid sequences constituting an epitope recognized by the MAb can be problematic. Producing ever and ever smaller fragments of the protein in vivo using expression systems, such as bacteria, yeast, or baculovirus, leads to problems of having the expression system influence the protein, i.e., correct folding, glycosylation, and degradation. In some cases,the foreign protein may in fact be toxic to the cell, thereby making synthesis impossible. In addition, each new fragment in the study has to be constructed, usually by PCR from the gene, cloned, and then sequenced to check for fidelity against the parent sequence to avoid PCR errors. To detect the newly expressed protein by immunoprecipitation, radiolabeled amino acids are usually incorporated into the new product, but at the same time this label goes into all other host proteins. This can be further complicated if MAb crossreactivity or coprecipitation occurs. Most of these problems can be avoided by epitope mapping using proteins synthesized by expression-PCR (E-PCR) (1). E-PCR is a rapid and simple method for the in vitro production of proteins without having to go through the rigors of cloning. The resulting radiochemically pure proteins are useful for a variety of purposes that include studies on the subunit structure of proteins, epitope mapping, and protein mutagenesis. E-PCR draws its power from that of PCR. Gene segments can be rapidly designed and synthesized requiring only specific oligonucleotides and PCR reagents. The gene segments then are linked to a universal promoter (Fig. I), which allows in vitro synthesis of translatable mRNA. New constructs can be From. Methods m Molecular Btofogy, vol 66’ Epltope Mapprng Protocols Edlted by. G E. Morris Humana Press Inc , Totowa, NJ

30s

UP-l: 5’ CCAAGC’ITCTAATACGACTCACTATAGGG~ATITITAA~~C UP-Z: 5’ CAGTGCCATGGTGGAG 3’ SINGLE

52

STRANDED

UNIVERSAL

PROMOTER

3’

:

5’ CCAAGCITCTAATACGAflCA~ATAGGG~A~AA~~CAAATAWCCACC T7 promoter (UTL from AIW)

ATG GCA ClJG 3

KS

Met Ala Leu

0

H3T7 PRIMER

5’ CCAAGCTTCTAATACGACTCACTATAGGG Hind IlI Site T’7 promoter

3’

Fig. 1. The sequence of the single-stranded Universal Promoter used m E-PCR and its 5’-specific H3T7 primer.

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made by making a new 3’-primer or using restriction enzymes to shorten the PCR product before transcription (see Fig. 2). The mRNA is translated using rabbit reticulocyte or wheat germ in vitro translation systems, avoiding the problems of cloning and host-cell expression. Once oligonucleotide primers are made, the process from amplification to protein production can be accomplished in <8 h. Ample radiolabeled protein products can be produced in this fashion, spanning distinct and defined amino acid residues, which can then be used to epitope map by immunoprecipitation as soon as the next day. Using this technique, epitopes have been mapped to as few as 10 amino acids in a myelin-associated glycoprotein (MAG) using an MAb (2) and a 52 amino acid region of human thyrotropin receptor (hTSH-R) using human polyclonal antisera from individuals with autoimmune thyroid disease (3). A discontmuous, disulfide-dependent epitope from a malaria surface protein, which could not be produced in a conformationally correct form in a bacterial expression system, was correctly generated by E-PCR and mapped using an MA\, (4). Additionally, we have made a small ligand (40 amino acids) by E-PCR of a malaria receptor and then immunized mice with this in vitro synthesized protein. The induced antibodies were used to show that the conformation of the epitope was reproduced by a second dimorphic allele encoding different amino acids in the same gene of another malaria clone (5). The key to E-PCR was the design of a small DNA cassettethat had all the functional regions needed by RNA polymerase in order to initiate transcription of a downstream DNA segment into an RNA molecule that could be translated into protein. Four functional regions were needed for this upstream segment (see Fig. 1): 1. 2. 3. 4.

An RNA polymerasebinding region. An untranslatedleader sequence. A Kozak sequence. A translation initiation codon.

We incorporated these four regions into a single unit, which we named the Universal Promoter (UP), because it permits the transcription of any DNA segment spliced to it. PCR primers can be used to amplify the DNA segment of interest, and many computer programs now exist that help select optimum primer pairs (e.g., Oligo 5.0, NBI, Plymouth, MA). The only requirement is that the forward or splicing primer has added to its 5’-end the last 9 bases (ATGGCACTG) of the UP with the first codon (ATG) positioned in the desired open reading frame (ORF). The 73-base UP can be made from two oligonuclotides, UP1 and UP2, or as a single

Lanar, Kain, and Butch

312

Make Universal Promoter

Make Gene Fragments with Spluang Primer by PCR

Link UP wdh Gene Fragments by SOE

III

UP43-

Synthesize mRNA with 17 Polymerase Iv*

NJG AC

AD AJZ AF

NJ’ NJG NJ0 NJUG

WA., A A

Fig. 2. Principles of E-PCR and its use for epitope mapping using immunoprecipitation. See text for details.

313

Expression-PCR

In vitro translation with 3JS-methionine *

Immunoppt with mAbv

0

PAGE Analysis

Fig. 2. (continued)

314

Lanar, Kain, and Burch

positive strand (SS-UP) (Fig. 1). The 29-nucleotide H3T7 primer 1smade as a single-stranded oligonucleotide. All reverse or antisense primers are selected in order to make successively longer or shorter gene fragments when combined with the forward splicing primer in the initial PCR reaction (Fig. 2, step II). All are made on a standard oligonucleotide synthesizer with purification of full-length products either by acrylamide gel electrophoresis and elution out of the gel or by using the “trityl on” step in the last synthesis cycle on the automated synthesizer (394 DNA Synthesizer, Perkin-Elmer Cetus, Norwalk, CT) and then oligopurification cartridge (OPC) column purification. 2. Materials 1. Primers: UPl, UP2, H3T7, forward and reverse gene-specific primers (Fig. 1). The forward primer has the sequence ATGGCACTG added to its 5’-end, making it the splicing primer with the UP. 2. PCR reagents: deoxynucleotides, buffer, Tuq polymerase, MgC&. 3. Low-melting-point agarose: Seaplaque (FMC Bioproducts, Rockland, ME). 4. T7-based in vitro transcription kit (Megascript, Ambion, Inc. Austin, TX, cat. #1334). 5. In vitro translation kit: rabbrt retlculocyte or wheat germ-based (Retie Lysate IVT Kit, Ambion, Inc., TX cat. #1200; Wheat Germ In Vitro Translation Kit, Promega, Madison, WI). 6. 35S-Methionine or 3H-Leu amino acids. 7. Protein A Sepharose 6MB (Pharmacia, Uppsala, Sweden). 8. Tris-buffered saline (TBS): 25 mM Tris-HCl, pH 7.4, 0.8N NaCl and TBS containing 1.O% Triton X- 100 (TBS-Tnton). 9. TCA precipitation reagents: 10% TCA, glass fiber filters, scintillation counting fluid, scintillation counter. 10. PAGE analysis reagents: 2X SDS sample buffer, acrylamide, bls-acrylamlde, ammonium persulfate (or precast SDS-PAGE gels [Novex, San Diego, CA]), Tris-borate buffer, gel plates, gel running tank, ENLIGHTNING (New England Nuclear-DuPont, Boston, MA), gel dryer, autoradiographic film.

3. Method If oligonucleotides are available, all radiolabeled proteins can be made in 1 d and immunoprecipitation analysis done the next. The technique is outlined in Fig. 2.

3.7. Sfage I: Synthesis of Universal Promoter from UP 1 and UP2 1. Mix primers UP1 and UP2 (100 pmol each) together in a volume of 100 pL with 2.5 U of Taq polymerase, dNTPs at 1.25 mM, and the 10X buffer supplied by the manufacturer. 2. Perform PCR amplification for 25 cycles (each consisting of 1 min at 94OC, 1 min at 3O”C, and 1 min at 72°C). 3. Electrophorese reaction products on 2% low-melting-point NuSieve agarose.

Expression- PCR

315

4. Excise the DNA band and store at 4°C. The DNA can be used directly from the low-melting-point agarose or purified with a DNA isolation system, such as GlassMAX (Gibco-BRL, Gaithersburg, MD) (see Note 1).

3.2. Stage II: PCR of Desired Fragments of Gene 1. Perform PCR on the gene of interest using the forward primer in combination with different reverse primers to make successively smaller gene fragments. The forward or splicing primer must have added to its S-end the last 9 bases (ATGGCACTG) of the UP with the first codon (ATG) positioned in the desired ORF. Because the forward primer is the only one that is unique to this technique, reverse primers that were made for other purposes, such as cloning or sequencing, may be used. 2. Electrophorese reaction products on 2% low-melting-point NuSieve agarose. 3. Excise the DNA band and store at 4’C. The DNA can be use directly from the low-melting-point agarose (see Note 1). Restriction enzymes can be used to shorten PCR products to generate further fragments (see Note 2). 4. A parallel series of PCR reactions can be set up to narrow down the epitope site (see Note 3).

3.3. Stage Ill: Spllclng Gene Products to the UP Using a Two-Step PCR 1. Mix approx 100 ng of the gene segment with approx 30 fmol of UP and link them together by splicing by overlap extension (SOE), essentially a PCR reaction without primers (see Note 4). 2. In the first step, mix the UP and specific gene fragments and perform PCR for 15 cycles without the addition of any forward or reverse primers. 3. In the second step, add a 3’-gene-specific reverse primer along with a forward primer specific to the UP (called H3T7 because it consists of the bases coding for the 5’-end of the UP where the Hind111 site and T7 promoter sequence lie). Add additional Taq polymerase and continue the PCR for another 20 cycles. 4. Extract the UP-linked gene produced with chloroform and precipitate, 5. Rinse with 80% EtOH and vacuum dry for 2 min. 6. Resuspend the DNA in 10 pL RNase-free water at a final concentration of about 100 ng/pL.

3.4. Stage IV: RNA Transcrlptlon 1. Using about 100 ng of secondary PCR product from Stage III, synthesize RNA using T7 polymerase in commercially supplied transcription kits. 2. The amount of RNA being made can be determined by incorporating [WEEP]UTP. Alternatively after the reaction is finished, mix 1 pL of the reaction with 1 yL of RNA gel-loading buffer and tun on a 1.2% agarose gel in an RNase-free apparatus (see Note 5). A sharp band should be obtained. 3. Compare RNA products from the different successive PCR reactions on the same gel; the difference in length of the RNA product should parallel the difference in DNA product lengths.

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3.5. Stage V: Protein Translation 1. Translate 1 yL of each individual RNA reaction into protein in 50 pL of cell-free translation mixture (see Note 6). During the synthesis, 35S-Met is incorporated into the first Met in the protein (the first AUG supplied in the splicing linker, step II) as well as any other Met in the gene. Alternatively, 3H-Leu can be used. The third codon in the splicing linker codes for Leu. 2. Analyze the production of protein by standard TCA precipitation of labeled protem or analysis of 5 pL of the reaction on a polyacrylamide gel.

3.6. Stage VI: lmmunoprecipttation

of Products

with Antibody

1. Mix 10 pL (100,000 cpm) of each translation reaction from step V with 2 pL of the antibody selected for epitope mapping in a final volume of 50 pL of TBS, and react for 1 h at room temperature (see Note 7). 2. Add 5 pL of protein A-Sepharose beads and react for a further 30 min at room temperature. 3. Rinse the beads two times with TBS-Triton by centrifugation.

3.7. Stage VII: PAGE Analysis 1. Resuspend the protein A-Sepharose beads in 10 pL 2X SDS sample buffer and boil for 5 min. 2. Centrifuge and load 5 uL on an SDS-PAGE gel for analysis. 3. After electrophoresis, fix the gel and enhance the radioactive protein signal with emersion in a fluorochrome, such as ENLIGHTNING (New England NuclearDuPont, Boston, MA). 4. Dry and expose to X-ray film.

4. Notes 1. If a plug of agarose is removed from the gel, soak it in 5 mL of 1X TE for 10 mm. Remove excess TE before melting the sample. This reduces the high EDTA from the gel running buffer, which could inhibit further cation-dependent reactions using this sample. 2. If unique restriction sites are located between the two primers, a portion of the PCR product can be digested after step III, but before transcription to yield an additional defined polypeptide after translation (see ref. 2). 3. It IS important to obtain a clean PCR band on this step. This may require the testing of several different thermophilic polymerase enzymes and/or buffer conditions. One can use a PCR Optimizer Kit (Invitrogen, San Diego, CA) if initial attempts do not yield acceptable results. If only a few extra bands are observed on gel analysis, use low-melting-point agarose to separate and excise the DNA band of interest. The DNA can be used directly from low-meltmgpoint agarose (see Note 1) or purified. 4. This has been determined experimentally, but in practice, about 2 yL of melted gel from step I and 2 pL of melted gel from step II are mixed in a new PCR

Expression-PCR

317

reaction, The low-melting-point agarose 1smelted at 65°C and then the tube transferred to 42O C. To aid m pipeting, a micropipet tip is cut off at about 2 mm with a razor blade to open the bore at the bottom. 5. Most gel rigs are contaminated with RNase from analysis of miniprep DNA procedures, which use RNase. A dedicated gel rig in a separate room is advised. If RNase is in the gel rig, the RNA will be digested as it runs, resulting in a smear or complete disappearance of the RNA. 6. It is absolutely imperative to titrate the potassium ion concentration for each different RNA. Sometimes a change of K+ ion by as little as 2 mm01 can change the production of protein from none, or low yield, to an abundant product. This has to do with the ability of the translation cocktail ribosomes to translate capped and uncapped mRNA. Either rabbit reticulocyte or wheat germ systems can be used. One usually works better than another, but this needs to be determined empirically. There are now linked transcription-translation systems that allow steps IV and V to be performed in a single tube (TNT Kit, Promega Corp., Madison, WI). 7. The volumes of translation reaction and antibody solution used depend on the specific activity of the translation reaction and the titer of the antibody. The volumes given here are a good starting point and should indicate whether the protein synthesized contams the desired epitopes. Adjustments of the volumes of reactants are needed for optimal results.

References 1. Kain, K. C., Orlandi, P. A., and Lanar, D. E. (1991) Universal promoter for gene expression without cloning: expression-PCR Biotechniques 10,366-374 2. Tropak, M. B. and Roder, J. C. (1994) High-resolution mapping of GENS3 and Bl IF7 epitopes on myelin-associated glycoprotein by expression PCR. J Neurochem

62,854-862.

3. Burch, H. B., Nagy, E. V., Kain, K. C., Lanar, D. E., Carr, F. E., Wartofsky, L., and Burman, K. D. (1993) Expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen: application to the human thyrotropin receptor. J. Immunol. Methods 158, 123-130. 4. Farley, P. J. and Long, C. A. (1995) Plasmodium yoelii 17XL MSPl-fine-specilicity mapping of a discontinuous, disultide-dependent epitope recognized by a protective monoclonal antibody using expression PCR (EPCR). Exp. Parasitol. 80,328-332.

5. Kain, K. C., Orlandi, P. A., Haynes, J. D., Sim, B. K. L., and Lanar, D. E. (1993) Evidence for two stage binding by the 175&D erythrocyte binding antigen of Plasmodiumfalclparum. J. Exp. Med. 178, 1497-1505.

27 Epitope Mapping on Extracellular Domains of Cell-Surface Proteins Using Exonuclease Ill Thomas Briimmendorf,

Antonius Plagge, and Ullrich Treubert

1, Introduction The knowledge of the distribution of ligand binding sites on extracellular domains of cell-surface molecules is important both in basic research and in the context of drug development. Mapping of binding sites of pathogens on the cell-surface molecules that they use for entry into the cell may be a first step toward the design of therapeutics. For example, the binding site of HIV on T-cells has been mapped to the first immunoglobulin-like domain of the CD4 molecule (id), the binding site of Plasmodium falciparum-infected erythrocytes on venular endotheliurn resides in the first immunoglobulin-like domain of ICAM- 1 (7,8), and the binding site of rhinovirus has been localized in the same domain of this molecule (9-Z I). Many of the interactions that have been characterized at the submolecular level in recent years have been studied by analyzing the binding behavior of truncated molecules expressed either in bacteria or by eukaryotic cell transfection. The transfection of eukaryotic cells with cDNAs encoding cell-surface molecules has been widely applied for the identification of ligands of such molecules and for the characterization of their binding properties. If the transfected cDNAs contain the complete open reading frame of a molecule comprising its signal peptide as well as either a transmembrane/cytoplasmic sequence or a carboxy-terminal glycosyl-phosphatidylinositol (GPI)-attachment signal, the expressed protein is usually targeted to the surface of the cells. One eukaryotic cell line that is well suited for such studies is the COS cell line, a derivative of African Green Monkey kidney cells. COS cells constitutively express the large T-antigen of the papova virus SV40, encoded by a fragment of the virus DNA that is integrated in the genome of the cells (12). From: Methods In Molecular Biology, vol. 66. Epltope Mapping Protocols Edlted by G E Morris Humana Press Inc., Totowa, NJ

319

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Briimmendorf, Plagge, and Treubert

COS cells can be transfected with plasmids that contain the SV40 origin of replication and an inserted cDNA cloned downstream of the SV40 early promoter. If such plasmids are introduced into the cells, the large T-antigen causes plasmid replication to a copy number on the order of lo5 molecules/cell (23). This high copy number and the SV40 early promoter lead to the accumulation of a comparatively high level of specific mRNA, which m turn causes translation of large amounts of specific protein. Because the cloned cDNA can be manipulated by means of recombinant DNA technology, it is possible to generate truncated mutants of the corresponding protein (deletion mutants). These can be expressed on the surface of the transfected cells and monoclonal antibody (MAb) epitopes (or ligand bindmg sites) can be mapped on the protein by comparing the binding behavior of multiple different mutants. In the present chapter we describe the new plasmid vector pDELF- 1, which was designed to map epttopes of MAbs or ligand binding sites on cell-surface molecules as well as on extracellular matrix molecules (Fig. 1). The plasmid pDELF- 1 allows the cell surface expression of proteins in transfected COS cells and 1s suited for the generation of deletion mutants of the protems (Fig. 2). Clones with inserts that are progressively shortened m a predetermined duection can be generated using exonuclease III. This enzyme is widely applied for the generation of unidirecttonal deletions in the context of DNA sequencing projects (14,15), and we have used it previously to map ligand binding sites on the multifunctional neural cell recognition molecule F11 (16). Progressive deletions of the cDNA insert in pDELF-1 can start either near the amino-terminus (N + C-mutants)or nearthe carboxy-terminus(C + N-mutants) of the encoded polypeptide. The polylinker m pDELF- 1 is followed by a genomic DNA fragment, which is coding for the CH2 and CH3 domains of human IgGl (Fig, 1). These domains, which have been widely applied as molecular markers (17), are fused to the carboxy-terminal end of the polypeptide encoded by the insert and can therefore be used as a positive control tag.

1.1. Brief Outline of the Protocol 1, The cDNA of interest is directionally subclonedinto the pDELF-1 vector (Fig. 1). To facilitate subcloning, appropriate restriction enzymecleavage sites are added to the cDNA via PCR amplification. The construct is checked using COS cell transfection and immunofluorescenceanalysis. The followmg steps are performed twice, once for the generation of a set of N + C-mutants and once for the generation of a set of C + N-mutants (Fig. 2). 2. The pDELF- 1 vector with the cDNA inser?is digestedsimultaneouslywith two restriction enzymes,one that generatesa 5’-overhang and one that generatesa 3’ overhang. 3. Progressive deletions of the cDNA insert are generatedusing exonucleaseIII, which degradesat the 5’-overhang,and a setof plasmid sizefractions is prepared.

Exonuclease IN Deletion Mutagenesis

327

pDELF-1

Fig. 1. Map of plasmid pDELF- 1. Plasmid pDELF-I is a derivative of plasmid pSG5, which contains the SV40 early promoter/origin of replication (39) and is therefore replicated in COS cells (12,13). The pDELF-I plasmid carries the signal peptide of the neural cell recogmtion molecule Fl 1 (black area), a multiple cloning site, and the CH2 and CH3 domains of human IgGl (43) followed by the GPI anchor attachment signal of the neural cell surface molecule Fl 1 (44), which is depicted as a black arrow. Numbering begins at the EcoRI site of the parental pSG5 plasmid. An insert that is cloned in pDELF- 1 can be progressively deleted by exonuclease III m a predetermined direction. As long as the deletion construct retains the sequences coding for the signal peptide and the GPI anchor attachment signal, which are provided by pDELF-1, the polypeptides they encode are attached to the surface of the COS cells and can be probed by MAb (or analyzed in ligand-binding assays). The two human IgGl domains serve as a positive control, since they can be easily detected using antibodies specific for human IgG, which are commercially available.

4. Each size fraction contains a population of shortened plasmids of similar lengths. The population of plasmids is analyzed as a pool with respect to MAb binding (or examined in ligand binding assays). 5. A crude map is assembled by size determination in agarose gels. 6. In-frame clones are identified in selected plasmid populations and are analyzed with respect to MAb binding (or examined in ligand binding assays). 7. A fine map is assembled by sequence analysis of characterized clones.

Briimmendorf, Plagge, and Treubert

322

linearize plasmid

t

exonuclease III / mung bean nuclease digestion

t

agarose gel

F

isolate size fraction recircularize plasmids, transform bacteria isolate DNA, transfect

COS cells

analyze COS cell transfectants anti human kG

MAb

.

A

A

e

analysis of plasmid pools

-

A A A

\1 single clone analysis

Fig. 2. Mapping of MAb epitopesusing exonucleaseIII-based deletion libraries. The plasmid pDELF- 1 containingthe cDNA under study is linearized in the polylinker region with appropriaterestriction enzymes.The linearizedplasmid is unidirectionally digested using exonucleaseIII, and size fractions are isolated in an agarose gel. COS cells are transfected with the recircularized plasmids and the polypeptides expressedon their surface are analyzedwith respectto MAb binding. Each deletion mutant containstwo immunoglobulin-like domainsof humanIgGl asa tag that canbe used as a positive control.

2. Materials 2.7. Subcloning info the pDEl.F-7 Vector 2.1.1. PCR Amplification of the DNA Fragment to Be Subcloned 1. RCRbtii (10X): 180rniWKl, 3 mMMgQ, 180mM Tris-HCl,pH 8.75-9.0,80mM (NH&S04, 16mM MgS04, 0.8% Triton X-100,0.8 mg/mL bovine serumalbumin (BSA).The~issterilizedby~~~(O2~)andstoredinsmallaliquotsat-2OoC.

Exonuclease III Deletion Mutagenesis

323

2. dNTP set (Pharmacia, Uppsala, Sweden; ultrapure dNTP kit). 3. Forward and reverse PCR primers. 4. Nujol Mineral oil (Perkin Elmer, Norwalk, CT) or Chill-Out 14TM liquid wax (MJ Research, Watertown, MA). 5. Enzyme-mix: 1 pL Pfu DNA polymerase (2.5 U/pL, Stratagene, La Jolla, CA) + 15 pL Taq DNA polymerase (5 U/PL, Pharmacia). 6. Thin-walled reaction tubes (Perkin Elmer). 7. Sterile-filter pipet tips to avoid contaminations by aerosols. 8. Thermal cycler.

2.1.2. Subcloning into Plasmid pDELF- I 9. The plasmid pDELF- 1 (see Note 1) is available from the authors on request (email: [email protected]). 10. Commonly used restriction enzymes can be obtained from most commercial suppliers (18’. Less common enzymes are supplied as follows: AgeI, AscI, AvrII, BspEI, BsrGI, and FseI by New England Biolabs (Beverly, MA, e-mail: [email protected]); SspBI and BseAl by Boehringer-Mannheim (Mannheim, Germany); Sun1 and NgoAIV by Gibco-BRL (Gaithersburg, MD); Bspl31, PspLl, and SbfI by SibEnzyme (Novosibirsk, Russia, e-mail: [email protected]); PspAI: Stratagene; BsiMI by Amersham Life Science (Arlington Heights, IL); Cfr91 by Fermentas (Vilnius, Lithuania) and Toyobo (New York, NY); PlnAI by Boehringer-Marmheim and Gibco-BRL; NgoMI by New England Biolabs and Promega (Madison, WI); FseI, Spll, and Sse83871 by Amersham and Takara Shuzo (Kyoto, Japan); Pfl2311 by Fermentas and AGS (Heidelberg, Germany); Asp7181 by Boehringer-Mannheim and Pharmacia; and Mm1 by BoehringerMannheim and Toyobo. 11. Examples of reagents for DNA isolation from agarose gels (or after enzymattc treatments) are the GeneClean reagents (BiolOl, La Jolla, CA) or the QiaexTM reagents (Qiagen, Chatsworth, CA). 12. Examples of reagents for plasmid isolation from bacterial lysates are the Qiagen Plasmid Kits (Qiagen) or the PurePrepa Kit (Pharmacia).

2.2. Preparation of a Deletion Library 13. T4 DNA ligase, exonuclease III, mung bean nuclease. 14. T4 DNA ligase buffer (5X): 330 mM Tris-HCl, pH 7.5, 25 n&f MgC12, 5 r&f DTE, 5 mM ATP. 15. Exonuclease III reaction buffer (2X): 120 mMTris-HCl, pH 7.6,6 mMMgC&. 16. Mung bean-nuclease reaction buffer (10X): 300 mA4Na-acetate, pH 5.0,500 mA4 NaCl, 10 mM ZnC&, 30% glycerol. Mung bean nuclease dilution buffer (IX): Add one part of mung bean nuclease reaction buffer (10X stock solution) to nine parts Triton X- 100,O.O 1% (v/v) in water. Mung bean nuclease stop solution (10X): 500 mMTris-HCl, pH 9.0,30 mMEDTA, and 0.1% SDS. 17. Standard equipment for agarose gel electrophoresis. 18. Reagents for DNA isolation from agarose gels (see point 11).

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2.3. Assembly of the Epitope Map 19. Standard equipment for agarose gel electrophoresis. 20. Standard equipment for DNA sequencing. A primer specific for the T7 promoter to sequence N + C mutants. Sequencing plvners AP90 (S-GGGTTTTGGGGGGAAGAG-3’) or AP91 (5’-CCACCACGCATGTGACCT-3’) for the C + N-mutants. The primers AP90 (which worked well m our hands) and AP9 1 have been chosen using the Oligo-program of National Biosciences (Plymouth, MN).

2.4. MA b Binding Analysis 2.4. I. Cell Culture Techniques 2 1. COS cells can be obtained from the American Type Culture Collection (ATCC, Rockville, MD; see Note 2). 22. DMEM/FCS.

23. 24. 25.

26. 27. 28.

29. 30.

Dulbecco’s

Modified Eagle’s Medium (DMEM,

Gibco-BRL)

con-

taming 2% fetal calf serum (FCS); see Note 3. Trypsin/EDTA: 0.05% trypsin, 0.02% EDTA, tissue culture grade (ICN, Costa Mesa, CA). Dulbecco’s PBS (Gibco-BRL). Tissue-culture-grade Petri dishes with g-cm diameter (Greiner, Frickenhausen, Germany), tissue-culture-grade six-well plates (wells with lo-cm* area, COSTAR, Cambridge, MA), and tissue-culture-grade 96-well plates (COSTAR). Chloroquine diphosphate (Sigma [St. Louis, MO] C6628), 100 mM, sterilized by filtration (0.2 pm), and store at -20°C. DEAE-dextran (Pharmacia; mol wt 500,000), 50 mg/mL in water, sterilize by filtration (0.2 pm) or by autoclaving, and store at 4°C up to 1 yr. Chloroquine/DEAE-dextran mix (50X): Mix 40 parts DEAE-dextran (50 mg/mL) and 1 part Chloroquine diphosphate (100 mM). Store at -20°C or at 4°C in the dark for up to 3 mo. Poly-L-lysine hydrobromide (Sigma P1399), 100 pg/mL, sterilized by filtration (0.2 pm). Eight-well Multitest glass slides, diameter of the well 6 mm (ICN). Rinse in 0.1% SDS and then thoroughly m tap water followed by tissue-culture-grade water. The slides are sterilized by autoclaving.

2.4.2. immunofiuorescence Analysis and Detection of Transfectants Using Alkaline Phosphatase 31. PBSCM: Dulbecco’s PBS, 0.02% (w/v) BSA (Sigma A2153), 0.1% NaNs. PBSCMBSA: Dulbecco’s PBS, 1% (w/v) BSA, 0.1% NaN,. TBS/BSA: 20 miU Tris-HCI, pH 7.4, 2O”C, 150 mil4NaC1, 1% (w/v) BSA, 0.1% NaNs. Clear by filtration through 0.2~pm filters and store at 4°C up to 3 mo. 32. MAbs to be tested. 33. Cy3TM- conjugated goat antibodies directed to human IgG, Fc y fragment specific (Jackson Immuno Research, West Grove, PA). For MAbs of the IgG class;

Exonuclease 111Deletion Mutagenesis

34.

35. 36.

37. 38.

325

Cy3TM-conjugated goat antibodies directed to mouse IgG, H + L chain specific (Jackson, see Note 33) AP-conjugated goat antibodies directed to human IgG, Fc y fragment specific (Jackson), For MAbs of the IgG class, AP-conjugated goat antibodies directed to mouse IgG, H + L chain specific (Jackson). Alkaline phosphatase buffer: 100 mM Tris-HCl, pH 9.5, 2O”C, 100 mA4 NaCI, 5 mA4 MgCl,. 5-bromo-4-chloro-3-mdolyl phosphate, p-toluidine salt (BCIP), 50 mg/mL in dimethylformamide, stored frozen in aliquots at -20°C. Nitro blue tetrazolium (NBT), 50 mg/mL in 70% dimethylformamide, stored frozen in aliquots at -20°C. Kaiser’s Glyzerin Gelatin (Merck, Darmstadt, Germany). Glycerol gelatin is stored as lo-mL aliquots in 50-mL tubes. Melting can be done in a microwave oven. Mowiol is prepared essentially as outlined in Harlow and Lane (19): Mix 2.4 g Mowiol 4-88 (Hoechst, Frankfurt, Germany) with 6 g glycerol and stir for 1 h. Add 18 mL 0.15M Tris-HCl, pH 8.5, and stir for 15 min at 5O’C. Clarify by centrifugation at 10,000 rpm (12,000g) for 15 min and store in small aliquots at -20°C.

3. Methods 3.1. Subcloning

into the @ELF-l

Vector

The cDNA of interest is directionally subcloned into the pDELF-1 vector (Fig. 1) using restriction enzyme cleavage sites, which have been added via PCR. The pDELF-1 polylinker contains six central restriction enzyme cleavage sites, which generate S-overhangs (BspEI to BssHII, Fig. 3). These sites are flanked by sites that generate 3’-overhangs, three at the amino-proximal end and three at the carboxy-proximal end (Figs. 3 and 4). Since exonuclease III degrades only S-overhangs (and blunt ends), but not 3’-overhangs, inserts cloned into the central region of the polylinker can be unidirectionally deleted (14).

3.1.1. PCR-Amplification of the DNA-Fragment to Be Subcloned The cleavage sites to be included in the PCR primers (see Note 4) must not be present in the cDNA itself. If none of the polylinker sites can be used, an alternative cleavage site that generates a compatible overhang may be included in the PCR primers (Fig. 4, third column). Care has to be taken to assure that the reading frame is maintained between insert and vector (the first codon within

a cleavage

site is underlined

in the second column

of Fig. 4). Since

the signal peptide and the GPI anchor attachment signal are provided by the pDELF-1 vector, the PCR primers should flank the extracellular part of the protein

under study without

its own signal

peptide

and transmembrane

sequence/GPI-anchor signal. 1. Combine the following m a 0.5~mL thin-walled PCR reaction tube (see Note 5): 5 pL 10X PCR-buffer, 5 pL dNTPs (each 2 mA4), 100 pmol of each primer, and 500 pg-5 ng of template plasmid. Add PCR-grade water to 50 yL.

Briimmendotf,

326 I-7 -->

pSG5

Plagge, and Treubert Fll

ir

TGGCAAAGAATTGTAATACGACTCACTATAGGGCGAATTC Fll

- slgnalpeptlde

ii-

CCATGAGGTTCTTCATCAGTCATCTTGTTACACTCTGTTTCATCTTCTGTGTGGCAGACTCTACCCATTT MRFFISHLVTLCFI FCVADSTHL

Sse83871 SpeI KpnI BstXI Sac1 BspEI Not1 XhoI Sun1 BssHII PstI FseI 1 GTCCAGGTACCTGGAGCTCTCCGGACTAGTGCGGCCGCTCGAGCGTACGCGCGCCCTGCAGGCCGGCCGGCCCA RTRALQAGP SRYLELSGLVRPLE

r

IgGl

CHZ-domaln

i-

AP90 -

CCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC~CCC~GGACACCCTCATGATCTCCC PELLGGPSVFLFPP KPKDTLMISR <-

AP91 -

GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG~GACCCTGAGGTC~GTTC~CTGG... T P EV T C VVV DV S H E D P E VK FN W .

Fig. 3. The polylinker of plasmid pDELF-1. The part of the pDELF-1 sequence comprising the Fl 1 signal peptide, the polylinker, and the beginning of the human IgGl CH2 domain are shown. The central part of the polylinker contains cleavage sites of restriction enzymes that generate S-overhangs (BspEI to BssHII). The aminoproximal and the carboxy-proximal parts of the polylinker contain cleavage sites of restriction enzymes that generate 3’-overhangs. Restriction enzyme cleavage sites are partially overlapping in the polylinker and are therefore indicated by underlining or by printing in bold. Sequencing primer annealing sites are depicted by arrows. Primers specific for the T7 promoter, which is present in the pSG5 part of the vector (39), are commercially available and the primers AP90 (worked well in our hands) and AP91 have been chosen using the Oligo-program of National Biosciences (Plymouth, MN).

2. 3. 4. 5.

Cover with overlay mineral oil (or liquid wax) to prevent evaporation. Denature for 3 min at 94’C. Add 1 pL of enzyme mix while the sample is kept at 80°C for 2 min (see Note 6). Perform 25 cycles, which may typically be as follows. Annealing, 1.5 min at 65’C; extension, 1 min/kb to be amplified at 68’C; denaturation, 0.5 min at 94°C.

3.1.2. Subcloning in to Plasmid pDEf. F- 1 1. Run the sample on an agarose gel and check that the band obtained is of the predicted size. 2. Excise the band from the gel, avoiding contamination with the parental plasmid (see Note 7).

327

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Recognltlon site, readlng frame

Compatible generated

overhang by

Is

3’ overhang BstXI

KpnI sacr,

CCmNNN/NTGG

GGTAC/C sst1

GAGCT/C

5’ overhang

BspEI,AccIII,MroI,Kpn2I, BseAI,BsiMI,Bspl31 SpeI Not1 XhoI SunI,SplI,BsiWI,Pfl2311, PSPLI BssHII

T/CCGGA AImGT

GmCCGC -GAG WACO

GlmGC

XmaI,PspAI,CfdI,AgeI, PxxAI,NgoMI,NgaAIV NheI,XbaI,AvrII,BlnI EagI (SalI) Asp718I,Acc65I,BsrGI, SspBI MluI

3’ overhana

Sse83871,SbfI PstI FseI

CaCAlGG =CNG GOCCGGICC

PstI,NsiI Sse8387I,SbfI,NsiI

Fig. 4. Restriction enzyme cleavage sites in the pDELF-1 polylinker. Enzymes are listed m the order of their cleavage sites in the polylinker. The names of the prototype enzyme and of commercially available isoschizomers are listed in the first column. In the second column, the recognition site is given in 5’ + 3’ direction, the cleavage position is indicated by a slash, and the first codon within each site is underlined. In the last column other enzymes are given that generate compatible overhangs and may therefore also be included in the PCR primers used for directional subcloning. Data were taken from the restriction enzyme data base REBASE (18,) established by R. J. Roberts at New England Biolabs. SalI is printed in brackets, since it may cleave inefficiently near the end of a DNA stretch (26).

3. Digest the PCR product using the appropriate restriction enzymes (see Notes & 10) and ligate with the pDELF- 1 plasmid, which has been cleaved with compatible enzymes (see Note 11). 4. Transform bacteria and isolate the plasmid DNA following standard protocols. 5. Transfect COS cells with the construct and check whether cell-surface expression of the appropriate polypeptide is detectable. To this end, polyclonal antibodies directed to human IgG as well as antibodies specific for the protein under study can be employed. We recommend following the protocol in Section 3.4.1.) which is given below for this purpose.

3.2. Preparation of a Deletion Library A library of plasmids with inserts that are unidirectionally deleted is prepared by:

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1 Linearization of pDELF-1 containing the insert with two restriction enzymes, which generate a 5’ overhang and a 3’ overhang, respectively. 2. Generation of progressive deletions beginning at the 5’ overhang of the linearized molecule. 3. Removal of the remaining single strand by mung bean nuclease. 4. Recircularization of the molecules. A deletion library representing a protein that comprises 1000 amino acid residues may consist of 18 plasmid size fractions, which can be obtained from a single exonuclease III digest of 50 pg plasmid DNA.

3.2.7. Closure of Random Sing/e-Strand Nicks Before cleavage with restriction enzymes and digestton with exonuclease III, random nicks, which may be present in the plasmid, are closed by T4 DNA ligase (see Note 12). 1. Mix in a total of 150 PL, using water to adjust the volume. 50 Pg of plasmid DNA, 30 l,tL ligase buffer (5X), and 2 U T4 DNA ligase. 2. Incubate at room temperature for 1-2 h. 3. Inactivate the ligase by heating to 65’C for 20 min and cool on ice. 4. Purify the DNA from the incubation mix and redissolve m 20 n,L TE (see Note 10).

3.2.2. Restriction Endonuclease Digestion Progressive deletions beginning near the amino-terminus (N + C-mutants) can be obtained by cleavage at one of the three amino-proximal 3’-sites (MXI, KpnI, or SacI) and at one of the 5’-sites present between the 3’-sites and the insert. Progressive deletions beginning near the carboxy-terminus (C + Nmutants) can be generated by cleavage at one of the three carboxy-proximal 3’-sites (Sse83871, MI, or FseI) and at one of the Y-sites present between the 3’-sites and the insert. 1. Digest the DNA in a reaction volume of 100 PL with restriction endonucleases to obtain a 5’-protruding end and a 3’-protruding end (see Notes 8, 13, and 14). 2. Inactivate the enzymes by heating to 65’C for 20 min. 3. Purify the DNA from the incubation mix and redissolve in 50 PL TE (see Note 10).

3.2.3. Exonuclease-Ill Digestion The optimal reaction temperature for the exonuclease-III digestion has to be defined for each individual plasmid, and depends on the length of the insert and the amount of DNA. The following protocol applies to pDELF-1 with a 2.9-kb insert and results in a degradation rate of approx 90 nucleotides/min (see Note 15).

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1. Adlust the following with water to a total volume of 450 yL: 50 i.tL DNA in TE, 225 l.tL exonuclease III buffer (2X), and 900 U exonuclease III (see Note 16). 2. Incubate at 26°C. 3. Remove 18 samples of 25 l.tL in intervals of 3.5 min. Stopthe reaction by adding

each sampleto a stop solution that has beenprepared in advanceby combining 20 pL mung bean nucleasebuffer (10X) and 155 pL water (seeNote 17). Store on dry ice until all samples are collected.

3.2.4. Mung Bean-Nuclease

Digestion

1. Heat the 18 samples to 75’C for 15 min, cool to 30°C, and add 2 pL mung bean

nucleasewith 5 U/pL, diluted in mungbeannucleasedilution buffer (seeNote 18). 2. Incubate at 3O“C for 60 min and inactivate the nuclease by addition of 20 l.tL mung bean nuclease stop solution.

3.2.5. Isolation of Size Fractions of Progressively Shortened Plasmids, Recircularization of Plasmids, and Transformation of Bacteria 1 Dilute 15 pL of the samples with 35 l.tL water (see Note 19). 2. Analyze by agarose gel electrophoresis

3. Isolate the DNA from the agarosegel, redissolve the pellet in 20 pL TE, and add the following to recircularize the molecules: 60 yL water, 20 pL ligase buffer (5X), and 1 U T4 DNA ligase.

4. Incubate at room temperaturefor 3 h. 5. Inactivate the ligase by heating to 65°C for 20 min and cool on ice.

6. Transform bacteria using standardprotocols.

3.3. Assembly of the Epitope Map The localization of an MAb epitope on the protein under study implies the identification of the shortest N + C-mutant of the protein and of the shortest C + N-mutant, which carries the MAb epitope. The following considerations apply to both types of mutants. The shortest epitope-carrying mutant is identified in a hierarchical way: First, the size fraction containing the shortest epitope-encoding plasmid is identified by analysis of plasmid populations as a whole. Second, the shortest plasmid encoding the epitope is identified in this size fraction by singleclone analysis. 3.3.1. Analysis of Plasmid Populations Pool 12-24 different bacterial colonies that arose from a single plasmidsize-fraction and process in a single-plasmid “mini’‘-preparation to prepare about 20 kg DNA (see Notes 20 and 2 1). Label the colonies before pooling in a way that allows you to refer to them later. Transfect COS cells with the plas-

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mid pool and characterize the expressed population of deleted polypeptides by immunofluorescence analysis as outlined in Section 3.4.1. It is recommended to check the protocol performance by monitoring the presence of the human IgGl domains, which should be contained in each pDELF- 1-derived molecule on the surface of the transfected cells. This can easily be achieved using CY~=~conjugated polyclonal antibodies directed to human IgG. A crude map can be assembled by determination of the mean length of the plasmids in each population using agarose gels. Depending on the length of the insert, either the plasmids can be linearized or the inserts can be excised from the plasmids to obtain a good length resolution. 3.32. Analysis at the Single-Clone Level In theory, one-third of the clones in a particular plasmid size fraction should have a continuous reading frame at the site of ligation/recircularization (step 3 in Section 3.2.5.) and two-thirds should have a frameshift. Only in-frame clones can be expected to code for polypeptides containing the two human IgGl domains with the carboxy-terminal GPI anchor attachment signal. The fraction of in-frame clones contains those folded properly and those with grossly disturbed tertiary structure and exposed hydrophobic surfaces. Whereas the former are expected to be targeted to and anchored on the cell surface, the latter most likely do not reach the surface, but are caught by chaperones and are ultimately degraded (20). Depending on the protein under investigation, less than onethird of the clones will therefore give rise to cell-surface expression on the transfected cells. Plasmid DNA is isolated from 12-24 single bacterial colonies, which arose from a particular plasmid size fraction. COS cells are transfected with single clones, and the expressed protein fragments are analyzed in MAb-binding assaysas described in Sections 3.4.1., 3.4.2., and 3.4.3. In our hands the protocol in Section 3.4.1. gives more reliable results than those in Sections 3.4.2. and 3.4.3., but it is more time-consuming. If the shortest plasmid that encodes the MAb epitope has been identified, the site of ligation/recircularization is determined by DNA sequencing. Clones of the N + C-series can be sequenced using primers specific for the T7 promoter, which is located upstream of the polylinker in the pDELF-1 vector (see Note 22). Clones of the C + N-set can be sequenced using the primer AP90 or AP9 1, which binds downstream of the polylinker in the region encoding the CH2 domain of human IgGl (Fig. 3).

3.4. MAb-Binding Analysis The following three alternative protocols can be used to identify the deletion mutants that are expressed at the cell surface and to analyze these proteins

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Fig. 5. Detection of transfectedCOS cells. The analysis of transfectantsusing the immunofluorescencemethod on glass slides (A) gives a better signal-to-noise ratio than analysis using alkaline phosphatasewith the substratesBCIP/NBT (23,241in tissue-cultureplates (B). The immunofluorescencemethod is thereforemore compatible with evaluationsusing image analysis systems.

with respect to the presenceor absenceof MAb epitopes (see Note 23). The procedureshave in common that COS cells are transiently transfectedwith the plasmids using the DEAE-dextran/chloroquine-protocol (see Note 24). They differ with respect to the duration of the protocols, the transfection efficiency (see Note 29, and the signal-to-noise ratio (with respect to the detection of transfectants), which can be expected. DEAE-dextran had been originally introduced to enhancevirus uptake by cells and is likely to facilitate binding of the negatively charged plasmid molecules to the negatively charged cell surface (21). Chloroquine is added, since it has been shown to enhancetransfection efficiencies most likely by inhibiting lysosomal DNA degradation (22). 3.4.1. Transfection in Tissue-Culture Plates and lmmunofluorescence Analysis on Glass Slides In this method, COS cells are transfected in a six-well tissue-culture plate and are transferred 1 d later to eight-well multitest glass slides for immunofluorescenceanalysis. Each of six different clones can be analyzed with at least 16 different antibodies. This 3-d protocol has a better transfection efficiency than the other two (Sections 3.4.2. and 3.4.3.). Additionally, a better signal:noise ratio with respect to the detection of the transfectants can be expected(Fig. 5A). However, the protocol is not well suited for the analysis of a large number of clones.

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3.4.1 .I. FIRST DAY 1. Wash COS cells, which have just grown to confluence in a g-cm Petri dish, three times with PBS (see Note 26). Aspirate the PBS, add 1.5 mL trypsin/EDTA solution, and swirl the dish to cover the cells evenly. Incubate for 5-10 min at room temperature until the cells appear rounded. Dislodge the cells by rocking the dish carefully. Block trypsin by addition of 10 mL DMEM/FCS (prewarmed to 37OC) and disperse the cells by repeated pipeting using a wide-mouth pipet to ensure an even cell suspension. Air bubbles should be avoided. 2. Transfer 1.5 mL of this cell suspension, which should have approx 3 x lo5 cells/ml to each well of a six-well tissue culture plate. 3. Incubate for 4-5 h at 37OC in a humidified incubator gassed with 5% CO,. 4. Prepare for each plate 10 mL of a premix containing 9.8 mL DMEM/FCS and 200 pL chloroquine/DEAE-dextran mix (50X). Aspirate the medium carefully and add to each well 1.5 mL of the premix containing 1.5 pg plasmid DNA (see Note 27). 5. Incubate for 2 h at 37°C in a humidified incubator gassed with 5% COz (see Note 28). 6. Remove the transfection mix by aspiration. Wash three times gently with DMEM (prewarmed to 37°C; see Note 26) and add 5 mL DMEM/FCS to each well. 7 Incubate overnight at 37°C in a humidified incubator gassed with 5% COz. 3.4.1.2.

SECONDDAY

1. Coat multitest glass slides with 40 pL poly+lysine/well (100 pg/mL), and incubate for 1-2 h at 37OC in a humidified incubator. (This can also be done on the first day with overnight incubation.) 2. Wash COS cells three times with PBS (prewarmed to 37OC; see Note 26). Aspirate the PBS, add 1 ml trypsin/EDTA solution/well, and incubate for 5-I 0 min until the cells appear rounded. Dislodge the cells by rocking the tissue-culture plate. Block trypsin by addition of 5 mL DMEM/FCS (prewarmed to 37OC). Disperse the cells by repeated pipeting avoiding air bubbles and using a widemouth pipet. Collect in a lo-mL sterile tissue-culture tube. Rinse the tissueculture wells with additional 4 mL DMEM/FCS and pool the suspensions (see Note 29). 3. Centrifuge for 20 min at 600 rpm (5Og). 4. Meanwhile, aspirate poly-L-lysine from multitest glass slides and wash three times with PBS. Slides must not dry out during washing. 5. Resuspend the cell pellet carefully (avoiding air bubbles) and adjust to 3 x lo5 cells/ml in 700-1400 pL DMEM/FCS. It is sufficient to count the cells of one well if six wells are being processed. 6. Aspirate the last PBS wash from the multitest glass slides and distribute 40 pL of the cell suspension/well, Slides must not dry out during this procedure, The suspension from a single IO-cm2 tissue-culture well should be sufficient for two to four multltest slides, depending on the yield. 7. Incubate overnight at 37°C in a humidified incubator gassed with 5% C02.

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3.4.1.3. THIRD DAY (SEE NOTE 30) 1. Aspirate the medium carefully and wash the cells once gently with 40 pL/well of PBSCM using a four-channel pipet (see Note 3 1). 2, Aspirate the PBSCM gently and incubate at room temperature for l-2 h with 50 pL/well of MAb culture supernatant or MAb IgG diluted in PBSCM/BSA (see Note 32). This and the following antibody incubation should be done in a wet chamber to prevent evaporation. 3. Wash three times with PBSCM using a four-channel pipet (see Note 26). 4. Aspirate the PBSCM carefully and incubate at room temperature for l-2 h with Cy3conjugated secondary antibody diluted in PBSCM/BSA as recommended by the supplier (see Note 33). 5. Wash three times with PBSCM using a four-channel pipet. 6. Mount the slides with one small drop of Kaiser’s Glyzerin Gelatine, molten in a microwave oven, and cooled to 30-4O”C in a glass Pasteur pipet. The slides can be observed after 10 min and are stable for several years if kept in a dark and cool place. Alternatively, mount slides with one small drop of Mowiol(19). Slides mounted in Mowiol can be evaluated for at least 4 wk if stored in a dark and cool place.

3.4.2. Transfection and lmmunofluorescence Analysis on Glass Slides In this procedure, COS cells are transfected and analyzed on eight-well multitest glass slides. Positive clones are identified using Cy3-conjugated secondary antibodies in immunofluorescence analysis. This 2-d protocol is suitable for the analysis of a medium number (e.g., 8-48) of clones, which are to be probed with one or two MAbs. However, the transfection efficiency and the reliability are lower than that of the protocol described in Section 3.4.1, 3.4.2.1. FIRST DAY 1. Coat multitest glass slides with 40 pL/well poly-L-lysine (100 p&/nil,) and incubate for l-2 h at 37OC in a humidified incubator. 2. Aspirate poly-L-lysine from multitest glass slides and wash three times with PBS, taking care that the slides do not dry out. 3. Trypsinize COS cells as outlined in Section 3.4.1 .l, (step 1) and adjust the suspension to approx 3 x lo5 cells/ml. 4. Aspirate the last PBS wash from the multitest slides and transfer 50 PL of cell suspension to each well of the slide. Slides must not dry out during this procedure. 5. Incubate for 4-5 h at 37OC in a humidified incubator gassed with 5% CO,. 6. Prepare for each slide 500 pL of a premix containing 490 I.IL DMEMKS and 10 pL chloroquine/DEAE-dextran mix (50X). Aspirate the medium carefully and add 50 pL premix to each well. Pipet 1 pL plasmid solution containing 50 ng DNA directly into the drop of premix on the slide (see Note 27). 7. Incubate for 2 h at 37°C in a humidified incubator gassed with 5% CO2 (see Note 28). 8. Remove the transfection mix by careful aspiration. Wash three times gently with DMEM (prewarmed to 37’C; see Note 26) and add 50 pL DMEM/FCS. 9. Incubate overnight at 37°C in a humidified incubator gassed with 5% CO,.

Brijmmendorf,

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Plagge, and Treubert

in Section 3.4.1.3.

3.4.3. Transfection and Characterization of Clones in 96-Well Tissue-Culture Plates In this method, COS cells are transfected and analyzed in 96-well tissueculture plates. Positive clones are identified using alkaline phosphatase-conjugated secondary antibodies with BCIP as a substrate and NBT as an enhancer (23,24). This protocol is suitable for the analysis of a large number of clones, especially if multichannel pipets are used for the washing steps. However, the transfection efficiency and the signal-to-noise ratio with respect to the detection of the transfectants (Fig. 5B) are lower than in Sections 3.4.1. and 3.4.2. 3.4.3.1. FIRST DAY 1. Trypsinize COS cells as outlined in Section 3.4.1.1. (step 1) and adjust the suspension to approx 1 x lo5 cells/ml. One 9-cm Petri dish with COS cells, which have just grown to confluence, should give enough cell suspension for two 96well tissue-culture plates. 2. Transfer 150 yL of the cell suspension to each well of a 96-well tissue-culture plate. 3. Incubate for 4-5 h at 37’C in a humidified incubator gassed with 5% COZ. 4. Prepare for each plate 10 mL of a premix containing 9.8 mL DMEM/FCS and 200 pL chloroquine/DEAE-dextran mix (50X). Aspirate the medium carefully and add 100 pL premix to each well. Pipet 1 yL plasmid solution containing 100 ng DNA directly into the premix in the well (see Note 27). 5. Incubate for 2 h at 37OCin a humidified incubator gassed with 5% CO1 (see Note 28). 6. Remove the transfection mix by careful aspiration. Wash three times gently with DMEM (prewarmed to 37“C; see Note 34) and add 150 pL DMEM/FCS. 7. Incubate overnight at 37OC in a humidified incubator gassed with 5% CO,. 3.4.3.2.

SECOND DAY

1. Aspirate the medium carefully and fix cells for 20 min using 150 pL PBS/4% formaldehyde (see Note 3 1). 2. Wash three times gently with 150 pL PBSCM (see Note 26). 3. Aspirate the PBSCM carefully and incubate for l-2 h at room temperature with 100 p.L MAb culture supematant or MAb IgG diluted in TBSiBSA (see Note 32) 4. Wash three times gently with PBSCM. 5. Aspirate PBSCM carefully and incubate for l-2 h at room temperature with alkaline phosphatase-conjugated secondary antibody diluted in PBSCMBSA as recommended by the supplier. 6. Wash gently twice with TBS/BSA and once with alkaline phosphatase buffer. 7. Develop at room temperature with 100 PL reaction mix/well until cells in the positive control are clearly darkened (this may take approx 10 mm). Reaction mix for one 96-well plate: 10 mL alkaline phosphatase buffer with 33 PL BCIP (50 mg/mL) and 66 pL NBT (50 mg/mL), added shortly before use.

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8. Wash twice with PBSCM and evaluate using a tissue-culture microscope. The plates can be stored for at least 4 wk in a cool, dark place with a wet atmosphere.

4. Notes 1. Plasmid pDELF- 1 has been generated as follows: The two exons encoding the CH2 and CH3 domains of human IgG 1 (including the intervening intron) of plasmid pIG1 (25) have been inserted between the sixth Ig-like domain of the neural cell recognition molecule F 11 and the GPI anchor attachment signal of F 11 in the plasmid pSGYIG (I 6). Afier the construct had been checked with respect to cellsurface expression using F 11 -specific antibodies, the six Ig-like domains of F 11 were excised and replaced by the polylinker sequence (Fig. 3). COS cells that have been transfected with pDELF-1 were stained with antibodies Qrected to human IgG, and the percentage of transfected cells as well as the staining mtensity were examined. Both parameters were indistinguishable from those obtained in F 11 -transfections (16). 2. We did not observe differences between the COS- 1 line (ATCC CRL 1650) and the COS-7 line (ATCC CRL 1651) with respect to transfection efficiency with pSG5-derived plasmids. 3. Previously, we kept COS cells in DMEM with 10% FCS (16). In order to save FCS, we culture the cells now in DMEM with 2% FCS with no discernible disadvantage. The cells are split 1:3 every third day or 1:5 every fourth day. 4. There are good programs available for the design of PCR primers; for example, PRIME of the GCG for UNIX (Madison, WI; e-mail: [email protected]) or OLIGO of National Biosciences for DOS or Wmdows (Plymouth, MN; e-mail: [email protected]) Primers are typically 20-30-mers with 50-60% GC content. Both primers of a pair should have similar annealing temperatures. There should be two to three additional nucleotides 5’ of the restriction enzyme cleavage site, since some enzymes may cleave inefficiently close to the end of a DNA stretch, especially SulI (26). 5. The high sensitivity of the PCR process requires that extreme care should be taken to avoid contamination of PCR reagents with template plasmid DNA, The following precautions are suggested: a. Use sterilfilter pipet tips. b. Change gloves frequently. c. Spin tubes before opening them, and open and close tubes carefully. d. Avoid aerosohzation. e. Special care should be applied when the enzyme(s) are added to the PCR mix, since the plasmid is already present in the mix. (If no hot-start protocol is followed, the plasmid should be the last component to be added to the PCR mix.) 6. Taq DNA polymerase lacks a proofreading activity and has an error rate of 1 x 1w5 to 1 x lti mutations/replicated nucleotide, depending on several variables, among them the pH, the Mg2* concentration, the dNTP concentration, and dNTP balance (27-32). We use a mixture of two DNA polymerases, as suggested

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8. 9.

10.

11.

12.

13.

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by Barnes (33). The mixture contains Tag DNA polymerase and Pfu DNA polymerase, which has a lo-fold lower error rate than Tag DNA polymerase (31). Selection against the template plasmid DNA may be achieved as follows: a. Use as little template plasmid DNA as possible; 500 pg to 5 ng should be sufficient if 25 cycles are applied. b. The PCR product should be separated from the template DNA in an agarose gel prior to ligation in pDELF- 1. If the supercoiled template comigrates with the PCR product, linearize the template plasmid prior to PCR. c Werner et al. propose selecting against the template DNA by digestion with DpnI. This restriction enzyme cleaves only DNA that has been isolated from Escherichia coli strains containing the Dam methylase (most common strains). The PCR product should therefore remain uncleaved (34). The optimal reaction temperature of some enzymes deviates from 37°C. AgeI: 25’C, BssHII: 5O”C, BstXI, BszWI, SunI, and Kpn21: 55°C AccIII and BsrGI: 60°C In theory, alkaline phosphatase treatment of the vector is not necessary after a double-restriction digest. It is nevertheless recommended in order to reduce the background that may be caused by traces of recirculanzed molecules, which have been cleaved by only one of the two enzymes. DNA can be isolated after enzymatic treatments by phenol/chloroform extraction followed by ethanol precipitation. Alternatively, most reagents suited for DNA isolation from agarose gels can also be used for the isolation of DNA from reaction mixtures. It is recommended to purify the linearized vector as well as the PCR product using agarose gel electrophoresis prior to ligation. This helps to get rid of residual PCR primers, of traces of uncleaved vector, of alkaline phosphatase, and of such restriction enzymes that cannot be completely inactivated by incubation at 60°C for 20 mm, among them AvrII, BspE 1, KpnI, and PstI Every plasmid preparation contains a certain number of molecules with singlestrand nicks. Since exonuclease III can inmate digestion from the nicks (35), closure of the nicks by the ligase prior to exonuclease III digestion helps to reduce background It is essential that the restriction enzyme cleavage has proceeded to completion prior to the exonuclease III digestion: Molecules that only have 5’-overhangs and are lackmg 3’ overhangs ~111 be shortened at both ends by the exonuclease, and molecules that only have 3’ overhangs will not be shortened at all (at least m theory). It is therefore suggested that long digestion times be used, 2-4 h for instance, or even overnight. Problems may arise if the restriction enzyme cleavage sites used to generate the 5’-overhang and the 3’ overhang, respectively, reside in close proximity or are even neighbors in the polylinker. If this is the case, the first cleavage of every single molecule occurs under standard conditions, whereas the second cleavage site is flanked only by a short sequence stretch that is known to be an unfavorable situation for some enzymes, especially PA (26). Under these circumstances, we recommend cleaving first with the enzyme generating the 3’overhang and then with the enzyme that generates the 5’-overhang.

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14. FseI seems to cleave different sites with different efficiencies, depending on the sequence context. It has been reported that one of the three sites m the Ad2 genome is kinetically very slow (36). In line with this, we found that FseI cleaves the pDELF-1 vector well, but cuts pDELF-1 less efficiently if it contains an Fll insert. 15. Exonuclease III activity is highly dependent on the temperature and doubles every 6% between 22 and 46°C (35). We have incubated 45 pg linearized DNA of up to 8.3 kb (which remained afier nick ligation/restriction digest of 50 pg DNA) with 900 U exonuclease III. Degradation rates of 30-90 nucleotides/min have been measured at 20-26’C. We are using sampling intervals of 2-5 min. 16. Commercial suppliers use different unit definitions for exonuclease III. We refer to the unit definition “1 U releases 1 nmol of acid-soluble nucleotides from sonicated calf thymus DNA within 30 min at 37’C.” 17. Whereas mung bean nuclease requires 0.1 mM Zn2+, exonuclease III is inhibited by this ion (37,38). Mung bean nuclease buffer is therefore used as an exonuclease III stop solution. 18. Since mung bean nuclease is unstable in diluted solution, prepare the dilution (in mung bean nuclease dilution buffer) shortly before use. 19. Time can be saved if-in a first experiment--only every second of the 18 different exonuclease III plasmid size fractions is analyzed by agarose gel electrophoresis, COS cell transfections, and MAb-binding assays. In a next step, one can focus on a subset of size fractions. 20 The pooling of single bacterial colonies to be processed in a single plasmid preparation can be avoided by growing the transformants, which arose from a single plasmid size fraction in one single liquid culture, rather than plating them on agar plates. However, control of the population complexity is lost in this case. 2 1. Time can also be saved if plasmid pools, which have each been prepared from hundreds of bacterial colonies, are screened in a pilot experiment. Pool several hundred colonies from an agar plate (but keep at least half of the colonies on a particular plate for later use), and process in a single plasmid preparation. Transfeet COS cells with the plasmid pool and analyze cell-surface expression following the protocol in Section 3.4.1. Narrow down the MAb epitope by comparing multiple plasmid pools and switch to single-clone level analysis (Section 3.3.2.) making use of the residual colonies that have been kept on the agar plates. 22. Single-stranded DNA for sequencing that contains the antisense strand of the insert can be produced making use of the Ml3 intergenic region, which resides in the pSG5 part (39) of the vector (Fig. 1). 23. Lack of MAb binding by a particular deletion mutant that is detectable at the cell surface by human IgG-specific antibody is not a formal proof of epitope absence in this polypeptide for at least two reasons. a. It is conceivable that deletion mutants with subtle conformational aberrations may escape chaperon surveillance and may reach the cell surface. These truncated molecules may have lost the ability to bind an MAb despite carrying the epitope m the wild-type molecule.

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24.

25.

26.

27.

Briimmendorf, Plagge, and Treubert b. The amino acid sequence encoded by the polylinker (or encoded by parts of the polylinker in the deletion mutants) most likely does not adopt a defined tertiary structure, depending on the structural environment, whrch is defined by the protein under study. It is, therefore, conceivable that this region may be susceptible to proteolytic degradation in some of the deletion mutants. This may lead to a release of the insert-encoded polypeptide from the surface of the COS cells. Conclusions concerning MAb-binding sites should therefore be based on positive rather than on negative data. If bacteria are transformed with plasmids under standard conditions, single cells usually take up only one single plasmid. By contrast, if COS cells are transfected with plasmids using the DEAE-dextran/chloroqume protocol, every cell most likely takes up several thousands of plasmid molecules. Transfection with a pool of different plasmids will therefore lead to the coexpression of different polypeptides in each transfected cell. If it is desired to transfect the cells under “one cell/ one plasmid” conditions, the COS cells should be transfected using the spheroblast fusion method (25,40). There are several suggestions m the literature to increase the transfection efficiencies of DEAE-dextran-mediated COS cell transfection: a. At the time of transfection COS cells should preferably be in the logarithmic phase of growth at about 50-75% confluence. b. Washing the cells after transfection followed by treatment with 10% DMSO in PBS for 2 min and an additional wash with PBS was reported to increase the transfection efficiency (25,40,41). c. Gonzales and Joly report that tightening the lid of the tissue-culture flask while exposing the COS cells to the transfection mix increases the transfection efficiency (42). The COS cells must not dry out during the washing steps, and osmotic stress, which may arise by partial evaporation of culture medium, wash solutions, or buffers, should be avoided. This can be achieved by processing only one well at a time if six-well tissue-culture plates are handled. The eight-well multitest slides should be processed row (4 wells) by row, and buffer that has been aspirated should be replaced by the next solution untnediately. Antibody incubations should be done in a wet chamber to avoid evaporation, In our hands, CsCl-purified DNA and DNA isolated by absorption techniques (e.g., using Qiagen reagents) do not significantly differ with respect to the transfection efficiency. We usually use 1 pg/mL of plasmrd DNA in the transfection mix, which means 1.5 pg DNA for each well of a six-well tissue-culture plate (Section 3.4.1.), 100 ng for each well of a 96-well tissue culture plate (Section 3.4.3.), and 50 ng for each well of a multitest slide (Section 3.4.2.). In Section 3.4.1.) a mix containing the transfection premix (medium/DEAE-dextran/ chloroquine) and the plasmid is prepared in advance and is then applied to the cells. By contrast, in the latter two protocols, the plasmid is pipeted directly into the small volume of premix that has already been applied to the cells. In our

Exonuclease III Deletion Mutagenesis

28.

29.

30.

3 1.

32.

33.

34.

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hands, diffusion of the plasmid is sufficient to obtain a good transfection efficiency, and time can be saved by this simplification. COS cells must not be incubated longer than 4 h in the transfection mix, since the toxicity of chloroquine may otherwise damage the cells. The incubation should be terminated if the majority of the cells shrink, look vacuolated, or round up. Since the cells are likely to detach after the transfection, washing after aspiration of the transfection mix has to be done very gently. The trypsinization and replating of the cells after transfection as recommended in Section 3.4.1. helps to remove chloroquine-damaged cells as well as DEAE dextran. By contrast, in Sections 3.4.2. and 3.4.3., damaged cells may remain present in the cellular monolayer, may unspecifically bind antibodies, and therefore may cause background fluorescence. The protocols in Sections 3.4.1 and 3.4.2. are compatible with double-immunofluorescence analysis. The human IgGl Fc domains can be detected in parallel with epitopes of MAbs on the same cells if DTAF- or FITC-conjugated goat antibodies directed to human IgG are added to the Cy3-conjugated secondary antibody directed to mouse antibodies (Section 3.4.1,3., step 4). The mounting of slides in Kaiser’s Glyzerin Gelatine is not compatible with immunofluorescence analyses using DTAF- or FITC-conjugated secondary antibodies because of a high background fluorescence of this mounting reagent. Therefore, Mowiol should be used for the double-immunofluorescence analysis (19). Protocols in Sections 3.4.1 and 3.4.2. work without formaldehyde-fixation of the COS cells on the slides prior to immunofluorescence analysis and are therefore suited for MAbs directed to formaldehyde-sensitive epitopes. In the protocol in Section 3.4.3 , the fixation of the cells helps to reduce the loss of cells during subsequent washing steps. Damaged cells may bind primary antibodies and/or secondary antibodies unspecifically. It is therefore recommended to include appropriate controls, e.g., a well lacking the primary antibody or a well with COS cells transfected with pSG5 (Stratagene) or another plasmid with the SV40 origin of replication. The Cy3 fluorochrome gives a brighter staining, has a better photostability, and can be used with the same filters as rhodamine derivatives. Cy3 is a trademark of Biological Detection Systems, Inc. If loss of cells during washing steps is a problem in Section 3.4.3. (96-well plates), we recommend seeding the cells 24 h before transfection, rather than 4-5 h, as suggested in the standard protocol, Seeding the cells 1 d before transfection helps to stabilize the monolayer, and therefore reduces the loss of cells durmg the transfection and subsequent washing steps. However, a lower transfection efficiency has to be expected because the cell density is higher than optimal.

Acknowledgments We would like to thank D. L. Simmons for providing the pIG1 plasmid, H. Volkmer for help&l discussions, H. Pachowsky for technical assistance,and F. Spaltmann for careful reading of the manuscript.

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References 1. Ryu, S. E., Kwong, P. D., Truneh, A., Porter, T. G., Arthos, J., Rosenberg, M., Dai, X. P., Xuong, N. H., Axel, R., Sweet, R. W., and Hendrickson, W. A. (1990) Crystal structure of an HIV-binding recombinant fragment of human CD4. Nature 348,419-426. 2. Truneh, A., Buck, D., Cassatt, D. R., Juszczak, R., Kassis, S., Ryu, S. E., Healey, D., Sweet, R., and Sattentau, Q. (1991) A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion. J. Biol. Chem. 266,5942-5948. 3. Camerini, D. and Seed, B. (1990) A CD4 domain important for HIV-mediated syncytium formation lies outside the virus binding site. Cell 60,747-754. 4. Arthos, J., Deen, K. C., Chaikin, M. A., Fornwald, J. A., Sathe, G., Sattentau, Q. J., Clapham, P. R., Weiss, R. A., McDougal, J. S., Pletropaolo, C., Axel, R., Truneh, A., Maddon, P. J., and Sweet, R. W. (1989) Identification of the residues in human CD4 critical for the binding of HIV. CelI 57,469-48 1. 5. Clayton, L. K., Hussey, R. E., Steinbrich, R., Ramachandran, H,, Husain, Y., and Reinherz, E. L. (1988) Substitution of murine for human CD4 residues identifies amino acids critical for HIV-gp120 binding. Nature 335,363-366. 6. Jameson, 13. A., Rao, P. E., Kong, L. I., Hahn, B. H., Shaw, G. M., Hood, L. E., and Kent, S. B. (1988) Location and chemical synthesis of a binding site for HIV-l on the CD4 protein. Science 240, 1335-1339. 7. Ockenhouse, C. F., Betageri, R., Springer, T. A., and Staunton, D. E. (1992)PZusmodium falciparum-infected erythrocytes bind ICAMat a site distinct from LFA- 1, Mac- 1 and human rhinovirus. Cell 68,63-69. 8. Berendt, A. R., McDowall, A., Craig, A. G., Bates, P. A., Sternberg, M. J., Marsh, K., Newbold, C. I., and Hogg, N. (1992) The binding site on ICAM- for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA- lbinding site. Cell 68, 7 l-8 1. 9. Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) The arrangement of the immunoglobulin-like domains of ICAMand the binding sites for LFA- 1 and rhinovirus. Cell 61,243-254. 10. Register, R. B., Uncapher, C. R., Naylor, A, M., Lineberger, D. W., and Colonno, R. J. (1991) Human-murine chimeras of ICAMidentify amino acid residues critical for rhinovirus and antibody binding. J. Virol. 65,6589-6596. 11. McClelland, A., deBear, J., Yost, S. C., Meyer, A. M., Marlor, C. W., and Greve, J. M. (199 1) Identification of monoclonal antibody epitopes and critical residues for rhinovirns binding in domain 1 of intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA S&7993-7997. 12. Gluzman, Y. (1981) SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23, 175-182. 13. Mellon, P., Parker, V., Gluzman, Y., and Maniatis, T. (1981) Identification of DNA sequences required for transcription of the human alpha- 1 globin gene in a new SV40 host-vector system. Cell 27,279-288.

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14. Henikoff, S. (1987) Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 155, 156-165. 15. Guo, L. H., Yang, R. C., and Wu, R. (1983) An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid. Nucleic Acids Res. 11,5521-5540.

16. Briimmendorf, T., Hubert, M., Treubert, U., Leuschner, R., Tarnok, A,, and Rathjen, F. G. (1993) The axonal recognition molecule Fl 1 is a multifunctional protein: specific domains mediate interactions with Ng-CAM and restrictin. Neuron 10,7 1 l-727.

17. Hollenbaugh, D., Chalupny, N. J., and Aruffo, A. (1992) Recombinant globulins: novel research tools and possible pharmaceuticals. CUM. Opin. Immunol. 4,2 16-2 19. 18 Roberts, R. J. and Macelis, D. (1994) REBASE-restriction enzymes and methylases. Nucleic Acids Res. 22,362&3639. 19. Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 20. Bergeron, J. J., Brenner, M, B., Thomas, D. Y., and Williams, D. B. (1994) Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem. Set. 19,124-128.

21. McCutchan, J. H. and Pagano, J. S. (1968) Enhancement of the infectivity of simian vu-us 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. N&l. Cuncer Inst. 41,35 l-357.

22. Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295-1308. 23. McGadey, J. (1970) A tetrazolium method for non-specific alkaline phosphatase. Histochemte 23, 180-l 84. 24. Mierendorf, R. C., Percy, C., and Young, R. A. (1987) Gene isolation by screenmg hgtl 1 libraries with antibodies. Methods Enzymol. 152,458-469. 25. Simmons, D. L. (1993) Cloning cell surface molecules by transient expression in mammalian cells, in Cellular Interactions in Development. A Practical Approach (Hartley, D. A., ed.), IRL, Oxford, UK, pp. 93-127. 26. Moreira, R. F. and Noren, C. J. (1995) Minimum duplex requirements for restriction enzyme cleavage near the termini of linear DNA fragments. Biotechniques 19,56-59.

27. Flaman, J. M., Frebourg, T., Moreau, V., Charbonnier, F., Martin, C., Ishioka, C., Friend, S. H., and Iggo, R. (1994) A rapid PCR fidelity assay. Nucleic Acids Res. 22,3259,3260. 28. Brail, L., Fan, E., Levin, D. B., and Logan, D. M. (1993) Improved polymerase fidelity in PCR-SSCPA. Mutat. Res. 303, 171-175. 29. Cariello, N. F., Swenberg, J. A., and Skopek, T. R. (1991) Fidelity of i?rermococcus litorulis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 19,4 193-4 198. 30. Ling, L. L., Keohavong, P., Dias, C., and Thilly, W. G. (1991) Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq and Vent DNA polymerases. PCR Methods. Appl. 1,63-69.

Briimmendorf,

342

Plagge, and Treubert

3 1. Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (199 1) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furzosus. Gene 108, l-6. 32. Eckert, K. A. and Kunkel, T. A. (1990) High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res. l&3139-3744. 33. Barnes, W. M. (1994) PCR amplification of up to 35-kB DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl. Acad. Sci. USA 91,2216-2220.

34. Weiner, M. P., Costa, G. L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J. C. (1994) Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151, 119-123. 35. Hoheisel, J. D. (1993) On the activities of Escherzchia colz exonuclease III. Anal Biochem. 209,238-246

36. Nelson, J. M., Miceli, S. M., Lechevalier, M. P., and Roberts, R J (1990) FseI, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5’ GGCCGGCC

3’. Nucleic Acids Res l&2061-2064.

37. Laskowski, M. (1980) Purification Methods Enzymol

and properties of the mung bean nuclease

65,263-276.

38. Rogers, S. G., and Weiss, B. (1980) Exonuclease III of E. colz K12, an AP endonuclease. Methods Enzymol. 65,201-2 11. 39. Green, S., Issemann, I., and Sheer, E. (1988) A versatile in vzvo and zn vitro eukaryotlc expresslon vector for protein engineering. Nucleic Aczds Res. 16,369. 40. Seed, B. and Aruffo, A. (1987) Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselectlon procedure. Proc Nat1 Acad Sci. USA 84,3365-3369.

41. Lopata, M. A., Cleveland, D. W., and Sollner-Webb, B. (1984) High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxlde or glycerol shock treatment. Nucleic Acids Res. 12, 5707-5717. 42. Gonzales, A. L. and Joly, E. (1995) A simple procedure to increase efficiency of DEAE-dextran transfectlon of COS cells. Trends. Genet. 11,2 16,2 17. 43. Ellison, J. W., Berson, B. J., and Hood, L. E. (1982) The nucleotide sequence of a human immunoglobulin c-y1 gene. Nucleic Acids Res. 10,407MO79 44. BrUmmendorf, T., Wolff, J. M., Frank, R., and RathJen, F. G. (1989) Neural cell recognition molecule F 11: homology with fibronectm type III and immunoglobulin type C domains. Neuron 2, 1351-1361.

28 An Improved Method for Mapping Epitopes of Recombinant by Transposon Mutagenesis

Antigens

Steven G. Sedgwick, Brian A. Morgan, Nguyen thi Man, and Glenn E. Morris 1. Introduction Epitope mapping of proteins defines the structural elements of a molecule needed for recognition by individual antibodies. X-ray crystallography of antibody-antrgen complexes (I) is perhaps the most precise means of defining an epitope. However, several quicker but less detailed methods are also available. These alternative approaches include fragmentation of the antigen by chemical or proteolytic cleavage (2), chemical synthesis of peptide fragments (3), competition between antibodies (4), protection by antibody against proteolytic cleavage (5) or against chemical modification (6), and natural variants in different tissues or species (7). With antigens expressed from cDNA in bacterial plasmids, antigenic fragments can be produced by deletion mutagenesis with restriction enzymes (8) or exonucleases (9), and by construction of epitope libraries of random cDNA fragments in bacteriophage (IO) or plasmid (II) vectors. Site-directed mutagenesis can be used to define important amino acid residues in epitopes (12). These methods, however, involve extensrve in vitro manipulations or are limited by available sites within the cloned sequence. A simple and inexpensive in vivo alternative (13), which is not constrained by restriction site availability, uses the bacterial transposon, TnlOOO (14), to interrupt a coding sequence. TnlOOO introduces early termination codons and, hence, generates a library of clones expressing N-terminal protein fragments with progressively fewer epitopes as they get shorter (Fig. 1).

From’ Methods m Molecular Biology, vol. 66 @Nope Mappmg Protocols Edtted by G E Morns Humana Press Inc. Totowa, NJ

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Transposon

mAb 2 3

1

clonea

4

m

u.

I

Epitope outer limits

--;--;:I: ++++++++++

Fig. 1. Epitope mapping by transposonmutagenesis.A hypotheticalprotein is diagrammedat the top of the figure. The coding sequenceis truncatedby transpositionand the sitesof insertion are determinedby transposon-primedDNA sequencing The predicted sizesof the N-terminal fragmentsof the target protein are ranked in decreasing size.The N-terminal fragmentsare expressedin E. coli and testedfor recognition by a panel of MAbs. Epitopesare mappedby the colinearity betweenthe increasingsizesof thetruncatedprotein fragmentsandthe increasingnumbersof MAbs that recognizethem. There are three stagesto mapping epitopes by transposition: 1, A bacterial expressionplasmid carrying the target cDNA sequenceis mutatedby random insertion of Tn1000. 2, Westernblotting is usedto identify coloniesthat are recognizedby someMAbs, but not others as a result of insertion of TnlOOOinto the cloned cDNA insert. 3. The precisepoints of truncation of the target sequenceby transposoninsertions are determined by DNA sequencingfrom universal primers complementary to sequencesat the termini of Tnl 000. TnlOOO is used because it transposes a wide range of vectors, it inserts only once per plasmid, and it has the least insertion site bias of Escherichia coli’s transposons (see Note 1). Stop codons in all incoming reading frames truncate target gene products. Short 3%bp inverted repeats allow transposon-primed

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nucleotidesequencing5’ and3’ from thepoint of insertion to fix the exactpoint of truncation of a coding sequence(15-17). Finally, with Tn1000,there is the major practical attraction of having a simple delivery systemthat producesa population of entirely transposedplasmids in one step (14). The transpositionapproachis bestsuitedto simultaneousmapping of a large panel of different MAbs against the samerecombinant antigen. In the form describedhere,it also requiresthat MAbs recognizeantigensafter exposureto SDS and attachmentto nitrocellulose; most MAbs suitable for Western blotting satisfy this criterion. It might be adaptable,however, to some “native” epitopesexpressedon the recombinantby modifying the method for screening the library. Since the first application of transpositionmutagenesisto epitope mapping (131,simpler but improved methodsof TnlOOOtranspositionhavebeendeveloped. The application of thesenew methodsof transpositionfor epitopemapping is describedhere. Future prospectsinclude the genetic manipulation of TnlOUOto deliver regulatablebacterial promotersclose to the point of transposition. Insertions of these transposonsin the correct orientation and correct reading frame will control expressionof the targetsequences3’ to the point of insertion, thereby producing a nestedseriesof C-terminal fragments that can also be screenedfor MAb reactivity. 7.7. Overview of Transposlfion System Transposonmutagenesisin E. coli might seemrather alien to many immunologists unfamiliar with bacterialgenetics.However, at the practical level, no specialist skills above thoseneededto perform a bacterial transformation are needed.The method of transpositiondescribedhere includes further simplifications comparedto earlier applications of this approach(13). It also usesthe TnXR deletion derivative of TnlOOOwhere the original 5890-bp element has beenreducedto 4888 bp (seeNote 2). The transposonsystemcomprisestransposondonor strainscarrying TnlOOO on the conjugative plasmid, R388. The R388 plasmid is stable and, for these purposes,is essentially “silent” in requiring no antibiotic selection. For transposition, the targetplasmid is introducedinto the appropriatetransposondonor strain depending on whether the expressionvector used is h repressible. A culture of the transformed “donor” cells is mixed with a second culture of “recipient” cells, where mating events occur. By plating on the appropriate selective agarplates,eachcolony arising from the mating containsan independently transposedtarget plasmid (14). For a kanamycin resistanceplasmid, selective plates would contain kanamycin and streptomycin. Similarly, transposition of an ampicillin resistanceplasmid requires ampicillin, methicillin, and streptomycin. Methicillin enforcesampicillin selection and without it the

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method is unlikely to succeed. A full understanding of the underlying in vivo events is not central to the application of this technique. Further details of this process can be found in Note 3. Two donor/recipient pairs of cells are described here. One pair of donor and recipients contains h repressor for the regulation &,.-based expression vectors. The second pair of donor and recipients is for use with other types of expression vectors and does not have h repression systems. 2. Materials 2.1. Transposition 1. MH1866 donor cells and MH15 12 (13) recrpientsareused for transpositionof h repressible expression vectors. Transposition of other types of expression vectors is done with MH 163 8 donor and HB 10 1 (IS) recipient bacteria (see Note 4). These strains are available from S. G S. 2. Streptomycin sulfate, ampicillin hydrochloride, and methicillin (Sigma, Poole, UK); “separate 10 mg/mL aqueous solutrons of each should be made fresh weekly and stored at 4°C. 3. L broth and agar are standard rich media for E. coli and contain per liter: 10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl adjusted to pH 7.5 with NaOH. L agar also contains 1.2% bacto-agar. Drugs are added to medra at the following concentrations: ampicillm and methicillin, 50 pg/mL; streptomycin, 100 pg/mL. Agar medrum is cooled to 48°C before adding drugs and plates are poured immediately afterward. 4. Sterile toothpicks.

2.2. Antibody

Screening

5. Nitrocellulose circles to tit agar plates (90~mm diameter; Schleicher & Schuell [Hempstead, UK] BA83 or BA85). 6. Electrophoretic transfer apparatus (e.g., Bio-Rad Transblot) and transfer buffer (192 mM glycine, 25 mA4 Tris, pH 8.3). 7. Peroxidase-labeled rabbit anti-(mouse total Ig) (DAKOpatts P260). 8. PBS: 0.9% NaCl, 25 mM sodium phosphate buffer, pH 7.2. 9. Incubation buffer (IB): PBS + 0.05% Triton X-100. It is convenient to prepare this using a 5% w/v stock of Triton X-100 m PBS. 10. IB + 3% low-fat skimmed milk powder (e.g., “Marvel,” and so forth, from any supermarket); IB + 0.3% BSA + 1% fetal calf serum + 1% horse serum. 11. Substrate buffer: Mix 25.7 mL of 0.2A4 sodium dihydrogen phosphate, 24.3 mL of O.lM citric acid, and 50 mL of water (final pH = 5.0). 12. Diaminobenzidine dihydrochloride (DAB) (Sigma) stock solution 80X: 32 mg/mL in water. Store in aliquots at -20°C and use only while they remain pale brown. This should be treated as a possible carcinogen and, in particular, the dusty powder should be weighed with appropriate containment and operator protection. 13. Hydrogen peroxtde (30% v/v) “100 vol.”

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Sequencing

14. Dldeoxy sequencing 1s done from primer sites in the S and y ends of Tn1000. Only one ollgonucleotlde pnmer, either one, is essential. 6 Primer 1sAGGGGAA CTGAGAGCTCTA. y Primer is CCTGAAAAGGGACCTTTGTATACTG (see Note 5). 15. Electrophoresis equipment (e.g., Sequi-Gen [40-50 cm length is sufficient] with 3000x1 Power Pack and Model 583 Gel Dryer, all from Bio-Rad Laboratories, Hempstead, UK). 16. Dideoxy sequencing k1t (e.g., Sequenase ~2.0 from United States Biochemicals, Amersham, UK) 17. a-35S-dATP (> 1000 Cl/mmol; 10 pCi/pL; Amersham International [Amersham, UK], No. SJ1304). 18. Kodak (Queensferry, UK) X-Omat AR X-ray film (this is available economically in 25-m rolls of 20-cm width, which is suitable for 2 1-cm wide gels; Sigma No, F-5138); Kodak GBX developer and fixer; Saran WrapTM for gel drying (available in the United Kingdom from Genetic Research International, Great Dumnow, UK; most “clmg-film” products are unsuitable). 19. Solutions for sequencing gels a. Acrylamide stock: 38 g/100 mL acrylamide and 2 g/100 mL N,N’-methylenebis-acrylamide made with deionized, distilled water and filtered. b. Acrylamlde/urea: 15 mL acrylamide stock solution plus 48 g urea made to 90 mL in deionized, distIlled water and filtered. c. 25X TBE buffer: 27 g Tris, 13.75 g boric acid, and 2.33 g EDTA/lOO mL, pH 8.3,

3. Methods 3.1. Transposition

with Tnl 000

1. Depending on the type of expression plasmid used, select donor cells with or without h repression. Throughout the transposition procedure, MH1866 and MH 15 12 with the h repression systems are cultured at 32°C; MH1638 and HB 101 are grown at 37°C. 2. Make donor cells competent using standard methods (29) and transform 1n the target plasmid. Altematlvely, introduce plasmid by electroporation. 3. Plate samples on L agar plates containing the appropriate selective drug and incubate overnight. 4. Pick a single colony of the transformed donor strain into 10 mL of L broth with drug selection. 5. Pick a single colony of the recipient strain into 10 mL of L broth with no antibiotics. 6. Grow both cultures overnight with shaking for aeration at either 32 or 37°C as explained above. 7. Inoculate 0.1 mL of donor and recipient strains into separate growth flasks containing 10 mL of fresh broth with selective drug(s) in the donor culture. 8. Grow with aeration to mid-log phase, again at 32 or 37°C depending on the type of vector used. The trming here is not critical. Normally a suitable growth phase

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9. 10. 11,

12. 13. 14.

15. 16.

17. 18.

is reached after approx 3 h, by which time the optical density at 600 nm is around 0.2. This phase can be judged by eye as approximately the period when the culture is becoming turbid and appears to swirl when shaken, but is before the point when it is uniformly turbid. Pellet 2 mL of donor culture in a bench-top centrifuge and wash by resuspending in 10X vol of fresh L broth with no antibiotics. Pellet again, this time using 1 mL of the recipient culture to resuspend the pellet of donor cells. Pour the cell suspension onto the surface of predried L agar plate with no antibiotics. It is not necessary for the liquid mating mix to soak into the agar of the mating plate. After 2 h of incubation at 32 or 37’C as appropriate, wash off the mating mixture by repeatedly pipeting 25 mL of L broth on and off the mating plate. Recover cells by bench centrifugation and wash again with an equal volume of L broth, finally resuspending the mating mixture in 1 mL L broth with no drugs Spread 10-100 pL samples of the mating mixture onto selective L agar plates For transposition of an ampicillin resistance plasmid, selective plates would contain 50 &mL amptcillin, 50 pg/mL methwillin, and 100 PglmL streptomycin (see Note 3). Store remaining mixture at 4°C for possible future use. Incubate plates overnight at 32 or 37°C as appropriate. Overnight plates should have 10-1000 single colonies on them (see Note 6). Pick single colonies onto fresh selective plates with sterile toothpicks. It is helpful to make a paper template marked into a grid of 50-100 squares. Place the template under the Petri dish and make a streak of each colony into each square. Incubate plates overnight at either 32 or 37°C. The collection of recipients with randomly transposed plasmids is ready for antibody screening.

3.2. Colony Screening forTn1000 and Loss of Epitopes

/metdon

There are two options for identifying plasmids encoding truncated target gene products with fewer epitopes. The first option simply uses Western hybridization to screen all the randomly transposed plasmids to identify those clones encoding truncated proteins that are recognized by some, but not all, MAbs. Alternatively, the DNA of the transposed plasmids can be first screened for hits in the target sequence (see Note 7). This gives an “enriched” collection of transposed target genes and eliminates plasmids with insertions of no interest from further analysis, With or without prescreening, cells with the transposed plasmids are subjected to Western hybridization as follows. 1. Streak up to 100 ampicillin/streptomycin-resistantcolonies onto replica agar plates andgrow ovemrght. Onereplica plate is required for every two annbodies to be screened (see Note 8)

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2 Replica plate streaked cells onto to dry nitrocellulose circles. Place the nitrocellulose circles colony side up on Whatman 3MM paper soaked in L ampiclllinmethtcillin broth. For h repressible vectors, the broth would be preheated to 32°C. For other systems, the L broth would be prewarmed to 37’C with the appropriate exogenous inducer added. 3. Induce expression of h repressible vectors protein by incubation for 2.5 h at 42°C. For other expression systems, induce by incubation at 37°C with chemtcal inducers, such as IPTG or indoleacrylic acid, in the medium soaking the 3MM paper. 4. Place a second nitrocellulose circle on top to form a sandwich (see Note 9), transfer to Whatman 3MM paper soaked with 5% SDS, and incubate for 30 min at 90°C to lyse the cells 5 Place the nitrocellulose sandwich between Whatman 3MM paper and subject to electrophoresis m a Transblot apparatus for 5 min in each current direction at 200 mA. Separate the blots and subject them to electrophoresls for a further 25 min at 200 mA with the colomes facing the cathode. 6. Block the blots in 3% skimmed milk powder m IB for 1 h at 20°C. 7. Wash twice in PBS for 5 min. Incubate blots with MAb culture supernatant (l/l00 dilution in incubation buffer/l% horse serum/l% fetal calf serum/0.3% bovine serum albumin) for 1 h at 20°C. 8. Wash three times in PBS for 5 mm. Incubate blots with peroxldase-labeled rabbit anti-(mouse Ig) (l/1000 m incubation buffer/l% horse serum/l% fetal calf serum/ 0.3% bovine serum albumin) for 1 h at 20°C. 9. Wash four times with PBS for 5 min. Add DAB substrate (0.4 mg/mL diammobenzidine in substrate buffer with 0.012% HzOz).

3.3. Identification of Transposon Insertion Sites by DNA Sequencing Plasmid DNA preparation and dideoxynucleotide sequencing are routine procedures detailed extensively elsewhere (19) and in users’ instructions furnished with commercially available DNA sequencing kits (see also Chapter 18). Only short electrophoresis times are required. Reading from top to bottom, find the sequence AAACCCC, which is the terminus of the transposon (see Note 5). All sequencesbelow this point are derived from TnlOOO and will be identical in all the transposed clones. Read a further 20-30 basesupward and compare with the known full-length cDNA sequence to determine the point of interruption of the cloned sequence.If there is no match, translate into the reverse complementary sequence(e.g., AATTTC becomesGAAATT) and searchagain (seeNote 10). 3.4. Assembly of the Epitope Map Having determined the point of transposon insertion in the nucleotide sequence, the last amino acid encoded by the cDNA insert can be identified. Note that the first 5 basesof any “downstream” sequenceare a duplication of the last 5 bases“upstream” to the point of insertion into coding cDNA (seeNote 10).

Sedgwick et al. Some transposon-encoded amino acids will also be added to the end of the truncated protein product. The number of theseadditional amino acids varies between 2 and 36 and is determined by the reading frame and orientation of insertion of the transposon (13). An epitope map is assembled by correlating the predicted lengths of a panel of truncated proteins with the MAbs that bind to them. Ideally, the amino acid sequence between the longest protein that fails to bind and the next longer protein that does bind an MAb will contain the contact residues of the epitope for that MAb (Fig. 1). It must be remembered, however, that for some conformational epitopes, the sequence thus determined may be essential for maintaining the epitope without being in direct contact with the antibody such that the actual contact amino acids are closer to the N-terminus. 4. Notes 1. Although transposition with Tnl 000 displays some predilection for AT richness (15,17), genes from GC-rich organisms can be transposed (20). Insertion is quasirandom, so that, for example, one-third of insertions will be in a cloned msert if the insert comprises one-third of the total plasmid. 2. TnXR was made from wild-type TnlOOO by the replacement of an internal EcoRIXbaI segment fragment with the EcoRI Sac1 KpnI SmaI BarnHI XbaI portion of the pUC 18 polylinker. The complete sequence of TnZOOO is designated ectnlOO0 in sequence data banks. 3. In brief, transposition from the transposon m the conjugative plasmid mto the target plasmid temporarily links the two molecules (22) As a result of this linkage, simultaneous mating events transfer both the conjugative plasmtd and the transposed target plasmid into the recipient cells. Transpositional linkage is the only way that the target plasmid can be transferred to the recipients during mating. This subclass of recipient cells can be readily selected by conditions that allow growth of recipient and not the donor cells coupled with selection for the drug resistance of the target plasmid. Usually, streptomycin-sensitive donors and streptomycin-resistant recipients are used, so that streptomycin in the selective plates eliminates all the donor cells. For transpositton of an ampicillin resistance plasmid, selective plates would therefore contain streptomycin, ampicillm, and methicillin. The streptomycin kills all the donor cells, and the ampicillm/ methicillin kills recipient cells not containing plasmids, leaving the rare subclass of recipients that received transposed plasmids as the only cells that can cope with the combined streptomycin and ampicillin/methicillin selection. Note that this system works without TnlOOO imparting any drug resistance marker of tts own. 4. The donor strains MH 1638’and MH1866 are derivatives of E. coli DH 1 (22). They contain TnXR on the low-copy-number conjugative plasmid, R388. They are recA mutant to eliminate recombination and so increase plasmid stability and prevent formation of dimeric plasmids. A L&857 prophage in MH1866 maintains repression of the ilpromoters in aPr expression vectors. Both MH1638 and MH1866 have the wild-type rpsL locus and are therefore sensitive to streptomycin. The recipient

Transposon Mutagenesis

5.

6.

7.

8.

9.

10.

351

strains, HBlOl (22) and MH1512 (131, have recA mutattons for plasmid stability and rpsL mutations, which confer resistance to streptomycin. Streptomycin resistance is a stable trait and there is no need to add streptomycin to routine cultures. MH1512 has a defective h prophage with a temperature-sensitive repressor to allow thermally induced expression. MH1512 also contains srZ*:TnlO as a byproduct of introducing recA1 and as a result is tetracycline-resistant. 6 Primer is homologous to bases 85-66 from the 6 end of Tnl000. The 3’ end of the y primer is 61 bp from the y terminus of TnZ000. The final 35 bases at 6 and y ends of TnZOOO are inverted repeats that give the sequence AACGTACGTTTTCGTTCCATTGGCCCTCAAACCCC with both 6 and y primers before entering the cloned DNA. If there are large patches of smeary growth, rewash mating mixture and spread fresh selecttve plates with either less mating mixture or with 100 pg/mL ampicillin-methicillm. If there are not enough colonies, replate with more mixture, again after rewashing. Some clones will be negative for all MAbs because of transposon insertion after the promoter, but before the first epitope. Similarly many clones may be positive for all MAbs because of transposition after the last epitope or, more likely, because of transposition into vector DNA. Transposed plasmids may be screened for insertion into the cloned insert by restriction site mapping or PCR analysis between flanking vector sites. A “miniblotter” apparatus (Immunetics or Bio-Rad) can be used to screen with larger numbers of antibodies. A sheet of nitrocellulose, cut to fit the miniblotter, is placed on L ampicillin agar. Using a ruler, test clones are drawn with a sterile toothpick across the full width of the sheet as horizontal lines 2 mm apart and grown overnight. Subsequent steps are the same, except that the MAbs are applied as vertical lanes across all clones in the miniblotter. In addition to generating an extra blot, this procedure also reduces sticking of the 3MM paper to the blot in Section 3.2., step 5. As much as possible, any of the paper that does stick to the nitrocellulose should be removed gently to avoid interference with antibody binding. TnlOUU can insert in either orientation so that sequencing with a single primer will read the “upstream,” noncoding strand in some isolates and the “downstream” coding strand in other cases.It is therefore necessary to compare the direct and complementary sequences of the target gene with the sequences obtained with y and 6 primers to fix the point of insertion. An alternative is to determine the orientation of the TnlOUO insertion by restriction enzyme digestion and use different primers for each orientation. The insertion position determined by DNA sequencing must take into account a 5-base duphcatton of the target sequence, which occurs during transposition. Thus, when using only one primer and sequencing downstream, the first 5 bases are duplicates of the 5 bases up to the point of insertion. Consequently, the truncated coding sequence on the other side of the transposon is actually 5 bases longer than might be naively expected. This correction does not apply to positioning the point of truncation by upstream sequencing into the noncoding strand.

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References 1. Amit, P., Mariuzza, R., Phillips, S., and Poljak, R. (1986) Three-dimensional structure of an antigen-antibody complex. Science 233,747-753 2. Morris, G. E. (1989) Monoclonal antibody studies of creatine kinase. The ART epitope: evrdence for an intermediate m protem folding. Blochem J 257, 461-469. 3. Geysen, H. M., Meleon, R. H., and Barteling, S J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino-acid. Proc Nat1 Acad. Sci. USA 81,399&4002. 4. Tzartos, S. J., Rand, D. E., Einarson, B. R., and Lmdstrom, J M. (198 1) Mapping of surface structures of electrophorus acetylcholme receptor usmg monoclonal antibodies. J Biol Chem. 256, 8635-8645. 5. Jemmerson, R. and Paterson, Y. (1986) Mapping epnopes on a protein antigen by the proteolysis of antigen-antibody complexes. Sczence 232, 1001-1004. 6. Burnens, A., Demo@ S., Corradm, G., Bmz, H., and Bosshard, H. R. (1987) Epitope mapping by chemical modtficatlon of free and antigen-bound protein antigen. Science 235,780-783. 7 Nguyen thi Man, Cartwright, A. J., Osborne, M., and Morris, G E. (1991) Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies. Biochzm. Blophys Acta 1076,245-25 1. 8. van Duijnhoven, H. L. P., Verschuren, M. C. M., Timmer, E. D. J., Vissers, P. M. A M., Groeneveld, A , Ayoubi, T. A. Y., van den Ouweland, A. M. W., and van de Ven, W. J. M. (1991) Application of recombinant DNA technology in epitope mapping and targeting. Development of a panel of monoclonal antibodies against the 7B2 neuroendocrme protein. J Immunol Methods 142, 187-198. 9. Gross, C. H. and Rohrmann, G. F. (1990) Mapping unprocessed epitopes using deletion mutagenesis of gene fusions. Biotechniques 8, 196-202. 10. Mehra, V., Sweetser, D., and Young, R. A. (1986) Efficient mapping protein antigenic determinants. Proc Nat1 Acad Sci. USA 83,7013-7017. 11. Bantmg, G. S., Pym, B., Darling, S. M., and Goodfellow, P. N. (1989) The MIC2 gene product: epitope mapping and structural prediction analysis define an integral membrane protein. Mol. Zmmunol. 26, 18 l-l 88. 12. Smith, A. M., Woodward, M. P., Hershey, C. W., Hershey, E. D., and Benjamin, D. C. (199 1) The MIC2 gene product: epnope mapping and structural prediction analysis define an integral membrane protein. J Immunol 146, 1254-1258 13. Sedgwick, S. G., Nguyen thi Man, Ellis, J. M., Crowne, H., and Morris, G. E., (1991) Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. Nucleic Acids Res. 19, 5889-5894. 14. Guyer, M. (1983) Uses of the transposon $ in the analysis of cloned genes. Methods Enzymol 101,362,363. 15. Liu, L., Whalen, W., Das, A , and Berg, C. M. (1987) Rapid sequencing of cloned DNA using a transposon for b&directional primmg: sequence of the E. colz K- 12 avtA gene. Nucleic Acids Res 15, 9461-9469

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16. Thomas, S. M., Crowne, H. M., Pidsley, S. C., and Sedgwick, S. G. (1990) Structural charactertzation of the Salmonella typhimurium LT2 umu operon J. Bacterial.

172,4979-4987.

17. Strausbaugh, L. D., Bourke, M. T., Sommer, M. T., Coon, M. E., and Berg, C. M. (1990) Probe mapping to facilitate transposon-based DNA sequencing. Proc. Natl. Acad. Scl USA 87,6213-62 17 18. Boyer, H. W. and Roulland-Dussoix, D (1969) A complementation analysis of the restriction and modification of DNA in Escherrchia coli. J Mol Biol 41, 459-472.

19. Maniatis, T., Fritsch, G. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratoly Manual, 2nd ed Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 20. Berg, C. M., Vartak, N. B., Wang, G., Xu, X., Liu, L., MacNeil, D. J., Gewam, K. M., Wiater, L. A., and Berg, D. E. (1992) The my6 element, a small y6 (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing. Gene 113,~16. 2 1. Reed, R. R. (198 1) Resolution of co-integrates between transposons y6 and Tn3 defines the recombinatron site. Proc. Nat1 Acad. Sci. USA 78,3428-3432. 22. Hanahan, D. (1983) Studies on transformation of Eschenchia coli with plasmids J. Mol. Biol. 166,557-580.

29 Incomplete Polypeptides of In Vitro Translation for Epitope Localization Lisa Djavadi-Ohaniance

and Bertrand

Friguet

1. Introduction In vitro protein synthesis often leads to the production of polypeptide chains of intermediate lengths in addition to the full-size polypeptide. This accumulation of nascent chains of discrete sizes has been observed for a variety of proteins and has been attributed

mainly

to ribosome

pausing at specific sites of

mRNA owing to rare codons (1,2) or to the secondary structure of the template mRNA (3). Recently, Tokatlidis et al. have presented evidence that mtermediate-length polypeptide chains obtained during cell-free translation of the P-subunit of Escherichia coli tryptophan synthase do not correspond to temporary pauses, but rather to incomplete N-terminal chains owing to mRNA cleavage at preferential sites (4). Whatever the reason leading to the discrete pattern of N-terminal nascent chains observed in cell-free transcription-translation systems, we have taken advantage of this situation to determine, during the in vitro gene expression of the P-subunit of tryptophan synthase, the size of the smallest N-terminal fragments recognized by monoclonal antibodies (MAbs) directed against this protein (5,. Since this method permits delineation of the C-terminal border of the epitope, it could be useful to apply this approach to identify an appropriate region for further epitope mapping by other methods, such as those using synthetic peptides. In addition to the discrete pattern generated spontaneously during gene expression, truncated protein synthesis can be obtained either by arrest of mRNA translation at predetermined sites by oligonucleotides complementary to defined coding sequenceswithin the mRNA (‘6)) by using DNA fragments generated with enzymes (7), or by PCR (8,9).

From

Methods II) Molecular Biology, vol 66 Epltope Mappmg Protocols Edlted by G E Morns Humana Press Inc , Totowa, NJ

355

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Djavadi-Ohaniance and Friguet

Fig. 1. SDS gel electrophoresisof nascentpolypeptide chains immunoadsorbed with different MAbs. After in vitro synthesisof E. coli tryptophan synthaseS-chains, the polypeptides were immunoadsorbedand the N-terminal polypeptides of different lengths analyzedby gel electrophoresisand autoradiographyas describedin Section 3. (A) Patternobtainedwith antinativeMAbs 68-1 (a), 19-1(b), 164-2(c), and 93-6 (d). (B) Patternobtainedwith antidenaturedMAbs 14-1 (a), 30-2 (b), B3B5 (c), andD4B6 (d). The regions recognizedby MAbs 164-2,93-l, and D4B6 were previously identified: the sequence273-283 for MAb 164-2,340-397 for MAb 93-6, and 363-397 for MAb D4B6. As expected,antibody 164-2 reacts with the N-terminal polypeptides carrying at least the first 280 amino acids. Antibodies 93-6 and D4B6, which are directed againstthe C-terminal end of the protein, recognizeonly N-terminalpolypeptideswith the sizeof me completeS-chain(397 residues,44 kDa).

The rationale of the method described in this chapter is the following: 1. The expressionof the coding DNA is achieved in vitro in a coupled transcription-translation systemcontaining 35S-methionine. 2. Aliquots of the mixture are incubatedwith eachMAb to allow the immunoreactive polypeptide fragmentsto bind to the antibody; if available, MAbs for which a region recognizedon the protein is known are usedas control. 3. Thepolypeptide-antibodycomplexesareadsorbedwith protein-G-Sepharose beads. 4. The radiolabelednascentchainsboundto the protein-G Sepharosebeadsare subjected to SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and their presencedetectedby autoradiographyof the gel. 5. The size of the chains is determinedby its electrophoreticmobility.

An example of such an experiment is shown in Figs. 1 and 2. A special situation that may be encountered is the lack of binding of an MAb to the generatedpolypeptides. Indeed, the possibility for an epitope to reach a conformation similar to the one in the cognate protein will depend on the ability of the nascentpolypeptides to undergo folding stepsnecessaryfor

Incomplete Polypeptides

357

Da L 80SO40. 30.

20'

10'

I Q25

I 0.5

I , 0.75 Ri

Fig. 2. Semilogarithmic plot showing the size of the smallest N-terminal fragments recognized by the different MAbs, as determined from electrophoretic analysis. The logarithm of the molecular mass of the different protem markers, including the p-subunit, is plotted vs their respective migration ratio (RJ. Arrows indicate the migration of the smallest N-terminal fragments recognized by the different MAbs: a: 93-6, D4B6; b: 164-2, c: 68- 1, d: 30-2, e: 14- 1. The point corresponding to the low-mol-wt marker 12.5 kDa is out of the straight line, since the lO-25% polyacrylamide gel used in thrs experiment does not permit the precise determination of small-mol-wt polypeptides. of the immunoreactive conformation. This could be the case, especially for epitopes formed by noncontiguous regions of the protein. For instance, the fact that in the experiment reported in Fig. 1, in addition to the antinative antibodies, antidenatured antibodies have recognized even the fulllength chains shows that all these nascent chains are not tightly packed. The antibodies referred to as antidenatured antibodies are those directed against epitopes that are hidden in the native P2-subunit of E. coli tryptophan synthase and exposed on nonnative forms of the protein, i.e., coated onto plastic or denatured on treatment by N-ethylmaleimide (see ref. 20). It is important to emphasize that in this method the antibody-polypeptide chains recognition is achieved in solution under native conditions. the acquisition

2. Materials 1. Expression plasmid encoding the desired protein and containing the appropriate prokaryotic promoter and ribosome binding sites.Template DNA purity is extremely important and influences the efficiency of the in vitro protem synthesis.

358

Djavadi-Ohaniance

and Friguet

2. E. coEi S30 extract system for circular DNA (Promega, Madison, WI). 3 35S-methionine (1000 Ci/mmol at 15 mCi/mL), for instance from Amersham (Little Chalfont, UK) (store at -70°C m ahquots, use each thawed ahquot once) 4. Nuclease-free 0.5- and 1.5-mL microcentrifuge tubes 5 Nuclease-free sterilized micropipet tips. 6 Nuclease-free water for plasmid preparation and in vitro protein synthesis. 7 Purified MAbs. 8 Protein G-Sepharose beads (Pharmacia). 9. Buffer A: 20 mM HEPES, pH 7 6, 100 mM potassium acetate, 10 mA4 magnesium acetate. 10. Buffer B* buffer A supplemented with 0.1% (w/v) Nonidet P-40. 11 Electrophoresis sample buffer: 10% (w/v) SDS, 20% (v/v) 2-mercaptoethanol, 16% (v/v) glycerol, 0.04% (w/v) bromophenol blue, 4 mA4 EDTA, and 40 mM Tris-HCl, pH 8.0. 12. Amplifier (NAMP 100, Amersham) 13. Whatman 3MM paper 14. Plastic wrap. 15 Film for autoradiography (for Instance, HyperfilmTM RPN 6 from Amersham) 16 Refrigerated microcentrifuge. 17. Electrophoresis equipment and reagents for SDS-polyacrylamide slab gels 18. Slab gel dryer. 3. Method

The following protocol is established for a standard reaction volume of 50 p-L of transcription-translation mixture (final volume) for testing five antibodies (with the controls). 3.1. Protein G-Sepharose

Bead Washing

1. Make the bead suspension homogeneous in the commercial vial before taking a 0.4-r& aliquot m a 1 5-mL microcentrifuge tube. 2. Add 1 mL of cold (4°C) buffer B to the 0.4-mL beads, mix, and centrifuge at 4OC for 1 mm at 10,OOOgin a microcentrifuge. 3. Discard the supernatant and repeat this washing procedure four times at 4°C with 1 mL cold buffer B. 4. Suspend the washed pellet of protein G-Sepharose beads in an equal volume of a 1 mg/mL casem solution m cold buffer A. 5. Incubate for 15 mm with gentle shaking at 4°C to saturate the hydrophobic surface of the beads. 6. Wash the beads again five times with buffer B. 7. Suspend the pellet of the last centrifugation at 50% (v/v) in cold buffer B; keep at 4“C.

359

Incomplete Polypeptides 3.2. In Vitro Transcription-Translation

1. Use a commerctally available E. coli transcription-translation system as the one from Promega (E colz S30 Extract System For Circular DNA), and follow the manufacturer’s recommendations with the exception of the amount of plasmid DNA. 2. Add the plasmid DNA (see Note 1) at a high concentration (see Note 2) to promote an enhanced production of intermediate-length nascent chains (m the experiment reported in Fig. 1, for 50 pL of transcription--translation mixture, 4 pg of plasmid were used).

3.3. lmmunoadsorption 1. In a series of 0 5 mL microtest tubes (see Note 3), put a constant volume of 100 pL of buffer B. 2 Add lo-pL aliquots of the transcription-translation mixture in each tube. 3. Add 30 pL of each MAb at 0.4 mg/mL in buffer B, mix and incubate the mtxture for 15 min at 4OC. 4. Add 50 pL of 50% (v/v) prewashed protein G-Sepharose beads (see Section 3.1.) and incubate for 15 min at 4°C under permanent mild shaking conditions to keep the beads in suspension. 5. Add 300 pL of cold buffer B and immediately centrifuge at 10,OOOgfor 1 mm in a microcentrifuge. Remove the supernatant and repeat this washing procedure twice with 0.5 mL of cold buffer B. 6 Add 10 pL SDS sample buffer on the pelleted beads and heat for 3 min at 100°C to dtssociate the radiolabeled polypeptides from the antibody and the beads.

3.4. Gel Electrophoresis

and Autoradiography

1. Run a polyacrylamide gel in the presence of SDS (see Note 4) according to Cabral and Schatz (II) or Schagger and von Jaggow (12) 2. Fix the gel by using the classical procedures of staining and destaining (see Note 5). 3. Incubate the gel for 1 h at room temperature in the dark m amplifier to increase the sensitivity of detection of 35S-labeled proteins. 4. Dry the gel as follows: Prepare a sheet of Whatman 3MM paper and put the gel on it; place the gel with the paper on a vacuum gel dryer; cover the gel with a plastic wrap; and dry for 1 h at 60°C. 5. Expose the dried gel to an autoradiography film overnight, or longer tf necessary, to visualize the labeled protein bands in the gel. 6. Determine the size of the chains by analyzing the electrophoretic mobility of the bands (see Note 6).

4. Notes 1. The template DNA purity is extremely important and influences the efficiency of the synthesis. Potential contaminants of purified DNA, such as sodium chloride

360

2.

3.

4 5

6.

Djavadi-Ohaniance

and Friguet

and polyethylene glycol, may inhibit in vitro expression. Residual NaCl can be removed from the ethanol pellet by washing with 70% ethanol and the polyethylene glycol can be removed by a chloroform extraction. Magnesium and potassium salts may also inhibit the system. The technical bulletin of Promega (No. 092, revised 7194) reports that an Increased amount of DNA can increase the number of internal translation starts or prematurely arrested translation products. Although usmg high amounts of DNA in our experiments had led to the production of C-terminally truncated products, no internal translation starts have been observed. For instance, m the experiment reported m Ftg. 1, it can be seen that the MAb D4B6 directed against the 22 C-terminal ammo acids of the P-chain has recognized only polypeptides with a size of the complete P-chain (44 kDa). If chams corresponding to internal initiation were present, (i.e., truncated at the N-terminal side), smaller nnmunoreactive products should be also visualized. The same concluston was reached by Lorenzo et al. (7) and Tokatlidis et al. (4), who have reported that almost all truncated chains produced during m vitro translation were immunoprecipitated by antibodies directed against the N-terminal end of the protein. When working with small amounts of protein G-Sepharose, 0.5-mL microtest tubes are preferred to reduce the wall surface and prevent, during shaking, beads stickmg to the walls of the tubes. Polyacrylamide gels of Schagger and von Jaggow (12) provide better resolution of low-mol-wt polypeptides (below 10 kDa). Coomassie staming is not sensitive enough to detect translation products. However, these staining and destammg steps will reveal the migration of the mol-wt markers and also help to wash out unincorporated labeled amino acids at the gel dye front. In interpreting the results in the original paper (5), we assumed that durmg antibody recognition, the nascent chains were still linked to the ribosome and that around 25 ammo acids at the C-terminal end were sheltered within the ribosome. Consequently, a length correspondmg to 25 ammo acids was subtracted from the length of the smallest immunoreactive chain determined in Fig. 2. However, experiments carried out recently with the m vitro translation of the P-chains of tryptophan synthase (4) have shown that 50% of the mcomplete nascent chains are released from the ribosome. If this is a common situation for in vitro synthesis systems (see also ref. 6), then the size of the smallest chain estimated from the gel corresponds to the real size of the smallest mnnunoreactive cham. An easy way to verify if incomplete nascent chains are released from the ribosome is to centrifuge (225,000g for 2.5 h at 4°C) the synthesis mixture through a glycerol cushion (0.5 mL of 40% glycerol in buffer A), and to submit the resulting pellet (ribosomal fraction) and the supernatant (free chains) to an SDS-PAGE and autoradiography. One should remember that this method is aimed at estimating the C-terminal border of the N-terminal fragment carrying the residues involved in the antigenic site and that this can be done with a precision of about 10-15 amino acids.

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361

References 1. Purvis, I. J., Bettany, A. J. E., Santiago, T. C., Coggins, J. R., Duncan, K., Eason, R., and Brown, A. J. P. (1987) The efficiency of folding of some proteins is increased by controlled rates of translation m vivo. A hypothesis. J. Mol. Biol 193,413-417. 2. Sorensen, M. A., Kurland, C. G., and Pedersen, S. (1989) Codon usage determines translation rate in Escherichla coli. J. Mol. Biol. 207,365-377. 3. Chaney, W. G., and Morris, A. J. (1979) Non uniform size distribution of nascent peptides: the effect of messenger RNA structure upon the rate of translation. Arch. Biochem. Biophys. 194,283-291.

4. Tokatlidis, K., Friguet, B., Deville-Bonne, D., Baleux, F., Fedorov, A. N., Navon, A., DJavadi-Ohaniance, L., and Goldberg, M. E. (1995) Folding and interaction with chaperones of nascent chains during their elongation on the ribosomes. Phil. Trans. R. Sot. Lond. 348,89-95.

5. Friguet, B., Fedorov, A. N., and Djavadi-Ohaniance, L. (1993) In vitro gene expression for the localisation of antigenic determinants: application to the E. coli tryptophan synthase p2 subunit. J. Immunol. Methods 158,243-249. 6. Haeuptle, M.-T., Frank, R., and Dobberstein, B. (1986) Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length. Nucleic Acids Res. 14, 1427-1447. 7. Lorenzo, F., Jolivet, A., Loosfelt, H., Hal, M. T. V., Brailly, S., Perrot-Applanat, M., and Milgrom, E. (1988) A rapid method of epitope mapping: application to the study of immunogemc domains and to the characterization of various forms of rabbit progesterone receptor. Eur. J. Biochem 176,53-&O. 8. Mackow, E. R., Yamanaka, M. Y., Dang, M. N., and Greenberg, H. B. (1990) DNA amphfication-restricted transcription-translation: rapid analysis of rhesus rotavirus neutralization sites. Proc. Natl. Acad. Sci. USA 87,5 18-522 9. Lesley, S. A., Brow, M. A. D., and Burgess, R. R. (1991) Use of ln vztro protein synthesis from polymerase chain reaction-generated templates to study interaction of E. coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies. J. Biol. Chem. 266,2632-2638. 10. Friguet, B., Djavadi-Ohaniance, L., and Goldberg, M. E (1984) Some monoclonal antibodies raised with a native protein bmd preferentially to the denatured antigen. Mel Immunol 21,673-677. 11. Cabral, F. and Schatz, G. (1979) High resolution one- and two-dimensional electrophoretic analysis of mitochondrial membrane polypeptides. Methods Enzymol. 56,602-613. 12. Schagger, H. and von Jaggow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins m the range from 1 to 100 kDa. Anal. Biochem. 166,368-379.

30 T-Cell Epitope Mapping with Synthetic Peptides and Peripheral Blood Mononuclear Cells Stuart J. Rodda 1. Introduction The epitopes recognized by the antigen receptors on T-lymphocytes (T-cells) consist of a molecular complex made up of a self-protein and a short linear peptide (11. The self protein is a product of the major histocompatibility gene complex (MHC), and is classified as either a Class I or a Class II molecule. Class I and Class II MHC molecules are specialized to hold and present peptides on the surface of the cell, making them available for interaction with the T-cell receptor (TCR). Despite the structural similarity of Class I and Class II MHC molecules, their functional roles are quite different. Class I MHC molecules are present on most cells in the body and are specialized for presentation of peptides derived from proteins synthesized within the cell. These Class Ipeptide complexes are recognized by cytotoxic T-cells (Tc or CTL), and are corecognized by the CD8 molecules present on the surface of CD8+ cells. In contrast, Class II MHC molecules are present only on certain specialized antigen-presenting cells (APC) and are recognized by helper T-cells (Th), which carry the CD4 molecule as the corecognition molecule for MHC Class II. As the names imply, Tc generally take the role of killer cells on recognition of an MHC Class I-peptide complex, whereas Th are programmed to help both the antibody and cytotoxic T-cell responses following contact with their specific MHC Class II-peptide complex. MHC Class I and Class II-peptide complexes are generated within the cell by quite different pathways, but in both cases, the peptide ends up held in a groove on the surface of the MHC molecule, and from the point of view of a TCR on the surface of a T-cell, it is the protein surface of the MHC-peptide complex that is being recognized, not a peptide as such. As a way of mapping From: Methods in Molecular Biology, vol. 66: Epltope Mapping Protocols Edited by: G. E. Morris Humana Press Inc., Totowa, NJ

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T-cell epitopes, it is possible to introduce synthetic peptides to a population of APC, whether presenting Class I or Class II MHC, and thereby to form MHC molecule-peptide complexes with the added peptide. These complexes are then recognized by the TCR as if they had been formed naturally. A major difference between the peptides recognized by Tc in assoctation with MHC Class I and peptides recognized by Th in association with MHC Class II is that the former (Class I) only binds short peptides of about nine residues, whereas the latter (Class II) usually associateswith peptides of 13-l 8 residues in length. As with monoclonal antibodies (MAbs), recognition of an “antigen” by a T-cell clone is very specific and is readily defined down to the role of individual amino acids within the peptide portion of the MHC-peptide complex (2). Thus, the same systematic approaches taken for total mapping of the linear (“continuous”) epttopes defined by antibodies (3) can be applied to T-cell clones, with allowance for the length of T-cell epitopes (see above). Owing to the wide variety of assays possible for measurement of responses of T-cell clones to peptide antigens, this chapter does not attempt to present specific methods for epitope mapping on clones (4). Rather, it presents a method for mapping of Th epitopes using one of the most readily available sources of T-cells, peripheral blood. The peptide synthesis and design strategies presented here for studies of peripheral blood mononuclear cells (PBMC) are equally applicable to, and easier to apply to, T-cell clones. PBMC contain a random selection of Th and Tc, along with various other cell types, especially monocytes and dendritic cells, both of which express MHC Class II and are good APC for Th cells. Although PBMC have been used in many studies of “cellular immunity” to whole antigens, they have rarely been successfully studied with peptide fragments as the antigen (5) because of the problems mentioned below. 1. The number of specific Th cells for any one epitope in a protein antigen is expectedto be low relative to the total numberof Th able to respondto the whole protein. PBMC are a small sample of all Th, and therefore replicate samplesof PBMC cannotbe expectedto contain a consistentnumberof cells responsiveto a particular epitope. 2. The quantity of peripheral blood available from human volunteers is very hmited, constrainingthe number of peptidesthat can be tested. 3. Spurious or “spontaneous” proliferation of PBMC in vitro often occurs. Conversely, PBMC from somedonors do not proliferate readily in somebatchesor formulations of culture media. Both factors make the interpretation of apparent antigen-driven proliferation lessreliable.

Offsetting theseproblems is the fact that PBMC readily proliferate in vitro in response to whole antigens or single helper epitopes. A culture of PBMC is

a self-amplifjring system,becausethe proliferation of one or a few specific Th

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cells triggers a cascade of recruitment of nonspecific T-cells (6). PBMC also represent a population of cells unselected by prior in vitro enrichment or selection, and may therefore represent a better “snapshot” of what is happening in an individual than can be found by cloning and characterization of single clones, no matter how carefully done (7). Thus, an aliquot of PBMC IS a selfcontained assay system for the Th repertoire, needing only a suitable culture medium and supply of antigen. The following approach has been used to tackle the problems of randomness of responses and the requirement for large numbers of peptides/replicates (8-1 I). 1. Sets of peptides covering an entire protein sequence are made by the Multipin method (see Chapter 14). Subsets of adjacent peptides are pooled for initial screening, and only those peptide pools that are stimulatory are exammed in further detail for the final identification of particular epitopes. 2. The usefulness of the available PBMC is maximized by performmg all assays with as many replicates of PBMC per test group as possible, with one hmtting factor being the need for sufficient cell numbers m each culture to provide both the APC and the nonspecrfic amplification fimction (IO). 3. A large bleed is obtained (e.g., >lOO mL), and any surplus of PBMC is frozen to allow a repeat assay or testing of individual peptides at a later date. 4. Mathematical methods and software have been developed to analyze the data from the proliferation assay. These take account of the Poisson nature of the responses observed in replicates and the need for appropriate controls to be performed in each experiment. The idea of peptide pooling, as implemented in the scanning of PBMC for Th epitopes, 1s equally applicable to testing of Th clones, and indeed the saving in effort can be much greater, since there are unlikely to be more than two adjacent active pools for each clone.

2. Materials 1. A set of overlapping peptides is designed for Th scanning (see Note 1). These may be synthesized using a kit from Chiron Mimotopes (Melbourne, Australia) or its distributors, purchased ready-made from Chiron Mimotopes, or can be made by one of the alternative methods for multiple synthesis of solution-phase peptides (12). 2. AR dimethylsulfoxide (DMSO) or HPLC grade acetonitrile for redissolving peptides. 3. A source of human PBMC: 50- 100 mL whole blood/donor or a fresh “buffy coat” from a blood bank (see Note 2). 4. Ficoll-paque (Pharmacia [Uppsala, Sweden] Cat, No. 17-0840-02) or Nycoprep 1.077 (Nycomed AS, Oslo, Norway) mononuclear cell-separation medium. 5. Sterile round-bottom mrcrotiter plates (Nunc [Roskilde, Denmark] Cat. No. 163320).

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6. Sterile 100-mL bottles contammg 10-15 glass beads (3-5 mm diameter) for defibrination of whole blood (see Note 3). 7. Culture media: “Incomplete” base medium is RPM1 1640 (ICN [Costa Mesa, CA] Cat. No. 12-602-54) with 2 wadded glutamine (ICN Cat. No. 16-801-49), 5 mMHEPES, pH 7.4, and 20 pg/mL gentanucin (KN Cat No. 10503 1) (seeNote 4) 8 A serum supplement, added to 10% (v/v), can be either autologous serum or screened, pooled human serum, preferably from type AB donors (ICN Cat No. 29-309-49) (see Note 5) 9. Specific positive control* whole protein antigen under study (see Note 6). 10. Nonspecific positive control: Concanavalin A mitogen (Sigma [St. Louts, MO] Cat. No. C0412) dissolved to 100 pg/mL m sterile distilled water. 11. High specific activity tritiated thymidme (40-60 Ci/mmol, e.g., ICN Cat. No. 24042) Store in the cold (Note: radiation hazard by ingestion) (see Note 7). 12. Cell harvester that collects cells from wells of a 96-well microtiter plate onto glass fiber filter paper (e.g., LKB Cat No. 1295-001) (see Note 8). 13. p-counter to count tritium-labeled DNA from individual wells (e-g , PharmaciaWallac Betaplate 1205). 14. Computer and analysis software, e.g., the “ALLOC” program, available free from Chiron Mimotopes Requires IBM-compatible computer with VGA display (see Note 9) 3. Method 1. Design a pepttde pooling/decoding strategy (I 0) (see Note 10) based on the number of peptides and on the total number of replicate wells that can be prepared with the number of PBMC available (see Note 11). 2. Dissolve the peptides in DMSO or in 40% (v/v) acetomtrtle/water to give solutions of at least 3 n-& (see Note 12). 3. Pool an aliquot of each peptide into pools of a size determined in step 1, keeping the stock solutions of the single peptides for later “decoding” of positive pools. 4. Dilute the peptide pools in salme or PBS, so that each peptide is at approx 10 @T, which is 10x the final concentration to be used m the assay (see Note 13). 5. Dispense 20 uL of each pool to replicate wells m a round-bottom mtcrotlter plate (see Note 14). Dispense 20 yL of diluent to the wells assigned as negative controls (Cells Alone), and 20 nL of each of the specific (protein antigen) and nonspecific (Con A) postttve controls. 6. Draw whole venous blood from the volunteer donors. We prefer to defibrmate whole blood with glass beads. Add 50 mL of freshly drawn whole blood immediately to a sterile lOO-mL bottle containing glass beads, and agitate gently endover-end for 10 mm or until the beads are firmly bound m the fibrin clot. Both serum and PBMC are recovered from the same blood sample (see Note 15). 7. Dilute the whole blood 1:l with “Incomplete” medium, and isolate the PBMC from this diluted blood by centrifugation over Ficoll-paque, following the manufacturer’s instructions. Recover the autologous serum from above the cell layer and immediately heat-inactivate (56°C for 30 min) (see Note 16).

T-Cell Epitope Mapping 8. After washing the PBMC band twice in incomplete medium to remove the Ftcollpaque (see Note 17), resuspend the cells in a “Complete” medium containing 10% v/v heat-macttvated autologous serum (see Note 18). 9. Adjust the PBMC concentration to 1.1 x 1O6viable PBMC/mL with the same medium and dispense 180 pL/well into all wells, mcluding the negative control wells (Cells Alone) and both positive controls 10. Incubate plates at 37°C and 5% CO, in a fully humidified atmosphere for 4 d (see Note 19). 11. For the last 6 h of the mcubation, add to each well 20 pL of medium (see Note 20) containing 0.25 pCi of tritiated thymtdine (see Note 7). 12. Using a multiple-well cell harvester, harvest all wells onto glass fiber filter paper, washing thoroughly with water to lyse the cells and capture the tritiumlabeled DNA. 13. Dry and transfer to scmtillant for P-counting in the usual way for the harvester and counter you have (see Note 21). 14. Count all wells, preferably to a precision of better than 5% (total count of >400) for all wells (see Note 22) 15. To analyze the data, begm by checking that sigmflcant responses (high cpm values relative to the Cells Alone controls) have occurred m the nonspectfic positive control (Con A) and the specific positive control (whole protein antigen). If no response has occurred, any data from the peptrde antigens must be treated with extreme caution. 16. Proceed to determine a cutoff cpm value, to be used to separate negative (unstimulated) cultures from positive cultures (see Note 23). A suitable basis for the calculation of the cutoff is usually the mean +3 SD of the cpm of the “no peptide” (Cells Alone) controls. 17. When each well has been scored as positive or negative, use Poisson statistics to calculate a precursor frequency for all experimental groups (e.g., each peptide pool), including the Cells Alone group and each of the positive controls. Estimate a confidence range for each precursor frequency value (see Note 24). 18. Test the significance of the difference between the precursor frequency value for each experimental group and the Cells Alone control group (see Note 25). Experimental groups that have a highly stgmficant precursor frequency can then be “decoded” in another proliferation experiment to identify the peptide responsible for stimulation of Th precursors.

4. Notes I. A general scheme for scanning for Th epitopes is to make 16-mer peptides offset along the protein sequence by four residues. This ensures that no continuous sequence of 13 residues or shorter is absent. Many other schemes are possible, e.g., 18-mers offset by 6,20-mers offset by 8, and so on. 2. Calculate on an expected yield of l-2 x IO6 PBMC/mL whole blood. Cells should be as fresh as possible, and should be used immediately or frozen m liquid nitrogen in 10% DMSO for later use.

Rodda 3. Beads can be recovered, washed, and sterilized for reuse. 4. Brands and batches of media should be carefully screened for ability to support proliferation and for absence of a nonspecific stimulatory effect (e.g., owing to endotoxin contamination). 5. Choice of serum is critical to the outcome. We have found autologous serum to be better than carefully selected pooled sera for many donors. If autologous serum is not to be used, then it is critical for success of this assay to screen and choose a suitable batch of pooled human serum. Serum from blood type AB donors is said to be superior to other types because of the lack of anti-A or anti-B antibodies. 6. Preliminary work to establish an effective dose level of the control antigen may be required. 7. A small dose of high specific activity thymidine is much more economical than a large dose of low specific activity thymidine, and gives equivalent cpm values. 8. The cell harvester and counter are a “matched pair;” a similar system is produced by Packard. 9. This software is not essential to the method, but greatly reduces the workload of data handling and analysis. 10. By testing pools of peptides, the amount of initial screening work is reduced. The saving in effort is maximized when the number of peptide pools that are negative (nonstimulatory) in the primary screen is maximal; no saving in testing effort accrues at all if all peptide pools are stimulatory. Following an initial scan with peptide pools, the peptides comprising any positive pools can be tested individually in a further experiment or, alternatively, can be tested as a set of smaller pools prior to the final testing of individual peptides. Frozen PBMC are a convenient source of cells for such repeat tests. 11. Total number of wells = (total no. of PBMC)/(no. of PBMC to be used/well). Each peptide or peptide pool should be tested in not less than eight replicate wells, preferably 16-24 replicate wells. 12. A final solvent concentration of up to 0.3% DMSO or up to 2% acetonitrile (HPLC-grade) is tolerable in a PBMC proliferation assay. Thus, as an example, 4 mA4 stock solutions of each peptide, used to make a pool of 10 peptides, can be diluted 400-fold to give a final concentration of 1 @4of each peptide and ~0.3% DMSO. Store the peptide solutions at -20 or -70°C between uses. Poorly soluble peptides may be warmed or sonicated to assist solubilization. 13. A small amount of dilution of the culture medium with this diluent occurs when the cell suspension is added. This is of minor consequence, but should be controlled for by preparing the “Cells Alone” controls and the positive controls in an analogous way. 14. Round-bottom wells keep the cells close together, enhancing antigen presentation and the uptake of cytokines released from activated cells. Flat-bottom plates give reduced responsiveness and V-bottom plates can cause toxicity from cell overcrowding. The positioning of replicates within the plate is not critical provided the plate is in a 100% humidified CO, incubator, which is rarely opened. (See Note 19.) Plates can be prepared in advance, stored frozen, and then thawed

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16.

17.

18. 19.

20.

21.

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just before use. Each plate should include replicates of the buffer (“Cells Alone” or “no peptide”) negative controls and two types of positive control: a whole antigen control to test antigen-specific responsiveness and a mitogen control to test nonspecific responsiveness on each plate. PBMC can also be obtained from heparinized or titrated blood, but the recovery of autologous serum suitable for culturing the PBMC is then more complicated. If whole blood has to be transported or the PBMC cannot be isolated immediately, a better cell recovery is obtained if the blood is held at room temperature rather than 4°C. Large numbers of leukocytes, obtained fresh from a blood bank as the “buffy coat” sample from a unit of blood, may also be used as the starting cell suspension for PBMC isolation. PBMC isolation seems to occur efficiently in tubes up to 50 mL in capacity but not in larger tubes, possibly owing to mixing effects. The fluid recovered from above the cell layer on Ftcoll-paque centrifugation 1s already diluted to approx 40% (v/v) serum by the initial dilution of whole blood with Incomplete medium. Heat inacttvatton does not improve the growth-supportmg properties of the serum for autologous cells, but prevents complement-mediated effects and makes the handling of unscreened serum safer. Immediate inactivation is necessary so that the autologous serum is available for resuspending the PBMC suspensron after washing (Section 3., step 8). The first centrifugation after diluting the cell layer containmg Ficoll-paque needs to be harder (higher speed or longer) than the subsequent ones, smce the medium in which the centrifugatron is occurring is still at higher density than the incomplete medium. Check for completeness of cell recovery, e.g., by examining the supernatant microscopically. Since the recovered serum is already diluted to 40% (see Note 16), a further 1:4 dilution of this with incomplete medium brings the serum concentration to 10% v/v. Although longer incubation times (up to 8 d) can give higher thymidine mcorporation, incubation for 3-5 d gives the best specific proliferation/background relationship. Incubation can be carried out inside a plastic lunchbox with a loosefitting lid, with a moistened tissue in the bottom, in a gassed, humidified incubator. This helps to minimize the effects of door opening. This is particularly important in order to avoid “edge effects” in 96-well plates. The precision of dispensing this radiolabel is vital for achieving high precision in the cpm values of replicates. Owing to the radiotoxicity of high specific activity tritiated thymidine, long labeling times are of little advantage in obtaining higher cpm values. Counters that count multiple samples simultaneously are more effective than single-channel counters, even if they do not have quite as high a counting efflciency as a single-channel counter. Thus, a counter that counts all 96 samples simultaneously could be as little as 10% as efficient as a six-channel counter and still be more effective, i.e., with a given level of radioactive isotope, the 96-well counter will still achieve the target counting precision in a shorter time than the six-channel counter.

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22. Aiming for higher precision than 5% is of little value owing to the variation in replicates and the errors arising from the thymidme dispensmg step (see Note 20). 23. The “ALLOC” software, available free from Chiron Mlmotopes, performs the cutoff analysis automatically after the user chooses from a menu of four algorithms. 24. The “ALLOC” software also calculates the precursor frequency and confidence range for each experimental group, usmg the Poisson distribution as the operational model. 25. The dtfference can be tested to the p < 0 0025 significance level (that the two groups are the same), which is the condition that applies when the upper 95% ltmtt of the control (Cells Alone) group 1s below the lower 95% limit of the experimental group.

Acknowledgments The method presented here was the product of a team effort involving inspiration by H. Mario Geysen, and contributtons from David Mutch, Jeanette Reece, Donna McGregor, Mary Benstead, Tom Mason, and Paul Bennell, all of whom I wish to acknowledge gratefully. I also wish to express my appreciation for the support given to the research effort by Nevtlle McCarthy. References 1. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R G , Strommger, J. L., and Wiley, D. C. (1993) Three-dimensional structureof the human classII histocompatibtlity antigen HLA-DRI . Nature 364,33-39. 2. Suhrbrer, A., Rodda, S. J., Ho, P. C., Csurhes, P , Dunckley, H., Saul, A., Geysen, H. M., and Rzepczyk, C. M. (1991) Role of single amino acids m the recognitton of a T cell epnope. J Immunol. 147,2507-25 13. 3. Geysen, H. M., Rodda, S J., Mason, T J., Tribbick, G., and Schoofs, P. G. (1987) Strategies for epitope analysis using peptrde synthesis. J. Immunol. Methods 102, 259-274. 4. Mutch, D. A., Rodda, S J., Benstead, M., Valeno, R. M., and Geysen, H. M. (199 1) Effects of end groups on the sttmulatory capacity of mmlmal length T cell determmant peptides. Pept. Res 4, 132-137. 5. Brett, S. J., Blau, J., Hughes-Jenkins, C. M., Rhodes, J., Liew, F. Y., and Tote, J. P. (1991) Human T cell recognition of influenza A nucleoprotem Specificity and genetic restriction of irnmunodominant T helper cell epitopes.J. Immunol 147, 984-991. 6. Via, C. S., Tsokos, G. C., Stocks, N I., Clenci, M., and Shearer, G. M. (1990) Human in vitro allogenelc responses. Demonstration of three pathways of T helper acttvatton. J Immunol. 144,2524-2528. 7. Gammon, G., Klotz, J., Ando, D., and Sercarz, E E (1990) The T cell repertoue to a multtdeterminant antigen. Clonal heterogenetty of the T cell response, vartation between syngeneic individuals, and in vitro selection of T cell specificities. J Immunol. 144,1571-1577.

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8. Fard, G. A. B. P., Latchman, Y., Rodda, S., Geysen, M., Roitt, I., and Brostoff, J. (1993) T cell epitopes of the major fraction of rye grass Lolium perenne (Lo1 PI) defined using overlapping peptides in vitro and in vivo. I. Isoallergen clone IA. Clin Exp Immunol. 94, 11 l-l 16. 9. Reece, J. C., Geysen, H. M., and Rodda, S. J. (1993) Mapping the major human T helper epitopes of tetanus toxin The emerging picture. J. Immunol. 151,6 175-6 184. 10. Reece, J. C., McGregor, D L , Geysen H M., and Rodda, S. J. (1994) Scannmg for T helper eprtopes with human PBMC using pools of short synthetic peptides. J. Immunol. Methods 172,241-254. 11. Mutch, D. A., Underwood, J. R., Geysen, H. M., and Rodda, S. J (1994) Comprehensive T cell epitope mapping of HIV- 1 env antigens reveals many areas recognized by HIV- 1-seropositive and by low risk HIV- 1-seronegative individuals J. AIDS 7,879-890. 12. Jung, G. and Beck-Stckinger, A. (1992) Multiple peptide synthesis methods and their applications Angew Chem. 31,367-383.

31 Use of Natural or Selected Mutants and Variants for Epitope Mapping Glenn E. Morris This brief chapter is less of a protocol and more of a reminder that valuable information on the location of epitopes can be obtained by studying antibody binding to naturally occurring variants of the antigen. These variants are usually antigens from different species or different isoforms of the antigen. Of course, amino acid sequences from different speciesmust be available, but this now applies to a large and growing number of proteins on the data bases. Since these natural variants retain their function (e.g., enzyme activity), the amino acid changes are unlikely to have affected antibody binding by causing global changes in protein conformation. However, in the case of assembled epttopes, caution is still required before concluding that the altered amino acid is a “contact” residue within the epitope, especially if no supporting evidence is available. With linear epitopes, the conclusion can often be confirmed by using synthetic peptides. For globular proteins, mutations that have no effect on protein function alter surface amino acids more frequently than amino acids in the protein “core,” and it is these surface amino acids that are also involved in antibody binding. Consequently, the chances of an MAb displaying species specificity are rather high, even when the antigen is fairly highly conserved overall. The use of neutralization escape mutants of viruses for epitope mapping of MAbs that inhibit viral infectivity is a similar approach, except that the high rates of viral replication and “evolution” make it possible to use selection methods to generate variants with altered MAb binding. In principle, cells are treated with virus in the presence of a neutralizing MAb. Those that escape neutralization replicate, are isolated, and their amino acid changes are identified by DNA sequencing. The assumption 1sthat an amino acid change within the epitope From. Methods m Molecular Biology, voi 66: Epltope Mapping Protocols Edited by. G. E. Morris Humana Press Inc , Totowa, NJ

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374 AVGAVFDISNADRLGFSEVEQVQMWDGVKLMVEMEKKLEQNQPIDDMIPAQK S s

v

G G G G SIY

V V V V

L L L

S S S L L L L

KG S KG S KG S LI LI LI LI

R QR QR QR R

KG S G G A G A NGKS

Chick

M

Rat M Rabbit M Human M LM LM LM LM L

Chick Rat Human Rabbit Torlsedo

B B B B

Fig. 1. Sequencesof the last53 aminoacidsof nine creatrnekinasesThe chick M-CK sequenceis shown in full, and only differences from this sequenceare shown for the others.Single arrows show four residuesthat areidentical m the four B-CK isoforms, but different m M-CKs and Torpedo CK. Double arrows show two residuesare identical in Torpedo CK and the four B-CKs, but Qfferent m all four M-CKs. has prevented MAb from binding to the escapemutant and enabled infection to occur. Because the mutant is infective and functional, it can be argued that the mutation is less likely to have caused anything more than just local conformational changes. Epitopes recognized by antibodies that inhibit viral infectivity are, of course, of particular interest to virologists m search of novel antiviral vaccines. A selection of recent references (14) will serve as an introduction to the literature on this approach. I will illustrate the use of antigen variants for epitope mapping with examples from studies of MAbs against the enzyme, creatme kinase (7,s). This dimeric enzyme has two identical subunits of 381 amino acids and exists as two isoforms; M-CK, found mainly in muscle, and B-CK, found m most other tissues,especially brain. There is also a mitochondrial isoform, and all three forms are produced from different genes. Ammo acid sequences for both M and B forms from several species have been available for some time. Two MAbs, CK-END 1 and CK-END2, were shown to bind to a proteinase K fragment that consists of the last 53 ammo acids (7). This sequence is shown in Fig. 1 for nine different CK variants. Binding studies by ELISA or Western blotting (purified antigen is not required for the latter method) showed that CK-END 1 bound only to the four B-CKs, whereas CK-END2 also recognized CK from the electric fish, Torpedo marmorata. There are four amino acid changes that could account for the species specificity of CK-END 1, but only two that could account for CK-END2 specificity (see arrows at the bottom of Fig. 1). Syn-

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thetic peptides revealed that both MAbs will recognize a peptide composed of the last six amino acids only, LMPAQK. To confirm the specificity, we synthesized the corresponding peptide in M-CKs, MIPAQK, and neither MAb would bind to it (7). The results suggest that the Leu residue is required for CK-END2 binding, whereas the Met residue and possibly the Leu residue as well are required for CK-END1 binding. To illustrate the application of this approach to a conformational epitope, I shall describe the mapping of CK-HTB, an MAb against human B-CK subunit, which is one component of the commercial Hybritech two-site ELISA for the human mixed dimer, MB-CK, a serum marker for cardiac infarction. Most MAbs against. CK recognize either native CK only or only the denatured enzyme, but CK-HTB is unusual in recognizing an epitope on native CK, which can survive denaturation. This enabled us to map the epitope initially to a C-terminal fragment of M,. 14,000 on Western blots, but any further fragmentation resulted in loss of binding (7). In this respect, it resembles our own chick M-CK-specific MAb, CK-ART, which also recognizes both native and denatured CK and binds to the same C-terminal region of the CK molecule (8). Unlike CK-ART, however, CK-HTB was found to display a very unusual species specificity; of the nine forms shown in Fig. 1, it recognized two brain forms (human and chick) and one muscle form only (rat). Within the 14,000 Mr fragment, only one amino acid change was found that might account for this strange specificity. The three forms recognized by CK-HTB MAb all have an Asn residue at position 300 in the sequence, whereas the other sequences have an His residue in this position (except chick M-CK, which has Lys) (7). References 1. Ping, L. H. and Lemon, S. M. (1992) Antigenic structure of human hepatitis-A virus defined by analysisof escapemutantsselected against murine monoclonal antibodies. J. Viroi. 66,2208-22 16. 2. Yoshiyama, H., MO, H. M., Moore, J. P., and Ho, D. D. (1994) Characterization of mutants of human immunodeficiency virus type 1 that have escaped neutralization by a monoclonal antibody to the GP120 V2 loop. J, Virol. 68,974--978. 3. Saito, T., Taylor, G., Laver, W. G., Kawaoka, Y., and Webster, R. G. (1994) Antigenicity of the N8 influenza-A virus neuraminidase. Existence of an epitope at the subunit interface of the neuraminidase. J. Virol. 68, 1790-l 796.

4. Zhou, Y. J., Bums, J. W., Morita, Y., Tanaka, T., andEstes,M. K. (1994) Localization of Rotavirns VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure. J. ViroE. 68,3955-3964. 5. Ciarlet, M., Hidalgo, M., Gorziglia, M., and Liprandi, F. (1994) Characterization of neutralization epitopes on the VP7 surface protein of serotype Gl 1 porcine rotaviruses.J. Gen. Virol. 75, 1867-l 873.

376 6. Shotton, C., Arnold, C., Sattenau, Q., Sodroski, J , and McKeating, J. A (1995) Identification and characterization of monoclonal antibodies specific for polymorphic antigenic determinants within the V2 region of the human immunodeficiency virus type 1 envelope glycoprotein. J. Virol. 69,222-230. 7. Nguyen thi Man, Cartwright, A. J., Osborne, M., and Morris, G. E (1991) Structural changes in the C-terminal regron of human brain creatine kinase studied with monoclonal antibodies. Biochim Biophys. Acta 1076,245-25 I 8. Morris, G. E. (1989) Monoclonal anttbody studies of creatme kmase. The ART epitope: evidence for an intermediate in protein folding. Biochem. J 257,461469.

32 Production of Panels of Monoclonal Antibodies by the Hybridoma Method Nguyen thi Man and Glenn E. Morris 1. Introduction Since the first description by Kohler and Milstein (I), many variations on this method for the production of monoclonal antibodies (MAbs) have appeared (e.g., 2-4, and it may seem superfluous to add another. The variation we describe here, however, includes a number of refinements that enable rapid (6-l 0 wk) production from a single spleen of large numbers (20-30) of cloned, established hybridoma lines producing antibodies of high affinity. We have applied this method to recombinant fusion proteins containing fragments of the muscular dystrophy protein, dystrophin (5-@, and dystrophin-related proteins (9,ZO); to hepatitis B surface antigen (II); and to the enzyme, creatine kinase (12). We have used the MAbs thus produced for immunodiagnosis, epitope mapping, and studies of protein structure and function (5-25). Epitopes shared with other proteins are common (e.g., dystrophin and a-actinin [Id]), so availability of several MAbs against different epitopes on a protein can be important in ensuring the desired specificity in immunolocalization and Western blotting studies (9). In the standard Kohler and Milstein method, Balb/c mice are immunized with the antigen over a period of 2-3 mo, and spleen cells are then fused with mouse myelomas using polyethylene glycol (PEG) to immortalize the B-lymphocytes secreting specific antibodies. The hybrid cells, or hybridomas, are then selected using medium containing hypoxanthine, aminopterin, and thymidine (BAT medium) (I 7). All unfused myelomas are killed by BAT medium, and unfused spleen cells gradually disappear in culture; only hybridomas survive. From

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Special features of the detailed method to be described here include: 1 2 3. 4.

A culture medium for rapid hybridoma growth without feeder layers

Screening early to select high-affinity antibodies. Cloning without delay to encourage hybridoma growth and survtval. The use of round-bottomed microwell plates to enable rapid momtormg growth with the naked eye.

of colony

In typical tislon experiments, cells are distributed over eight microwell plates (768 wells), and 200-700 wells show colony growth. Of these hybridomas, up to 50% (100-300) produce antibodies that show some antigen binding, though many may be too weak to be useful. After further screening for desired affinity and specificities, we normally select 25-30 as a convenient number to clone. Only occasionally, in our experience, are two or more MAbs produced that are mdls-

tmguishable from each other when thetr epitope specificities are later exammed m detail (18). Table 1illustratesthe resultsobtained from eight fusions by this method. In the early stages after fusion, a particularly good cell culture system IS needed to promote rapid hybridoma growth in HAT medium. Horse serum, selected for high cloning efficiency, is a much better growth promoter than most batches of fetal calf serum, and HAT medium is prepared using myelomaconditloned medium and human endothelial culture supematant, a hybrldoma growth promoter.

Under these condttions,

colonies are vlslble with the naked

eye 7-10 d after fusion, and screening should start Immediately. Even ummmunized mouse spleens will produce large numbers of colonies, so the use of hyperimmumzed mice, with high serum titers in the screening assay, is essential for generating a high proportion of positive colonies. The screening assaysare also of critical importance, because they determine the characteristics of the MAbs produced. Both these points are illustrated in Fig. 1. Creatine kinase (CK), a fairly typical “globular” enzyme, is denatured and inactivated when attached directly to ELISA plastic, but It can be captured onto ELISA plates in its native form by using Ig from a polyclonal antiserum (12). When mice were nnmunized with untreated CK, antibody titers against denatured CK were high, whereas titers of antibody against native CK were insufficient to produce a plateau in the capture ELISA, even at high serum concentrations (Fig. 1A). Seven fusions from such mice (over 1000 colonies screened) produced only two low-affinity MAbs specific for native CK. In contrast, when we immunized with CK aggregated by glutaraldehyde treatment (Fig. lB), titers were higher for native CK antibodies than for those against denatured CK. In a fusion performed with one of these mice, only 2% of the fusion wells were positive

in the direct ELISA

(denatured

CK), but 13% recogmzed

only

native CK in the capture ELISA (12). This illustrates the importance of having both high-titer mice and a suitable screen.

An Efficient Method for MAb Production Table 1 Examples

of Colony

Antigen 108-kDa rod fragment of dystrophin in fusion protem (5) 55 -kDa C-terminal fragment of dystrophin m fusion protein (6) 59-kDa rod fragment of dystrophm in fusion protein (7) 34-kDa rod fragment of dystrophin in fusion protein (8) 37-kDa C-terminal fragment of a dystrophm-related protein m fusion protein (9) 12-kDa C-terminal fragment of dystrophin m fusion protein (20) 8-kDa N-terminal fragment of dystrophin in fusion protein (21) 15-Amino acid peptide from /3-dystroglycan conjugatedto BSA (22)

379

and MAb Yields Using This Method

Wells wrth Wells binding to Wells with desired Final no. growth antigen strongly affinity/specificity of MA\,

587

136

27

16

700+

42

20

17

669

147

30

25

300+

47

22

12

186

54

24

19

791

252

23 (out of 143tested)

19

694

64

3

2

594

69

8

4

Finally, although the method is rapid and efficient, it is also labor-intensive, especially during screening and cloning. When embarking on the method, one should not be daunted by the prospect of having over 70 culture plates in the incubator simultaneously by the time that the later cloning stagesof a single experiment are reached.

Nguyen thi Man and Morris

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6

A Denatured

Denatured (dotted llnesj NathJe (solld lines)

B 0.0

10”

Serum

dilution

Aggregated

IO’”

CK

lmmunoget

ia-’

10’”

Serum

dilution

Fig. 1. Effect of aggregation of CK on the immune response m Balb/c mice. Test sera were taken from three mice immunized with untreated CK ([A] circles, squares, and triangles represent individual mice) or with glutaraldehyde-aggregated CK (B), and serial dilutions were used as primary antibody in either direct ELISA for denatured CK (dotted lines) or sandwich ELISA for native CK (sohd lines). (Reproduced with permission of Elsevier Science Publishers from [12/).

2. Materials 1. One or two vertical laminar flow sterile hoods (e.g., Gelaire BSB4, ICN/Flow, Costa Mesa, CA). 2. 37°C CO2 incubator (e.g., LEEC, Nottingham, UK; see Note 1) 3. Transtar 96 (Costar, Cambridge, MA) for plating fusions and removing hybridoma supernatants. 4. Round-bottomed 96-well tissue-culture microwell plates (NUNC /Gibco [Gaithersburg, MD] Cat. No. I-63320) (see Note 2). 5. Sterile tissue-culture flasks, 50 mL (NUNC/Gibco Cat No. l-63371), 260 mL (NUNC/Gibco Cat. No. l-53732), and 24-well plates (NUNCYGibco Cat No. l-43982); 1.8~mL cryotubes (NUNC/Gibco Cat No. 368632); sterile 30-r& umversa1 bottles. 6 Freund’s adjuvant, complete (Sigma [St. Louis, MO] No. F-4258) stored at 4°C. 7 Freund’s adjuvant, incomplete (Sigma No. F-5506) stored at 4°C. 8. Pristane (2,6,10,14 tetramethylpentadecane; Aldrich Chemical Co. [St. Louis, MO], No. T2280-2) stored at room temperature. 9. 500X HT is 6.8 mg/mL hypoxanthine (Sigma No H-9377) and 1.95 mg/mL thymidine (Sigma No. T-9250) (see Note 3). Filter to sterilize. Store in 5-mL aliquots in sterile plastic universals at -20°C (stable for a few years). 10. 1000X Aminopterin (Sigma No A-2255). 4.4 mg are dissolved in 25 mL of water (see Note 3). Since the chemical is toxic and teratogenic, it must be weighed with

An Efficient Method for MAb Production

11.

12 13.

14. 15. 16. 17. 18. 19.

20.

381

appropriate contamment and operator protection, Filter to sterilize. Store in 2-mL aliquots at -20°C, wrapped in aluminium foil to protect from light (stable for years). 50% polyethylene glycol (PEG), 1500 mol wt (BDH Chemical Ltd. [Poole, Dorset, UK] Prod. 29575): make up by weighing 10 g of PEG in a glass universal and autoclaving. While the solution is still hot (about 60°C), add 10 mL of DMEM/25 mM HEPES prewarmed in a 37°C water bath. Mix well and store m 5-mL aliquots at room temperature in the dark; it IS stable at least for 6 mo. The pH should be slightly alkaline (as judged by phenol red) when used for fusion. DMEM/25 mMHEPES. (Gibco Cat. No. 041-2320H). This IS used as a “physiological saline,” not for growing cells. DMEMRO% HS is prepared by adding to each 100 mL of 1X DMEM (Gtbco Cat. No. 041-1885): 1.3 mL L-glutamine (200 miV) (Gibco Cat. No. 043-5030H), 1.3 mL sodium pyruvate (100 mM) (Gibco Cat. No. 066-01840E), 1.3 mL penicillm-streptomycin (Gibco Cat. No. 043-5140H), 0.6 mL nystatin (Gibco Cat, No. 043-5340H), 1.3 mL nonessential amino acids (MEM) (Gtbco Cat. No. 043-I 14OH), and 25 mL selected horse serum (HS). Forroutme growth and maintenance of both myelomas and established hybridomas, serum is reduced to 5% Cloning medium: DMEM/20% HS supplemented with 1X HT and 5% human endothelial culture supernatant (HECS) (Costar No. M712). HAT medium: myeloma-conditioned DMEM/20% HS with 1X HAT and 5% HECS. Medium for feeding the fusion: DMEM/20% HS with 5% HECS and 5X HT Medium for freezing down cells: 92.5% HS/7.5% dimethyl sulfoxide (DMSO) (Sigma No D-5879). Trypan blue (0.1%) (Sigma No. T6 146) in PBS (25 mM sodium phosphate, 0.9% NaCl, pH 7.2). NSO/l (2) and Sp2/0 (19) myeloma lines: These grow very fast and do not synthesize immunoglobuhns They should be kept in logarithmic growth m DMEM/ 5% HS for at least a week before fusion by diluting them to 5 x lo4 /mL when the cell density reaches 4 or 5 x 105/mL. If thawing myelomas from liquid mtrogen, it is advisable to begin l-2 wk before fusion (see Note 4). We have never found it necessary to treat lines with azaguanine to maintain their ammopterin sensrtivity, which can be checked by plating in HAT medium at 100 cells/well. Dissection instruments: scissors, forceps, and a scalpel with a large broad blade (Swann-Morton No. 21). Sterilize by dry heat (16O’C for 5 h), autoclave (120°C for 30 min), or dip m 70% ethanol and flame m a Bunsen burner.

3. Method

3.1. Immunization

of Balb/c Mice

1, Purify the protein antigen. With purified antigen, screening is faster and easier, and the yield of MAbs is higher, but proteins of 50% purity or less on SDSPAGE may still give good results. 2. Each of three Balb/c mice (6-8 wk-old) is given a subcutaneous (SC) injection of 50-100 pg of antigen in 0.1 mL PBS emulsified with an equal volume of Freund’s

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complete adjuvant by sucking up and down many times into a disposable sterile plastic 2-mL syringe with a sterile 2 l-gage 1l/z 0 8 x 40 No. 2 needle (see Note 5). 3 Four weeks later, an SCboost of 50-l 00 pg antigen in Freund’s mcomplete adjuvant is given. 4. Ten days later, blood (approx 0.1 mL) is taken from the tall vein, allowed to clot, and centrifuged at 10,OOOgfor 5 mm. Test the serum by the method to be used for screening hybridomas. If the titers are high in this assay (see Fig. l), the mice should be left to rest for a further month before fusion. 5. Boost the best mouse again with 50-100 pg of antigen m PBS, ip and/or iv in the tail vein, 4,3, and 2 d before fusion

3.2. Selection

of Horse Serum

Most supplrers will provide test samples of different serum batches and keep larger amounts on reserve. 1. Thaw serum m a 37°C water bath, and swirl to mix before incubating m a 56°C water bath for 30 min with occasional swirling (this heat-mactivation step 1snot known to be essentral). 2. Prepare cells of any hybrrdoma or myeloma line at 1000 cells/ml m DMEM, and dilute to 40,20, and 10 cells/ml in DMEM/20% HS of different batches (cloning of established cell lines does not require feeder layers or HT and HECS). For each test serum, plate one 96-well round-bottomed microplate with four rows at four cells in 0.1 mL/well, four rows at two cells m 0.1 mL/well, and four rows at one cell in 0.1 mL/well. 3. From the fifth day onward, note the number of colomes at each cell density for each test serum, as well as the size of the clones After 2 wk, choose the batch of serum wrth the highest number and the largest clones. Serum may be stored at -70°C for at least l-2 yr without loss of activity.

3.3. Fusion of Spleen Cells and Myeloma Cells 1. Do two separate fustons with each spleen as a precaution against accidents. 2. Two days before fusion, set up 2 x 260 mL flasks with 40 mL each DMEM and 20% HS and 1 x lo5 myeloma cells/ml (NSO/l or Sp2/0). 3. On the day of fusion, place m a 37°C water bath 100 mL of DMEM/25 mM HEPES, 10 mL of DMEM/20% HS, and one bottle of PEG1500. 4. Count the myeloma cells in a hemocytometer after mixing an ahquot with an equal volume of 0.1% trypan blue in PBS; cell density should be 4-6 x 105/mL with a viability of 100% and no evidence of contamination. 5 Transfer the myeloma cells to four universals, and centrifuge for 7 mm at 500g (MSE 4L). Remove the supernatants with a lo-mL pipet and retain them for plating the fusion later (myeloma-conditioned medium). Resuspend each pellet in 5 mL of DMEMHEPES, and combme m two universals (2 x 10 mL). 6. The immunized mouse is killed by cervical dislocation outside the culture room and completely unmersed in 70% ethanol (15-30 s). Subsequent procedures are performed m sterile hoods, Pin the mouse onto a “sterile” surface (polystyrene

An Efficient Method for MAb Production

7.

8.

9.

10

11. 12.

13.

14. 15.

383

covered with aluminum foil and swabbed with 70% ethanol), and a small mcision m the abdominal skin ts made with scissors. The peritoneum is then exposed by tearing and washed with 70% ethanol before opening it and pinning it aside The spleen is lifted out, removing the attached pancreas with scissors, and placed m a sterile Petri dish (see Note 6). After removing as much surroundmg tissue as possible, the spleen is placed in 10 mL of DMEM/25 mM HEPES, and held at one end with forceps while making deep longitudmal cuts with a sterile, curved scalpel to release the lymphocytes and red blood cells. This process is completed by scraping very gently with the scalpel blade until only connective tissue IS left (2-3 mm) The spleen cell suspension is transferred to a 25-mL universal, pipeting up and down five to SIX times to complete the drspersal. Large lumps are allowed to settle for 1 mm before transferring the supernatant to a second universal and centrifugmg for 7 mm at 5OOg. Resuspend the pellet in 5 mL of DMEM25 mA4 HEPES and keep at room temperature. Usually about 1 x10’ cells/spleen are obtamed (see Note 7). Add 2.5 mL spleen cell suspension to each 10 mL of myeloma cell suspension, and mix gently. Centrifuge at IOOOg for 7 min at 20°C. Remove supernatants with a pipet, and resuspend pellets m 10 mL DMEM/25 rmV HEPES using a pipet. Centrifuge at 1OOOgfor 7 min. Remove the supernatant completely from one pellet (use a Pasteur pipet for the last traces), and then loosen the pellet by tapping the universal gently (see Note 8). Remove the DMEM/25 mM HEPES and the 50% PEG from the water bath just before use. Take 1 mL of PEG in a pipet, and add dropwise to the cell pellet over a period of 1 mm, mixing between each drop by shaking gently m the hand. Contmue to shake gently for another mmute. Add 10 mL DMEM/25 mA4HEPES dropwise with gentle mixing, 1 mL during the first minute, 2 mL during the second, and 3.5 mL during the thrrd and fourth minutes. Centrifuge at 1OOOg for 7 min. (This procedure can be repeated with the second cell pellet, while the first is spinning.) Remove supernatant and resuspend pellet gently in 5 mL of DMEM/20% HS. Place both 5-mL cell suspensions in their universals m the CO2 incubator for l-3 h. Durmg this time, take the approx 80 mL of myeloma-condrtroned medium and add 4.5 mL of HECS, 90 pL of aminopterin and 180 pL of hypoxanthme/thymidine solutions. Filter 2 x 40 mL through 22-pm filters (47 mm, Millipore) to resterilize and remove any remaining myeloma cells. To each 40 mL, add the 5-mL fusron mrxture from the CO* incubator and distribute in 96-well microtiter plates (4 plates/fusion; 100 yL/well) using a Transtar 96 or a plugged Pasteur pipette (3 drops/well) (see Note 9). Put the plates from each fusion in a separate lunchbox in the COZ incubator and leave for 3 d. On d 4, add to each well 80 uL of DMEM/20% HS supplemented with HECS and 5X HT. Replace m the CO1 incubator as quickly as possible. By d 10-14, a high proportion of the wells should have a clear, white, central colony of cells, easily visible wrth the naked eye. If you want to select for hrgh-

Nguyen thi Man and Morris

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affinity MAbs (and you would be well advised to do so, unless you have some very special objectives), do not delay screening. If you are usmg a sensitive screening method, such as ELISA, nnmunofluorescence, or Western blotting, you will detect high-affinity antibodies from even the very small colonies. Do not wait for the medmrn to turn yellow, or your cells may start to die (if you are using a less sensitive screen, such as an inhibition assay, you may need to wait longer). A high proportion of the 768 wells (X)-100%) should have colony growth by 14 d. If it is
3.4. Screening It would be quite wrong to describe one screening method in detail, since this must be chosen to suit the purpose for which MAbs are required. We recommend ELISA (see Chapters 5, 6, 16, and 24) as the simplest technique for screening 8 x 96 wells in 1 d, but for coatmg ELISA plates directly, 1O-50 l.tg of reasonably-pure antigen are required. Alternatively, plates can be

coated with a capture antibody; we have used this approach to CK kinase in its native

conformation

(12) and to capture hepatitis

B surface antigen

from

human plasma (11). The availability of multichannel miniblotters makes direct screening by Western blotting feasible and with two miniblotters, 224 microwells can be screened in one long day. Immunohistochemistry on cultured cells grown in microwells is also feasible, although we have not used it ourselves. A Transtar 96 apparatus is a rapid and sterile way to remove lO-50 uL of culture supernatant from all 96 wells of a microwell plate. The culture plates have to be open inside the hood for only a few seconds. They should always be wiped with paper tissue soaked in 70% ethanol before opening, Screening the whole plate, including wells without cell growth, by ELISA, avoids possible errors when trying to keep track of individual wells. If ELISA-positive wells are to be screened further (e.g., by Western blotting using a multichannel miniblotter), this first 50 uL of supernatant may be diluted into 100 PL or more of PBS; multiple sampling of fusion wells should be minimized to avoid contamination. When best-quality horse serum is available, we usually screen by ELISA on d 10, carry out further screening of ELISA-positive wells on d 11 and 12, and clone about 30 of the best wells on d 12, 13, and 14.

3.5. Cloning by Limiting Dilution 1. Using a plugged Pasteur pipet, transfer the positive clone to a 24-well plate contaming 0.5 mL of cloning medium (see Note 11). 2. Take an aliquot of these cells, dilute with an equal volume of 0.1% Trypan blue in PBS, and count cells on at least two chambers of a hemocytometer.

An Efficient Method for MAb Production

385

3. Prepare 6 mL of 160 cells/ml, and perform serial dilutions to 40 and 10 cells/ml. Plate four rows at 16 cells in 0.1 n&/well, four rows at four cells in 0.1 mL/well, and four rows at 1 cell in 0 1 mL/well. Three drops from a plugged Pasteur pipet are about 0.1 mL. 4. Eight to ten days after plating, clones at 1 cell/well are visible and screening can start immediately. It is best to screen at least 16-24 wells from each cloning plate. As soon as positive clones have been identified, they should be cloned a second time, but at 4, 1, and 0.5 cells/well instead of 16,4, and 1. 5. When these plates are screened again after another 8-10 d, at least one dilution should have ~50% of the wells with growth, and all wells with growth should be positive in the screenmg. If any colonies are negative, cloning should be repeated until all wells are positive (see Note 12).

3.6. Preservation of Hybridoma Lines 1. Hybridoma clones should be expanded slowly from microwells, first into 0.5 mL in 24-well plates, feeding to l-2 mL before transferring to a 50-mL flask in 5 mL of medium. 2. To preserve cell lines mdefinttely, centrifuge l-3 x lo6 growing cells in a universal, remove all supernatant, suspend cell pellet in 0.3-0.5 mL. of ice-cold 7.5% DMSO/HS, and transfer the suspension into a 1.8-mL cryotube. 3. Surround the cryotube with 1 m of polystyrene, and place in a -70°C freezer. Transfer it the next day to a hquld nitrogen container. 4. It 1sadvisable to freeze several vials of the same line and thaw one after a week to ensure viability, while maintaining the cells in culture. 5. To recover cells from liquid nitrogen, thaw the cryovial quickly in a 37’Y! water bath until only a tiny piece of ice is visible. Transfer the cell suspension nnmediately to 10 mL of ice-cold DMEM/20% HS. Centrifuge at 500g for 7 min, remove supernatant completely, and resuspend the cell pellet in 3-5 mL of cloning medium. Transfer the cell suspension into a 50-n& flask, reserving about 0.1 mL to count viable cells, and place flask m the CO, incubator. If the viable cell count is
3.7. Antibody Production 1. To generate antibody as culture supematant, start a flask culture at 1 x 105/mL in DMEM/20% HS and, when the cell density reaches 4-6 x 105/mL, dilute to 1 x 105/mL. Reduce the concentration of horse serum gradually to 10 and then 5% in the process of reaching the volume required (see Note 14). Then leave the cells to grow undisturbed until they are all dead (see Note 15). Harvest the culture supernatant by centrifugation and store in aliquots at -20 or -8OOC until required. For routine use, keep an aliquot of l-2 mL with 0.1% sodium azide at 4°C. 2. For ascites fluid, prime an adult Balb/c mouse with 0.2 mL of pristane ip. 3. Seven to ten days later, harvest l-3 x lo6 hybrtdoma cells in logarnhmic growth (100% viability) by centrifugation at 500g for 7 min at 2O”C, and wash once with

386

Nguyen thi Man and Morris

DMEM/25 mMHEPES to remove all serum. Resuspend the cell pellet in 0.2-mL of DMEMHEPES, and inject ip into a primed mouse using a 21-gage No. 2 needle (green). 4. When the asctteshave developed, kill the mouse and harvest the tp fluid (seeNote 16). 5. Allow the fluid to clot, and then centrrfuge at 3000 rpm for 20 min. Store in ahquots of 200 pL at -20 or preferably -70°C.

4. Notes 1. Fusions from a single mouse spleen aimed at producing 20-30 cloned hybridoma lines by the method described here can eventually fill a standard-size bench-top incubator completely. The floor of the incubator is tilled with autoclaved, doubledistilled water. Do not use the heater on the inner glass door; a soakmg wet door is a good check for 100% humrdity The interior of the mcubator must be kept as free of contaminatton as possrble; this may necessitate removmg the contents, changing the water, and wiping the interior with 70% ethanol on several occasions during hybrtdoma growth and clonmg. Even so, we use large plastic lunchboxes to insulate unsealed culture vessels (e.g., mrcrowell plates) from the turbulent incubator environment (a fan is desirable for uniform temperature and humidity). Holes plugged with cotton wool at each end of the lunchbox to admrt CO2 are optional; an alternative is to leave the hds ajar for 1-2 min to equtlibrate before closing them. They can be wiped regularly, instde and out, with 70% ethanol Never use toxic chemicals (e.g., bleach) to decontammate an incubator. Never handle culture plates without gloves. 2. Cells roll to the bottom giving a white colony in the exact center of each well, visible even at early stages; flat-bottomed plates have initially transparent colonies, often at the side of the well and easily overlooked. Use of round-bottomed plates makes it very easy to monitor colony growth after fusion without using a microscope. 3. The mixture does not dissolve by itself, so add 1NNaOH dropwise while stirrmg until the solution becomes clear. 4. The myeloma cells should look uniform and healthy by this time with no srgn of cell debris. 5. Avoid syringes with rubber plunger tips; when the emulsion thickens, they come off the plunger. 6. We normally perform all steps up to this point in a separate sterile hood The mouse IS nonstertle externally, and particular care is taken to avoid hairs; the outer skin is torn, rather than cut, for this reason. Mice should not be Introduced into the hood used for cell culture. 7. Some protocols remove red blood cells by differential lysis at this point, but we have not found it necessary. 8. The final concentration of PEG during fusion is thought to be critical for the yield of colonies. If mitral yields are low, try adding increasing (but small) amounts of DMEM/25 mIi4 HEPES to the pellet before adding PEG. 9. Never use unplugged mtcroprpet tips for addmg to culture plates; sterile tips can be used for removing culture supernatant only.

An Efficient Method for MAb Production 10. Bacterial and yeast contaminations rarely occur and are usually the result of a major failure in sterile technique (e.g., inadequate sterilization of culture medium or glassware). Sources of any sporadic fungal contamination should be tracked down and eliminated. 11. Some protocols recommend freezing down uncloned or partly cloned cells as a precautionary measure. Following our rapid cloning protocol, however, the first round of cloning and screening is otten complete before the original colony is sufficiently expanded for freezing. It is certainly advisable to keep the original culture alive, however, by adding 0.1 mL of cloning medium back to the fusion well and by feeding the 24-well culture, if necessary. Cloning should not be regarded as an ordeal suitable only for healthy cells, but rather as a means of envigorating a failing culture. We always clone twice for this reason, even if the line is evidently clonal after the first round. As a general maxim, if in doubt or trouble, clone immediately. 12. There are rare exceptions to the general principle that cloning should be continued until all colonies are positive in the screens. We were once performmg an initial screen by ELISA and then testing ELISA-positive wells by mnnunofluorescence microscopy (IMF) on muscle sections. After cloning one well that was positive in both assays, we found that only half the clones were ELISA-positive, and very few of these were also IMF-positive. We recloned wells that were positive in both assays again with the same result. We thought we had come across our first “unstable” hybridoma, but by chance we tested ELISA-negative wells in IMF and found they were all IMF-positive. Only then did we realize that the original fusion well had contained two different hybridomas, one ELISA-positive and one IMF-positive. The purpose of cloning is to separate such lines, but by selecting wells that were positive in both assays (and hence still had two clones), we had been systematically defeating this objective. 13. When very few cells survive after being kept in liquid nitrogen, “cloning” by limiting dilution at about 100 total cells/well may be the only way to recover the cell line. 14. Cells may also be collected by centrifugation and resuspended m 5% fetal calf serum at this stage, if a culture supernatant with low levels of nonmouse Ig is required. 15. Most MAbs are stable for long periods in sterile culture medium, but there are undoubtedly some that lose activity rapidly even at 4°C. These are perhaps best avoided, but if required, bulk culture would have to be monitored regularly and supernatants harvested when their antibody activity is still high. 16. Ascites development becomes evident by external examination within 7-14 d, and mice must be examined twice a day over this period. Mice are killed by cervical dislocation as soon as they show signs of discomfort (advice should be sought and followed). The peritoneum is exposed as in Section 3.3., step 6, and a 2 l-gage needle on a 5-mL syringe is inserted about 1 cm so that the tip remains visible through the peritoneum and fluid can be withdrawn without blockage. The last traces of fluid are removed after opening the cavity with scissors. The final volume obtained is usually 2-3 mL, although blood from the heart and thoracic cavity can also be collected (about 0.5 mL) and processed separately as a source of antibody.

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Acknowledgment We thank C. J. Chesterton (Kmg’s College, London) for sharing with us his enthusiasm for, and experience of, hybridoma technology in I98 1.

References 1. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predetined specificity. Nature 256,495-497. 2. Galfre, G. and Milstein, C. (198 1) Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 73,346. 3. Fazekas de St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1-21. 4. Zola, H. and Brooks, D. (1982) Techniques for the production and characterization of monoclonal hybndoma antibodies, in Monoclonal Antibodies* Techniques and Appltcations (Hurrell, J. G., ed. ), CRC, Boca Raton, FL, l-57. 5. Nguyen thi Man, Cartwright, A. J., Morris, G. E., Love, D. R., Bloomfield, J. F., and Davies, K. E. (1990) Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin. FEBS Lett 262,237-240. 6. Nguyen thi Man, GinJaar, I. B., Van Ommen, G. J. B., and Moms, G. E. (1992) Monoclonal antibodies for dystrophin analysis--epitope mapping and improved binding to SDS-treated muscle sections. Biochem. J. 288,663-668. 7. Nguyen thi Man and Morris, G. E. (1993) Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy. Amer. J. Hum. Genet. 52, 1057-1066. 8. Le Thiet Thanh, Nguyen thi Man, Hori, S., Sewry, C. A., Dubowitz, V., and Morris, G. E. (1995) Characterization of genetic deletions in Becker Muscular Dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin. Am. J. Med. Genet. 58, 177-186. 9. Nguyen thi Man, Ellis, J. M., Love, D. R., Davies, K. E., Gatter, K. C., Dickson, G., and Morris, G. E. (1991) Localization of the DMDL-gene-encoded dystrophinrelated protein using a panel of 19 monoclonal antibodies. Presence at neuromuscular junctions, in the sarcolemma of dystrophtc skeletal muscle, in vascular and other smooth muscles and in proliferating brain cell lines. J Cell Btol. 115,1695-l 700. 10. Nguyen thi Man, Helliwell, T. R., Simmons, C., Winder, S. J., Kendrick-Jones, J., Davies, K. E., and Morris, G. E. (1995) Full-length and short forms of utrophin, the dystrophin-related protein. FEES Lett. 358,262-266. 1 I. Le Thiet Thanh, Buu Mat, Phan Ngoc Tran, Nguyen thi Vanh Ha, and Morris, G. E. (1991) Structural relationships between hepatitis B surface antigen in human plasma and dimers of recombinant vaccine: a monoclonal antibody study. Virus Res. 21, 141-154. 12. Nguyen thi Man, Cartwright A. J., Andrews K. M., and Morris G. E. (1989) Treatment of human muscle creatine kinase with glutaraldehyde preferentially increases the immunogenicity of the native conformation and permits production of high affinity monoclonal antibodies which recognize two distinct surface epitopes. J. Immunol. Methods 125,25 l-259.

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13. Nguyen thi Man, Cartwright, A. J., Osborne, M., and Morris, G. E. (1991) Structural changes in the C-termmal region of human brain creatine kmase studied with monoclonal antibodies. Blochlm. Bzophys. Acta 1076,245-25 1. 14. Morris, G. E. and Cartwright, A. J. (1990) Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase. Biochim Biophys. Acta 1039,3 18-322 15. Sedgwick, S. G., Nguyen thi Man, Ellis, J. M., Crowne, H., and Morris, G. E. (1991) Rapid mapping by transposon mutagenesis of epnopes on the muscular dystrophy protein, dystrophin. Nucleic Acids Res. 19, 588!+5894. 16. Nguyen thi Man, Ellis, J M., Ginjaar, I. B., van Paassen, M. M. B., van Ommen, G.J. B., Moorman, A. F. M., Cartwright, A. J., and Morris, G. E. (1990) Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin. FEBStett. 272, 109-l 12. 17. Littlefield, J. W. (1964) Selection of hybrids from matings of fibroblasts m-vitro and their presumed recombinants. Science 145,709,710. 18. Morris, G. E., Nguyen, C., and Nguyen thi Man (1995) Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin. Bzochem. J. 309,355-359 19. Shulman, M., Wilde, C. D., and Kohler, G. (1978) A better cell lme for makmg hybridomas secreting specific antibodies. Nature 276,269,270.

33 Production of Phage-Display Antibodies for Epitope Mapping Jenny Walker and George Banting 1. Introduction The development of genetic engineering has enabled the production of antibodies in Escherichia coli (I,2). An essential requirement for a good antibody expression system is that an antibody fragment is folded and in a functional state so that the selection or purification procedure can make use of its antigenbinding properties. The application of filamentous phage-display technology to the expression of antibody fragments enables the rapid cloning of an immunological repertoire to produce large libraries that can be screened for appropriate antigen-binding specificities. New expression systemshave been developed, inspired by a method where diverse peptides are displayed on the surface of bacteriophage (3,4). In these systems, an antibody fragment is cloned into a phagemid vector next to a gene encoding a bacteriophage coat protein and subsequently expressed attached to the coat protein on the surface of the bacteriophage (.5,6). Such surface expression allows the selection of specific antibodies based on their ability to bind to immobilized antigen. Those phage particles displaying a particular antibody fragment contain the gene encoding that fragment, thus creating a “genetic display package” (Fig, 1). This mimics the immune system where the rearranged gene encoding an antibody is found within the B-lymphocyte on whose surface the antibody is displayed. Several variations of antibody phage-display vectors have been described (5-7). Each system involves polymerase chain reaction (PCR) amplification of the relevant regions of the antibody gene. PCR primers have been designed based on conserved nucleotide sequences of antibody genes extracted from the Kabat database (8). The oligonucleotide primers used have included rare From

Mefhods m Molecular Biology, vol 66. fpltope Mapplng Protocols Edlted by G E Morris Humana Press Inc , Totowa, NJ

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392 Fab

Fig. 1. Cartoon representing a bacteriophage particle containing phagemid DNA that encodes for the Fab expressedon its surface. The Fab fragment is expressed attached to a gene 3 bacteriophage coat protein.

restriction sites in order to reduce the chances of digesting the imported immunoglobulin gene when restricting PCR products prior to cloning. The most complex proteins to be heterologously expressed on the surface of fllamentous phage are antibody Fab fragments (5). The cloning of these fragments is the basis of the system described in this chapter for the production of phage-display antibody fragments. Antibody Fab fragments are composed of the complete light chain and the heavy-chain Fd region, which consists of the variable domain and the first constant region of the heavy chain, The heavy- and light-chain variable domains alone make up the antibody Fv fragment. The constant region domains present in Fab antibody fragments facilitate strong association between the heavy and light chains relative to their smaller Fv counterparts (9). The binding between the heavy and light constant domains is both covalent (with the formation of a disulfide bridge) and noncovalent in nature. The resultant additional stability in the structure overcomes the need for an artificial covalent link between the chains, such as the peptide linker in single-chain Fv fragments. The phagemid vectorpComb3H-SS is a modified versionof pComb3, which was derived from the phagemid pBluescript (510) (see Fig. 2). In this system, unique restriction sites are provided for the independent cloning of the heavychain variable domain, and first constant domain (Fd) sequences (X401 and SpeI) and light-chain sequences(Sac1and XbaI). The SS designation (see Fig. 2)

393

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pCombSH-SS

on

Fig. 2. pCornb3H-SS phagemid cloning vector. See text for details.

representsthe presenceof stuffer fragments in the antibody fragment cloning sites. On expression,both the heavy- and light-chain antibody fragments are targeted to the periplasmic spaceby appropriateleader peptides.The cDNA encoding the light chain is inserted immediately 3’ of sequenceencoding the outer membraneomp A leaderpeptide of E. coli, whereasthe cDNA encoding the Fd chain is insertedbetween sequencesencoding the pel B leaderpeptide of Erwinia caratovora and the C-terminal domain of the phage coat protein g3p. The g3p portion of the resultant expressedfusion protein accumulatesin the inner membraneof the E. co&, with the Fd region in the periplasmic space, where it can associatewith a light chain to form a functional Fab fragment (Fig. 3). The expressionof the heavy and light chain sequencesis under the control of a lac promoter/operatorsequence.The phagemid vector is propagated in XLl-blue cells (II), thus allowing the induction of transcription by the addition of IPTG (22). Inclusion of the bacteriophageintergenic region in the vector allows helper phage initiated transcription of single-strandedphagemid DNA. The helper phage infection leads to the expression of native g3p as well as phagemidderived Fab-g3p. The resultant packagedphage carries native g3p, which is

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n

pelB Leader

Sequence

H chain Antibody

I

@P

Fig. 3. Cartoon representingthe pathway for the assemblyof an Fab antibody fiagment in the bacteriophageexpressionsystem.The antibody heavy and light chainsare directedto the periplasmic spaceby a leadersequence,which is subsequentlycleaved. The heavy and light chainsassemblein the periplasmic spaceto form a functional Fab fragment,which is secretedfrom the cell attachedto a gene3 bacteriophagecoatprotein.

necessaryfor infection, and the encodedFab-g3p, which is displayed for selection. According to published reports, Fab fragments expressedwith this system are monomeric in nature, thereby facilitating the isolation of specific, high-affinity antibody clones (13,14). The random combinatorial principles of the antibody library construction result in a complete scrambling of association between protein sequences encodedby the heavy- and light-chain genes,i.e., any heavy chain can associate with any light chain. As a result, the original heavy- and light-chain pair is

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unlikely to be recovered, and high-affinity (> 1OSmol-I) antigen-binding heavy and light chain combinations are likely to be rare. Two factors that improve their frequency in antibody libraries are (1) chain promiscuity-the ability of particular heavy chains to bind effectively antigen when combined with different light-chain partners (61, and (2) using RNA from an immune source in library constructton (5,6). Affinity purification of phage antibodies by “panning” enables the selection of even rare antigen-binding antibody combinations from a library. Antibody fragments can be displayed to antigen in the following ways: 1. Antigen adsorbedto a plastic surface ($15). 2. Columns of antigen linked to a matrix (26). 3. Biotinylated antigen in solution, subsequentlycaptured on streptavidm-coated magneticbeads (17). 4. Antigen expressedon the surface of an immobilized cell (18). Nonbinders can be removed by washing and the bound phage can then be eluted at low pH (5), high pH (Z8), or by addition of excess antigen (6). Successive rounds of selection can be achieved by infecting bacteria with the enriched phage and panning the phage prepared from the culture. The enrichment resulting from repeated rounds of selection should be sufficient to isolate specific phage occurring only singly in the initial library (i.e., about 1 in 107). Once phage-displayed antibody fragments have been selected against a specific antigenic target, they can be expressed without the g3p and can be screened for reactivity with antigen, usually by conventional enzyme-linked immunosorbent assay (ELISA). In this chapter, detailed methods for the production and panning of Fab antibody libraries will be described. In addition to the obvious advantage of obtaining a number of epttope-specific antibodies from a single library, the smaller size of Fab fragments (relative to whole antibody molecules) produced with this system may facilitate greater accessibility than whole antibodies to the target antigen, Antibody library construction circumvents the laborious process of hybridoma production and allows new approaches to antibody selection and design. With antibody cloning, one has immediate accessto the DNA encoding the Fab fragment of interest. The sequence encoding a Fab fragment that binds to an antigen with low affinity can be manipulated in order to encode a higher affinity antibody (19).

2. Materials 1. Mice immunizedwith the required irnmunogen(following protocols usedfor the production of monoclonal or polyclonal antibodies [20]; seealso Chapter 32). 2. Mechanical homogenizer,e.g., Polytron. 3. RNA extraction kit (Phannacia, Cat. No. 27-9270-01). 4. mRNA purification kit (Pharmacia,Cat. No. 27-9258-01).

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5. cDNA synthesis (Pharmacia, Cat. No. 27-9661-01) kits obtained from LKB Pharmacia. 6. The mouse immunoglobulin primers required for the amplification of heavy- and light-chain antibody fragments are given below (21). Heavy-chain Fd 3’-primers (reverse): IGgl S’-AGGCTTACTAGTACAATCCCTGGGCACAAT-3’ IGg2a 5’-GTTCTGACTAGTGGGCACTCTGGGCTC-3’ Heavy-chain variable domain 5’ primers (forward)* Hc 1 5’-AGGTCCAGCTGCTCGAGTCTGG -3’ Hc2 5’-AGGTCCAGCTGCTCGAGTCAGG -3’ Hc3 5’-AGGTCCAGCTTCTCGAGTCTGG-3’ Hc4 5’-AGGTCCAGCTTCTCGAGTCAGG-3’ Hc5 5’-AGGTCCAACTGCTCGAGTCTGG-3’ Hc6 5’-AGGTCCAACTGCTCGAGTCAGG-3’ Hc7 5’-AGGTCCAACTTCTCGAGTCTGG-3’ Hc8 5’-AGGTCCAACTTCTCGAGTCAGG-3’ Hc9 5’-AGGTIIAICTICTCGAGTC(TA)GG-3’ (see Note 1) Murine K light-chain 3’-primer (reverse): 5’-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3’ Murine light-chain variable-domain 5’-primers (forward): Lcl S’CCAGTTCCGAGCTCGTTGTGACTCAGGAATCT-3’ Lc2 5’-CCAGTTCCGAGCTCGTGTTGACGCAGCCGCCC-3’ Lc3 5’-CCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA-3’ Lc4 5’-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3’ Lc5 S’CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3’ Lc6 5’-CCAGATGTGAGCTCTCATGACCCAGTCTCCA-3’ Lc7 S’CCAGTTCCGAGCTCGTGATGACACAGTCTCCA-3’ 7. Tag Polymerase with supplied buffer and dNTP solution (5 mM stock solution). 8. Preparative gels should be made with Genetic Technology Grade (GTG) agarose available from Flowgen. Seakem or Nusieve GTG agarose is recommended. 9. Electrophoresis buffer (TAE): 0.2M Tris base, 0.0005M EDTA, and 0.12% acetic acid adjusted to pH 7.0. 10. DNA mol-wt standard markers. 11. Electroelution apparatus or see Note 2. 12. Restriction enzymesXba1, SacI,xhoI, S’eI, and NheI (Boehringer Mannheim, to be used with the supplied buffers). 13. Chromaspin 100 column (Clontech Laboratories Inc.). 14. The phagemid vector pComb3HSS (5) can be obtained from: Carlos Barbas III at The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037. 15. XLl-blue MRF cells (Strategene [II]). 16. LB agar: 10 g/L bacto-tryptone, 5 g/L bacto-teast, 5 g/L NaCl, 16 g/L agar. 17. Carbenicillin (carb) made up into 100 mg/mL stock. 18. Super broth (SB): 30 g/L bacto-tryptone, 20 g/L bacto-yeast extract, 10 g/L MOPS.

Phage-Display Antibodies 19. 20. 2 1. 22. 23. 24. 25.

26. 27. 28. 29. 30. 3 1. 32. 33. 34. 35.

36. 37. 38.

397

Preparative DNA kit with tip-500 columns (Qiagen). T4 DNA ligase (Gibco BRL recommended, to be used with 4X buffer supplied). 0.025pm microdialysis membranes (Millipore). SOC: 20 g/L bacto-tryptone, 5 g/L bacto-yeast extract, 0.5 g/L NaCl, 2.5 mMKC1, 10 mMMgCl,, and 20 mA4glucose (filter-sterilized and added after autoclaving). Tetracycline (tet): 10 mg/mL stock solution. Ml3 helper phage M13K07 (Pharmacia Cat. No. 27-1525-01). Stocks of the helper phage should be prepared and titered following standard procedures (15). Kanamycin (Kan): 100 mg/mL stock solution. PEG8000. PBS: 0.9% NaCl, 25 mM sodium phosphate buffer, pH 7.0. Blocking buffer: 3% BSA in PBS. Microtiter plates; any high binding capacity plate suitable for ELISA can be used. Relevant antigen diluted in coating buffer, e.g., O.lM bicarbonate, pH 8.6 (optimal coating buffer may vary with antigen). Humidified incubator at 37°C. PBS containing 0.5% Tween-20 solution (PBST). Elution buffer: 0. IM HCl (adjusted with glycine to pH 2.2), 1 mg/mL BSA. 2M Tris base neutralizing buffer. IPTG: O.lM stock solution m HzO. 10 mA4Tris, pH 8.0. Appropriate enzyme-conjugated anti-Fab second antibody, e.g., alkaline phosphatase-conjugated rabbit antimouse Fab (Pierce or Sigma). Appropriate developing substrate solutions, refer to ref. 20 for details.

3. Methods 3.1. Obtaining

cDNA from an Immunized

Source

The preferred and reliable method is to use Pharmacia kits, although other appropriate methods could be adopted. RNA can be quickly and efficiently obtained from the spleen of an immunized mouse (see Note 3) using a Pharmacia RNA extraction kit, following the manufacturer’s protocol. Precautions should be taken to minimize RNA digestion by ribonucleases present in the spleen cells (see Note 4). 1. Remove the spleen aseptically, directly transfer it to an aliquot (3 ml/spleen) of extraction buffer, and immediately homogenize. 2. Isolate polyadenylated mRNA on oligo(dT)-cellulose spun columns supplied in a Pharmacia mRNA Purification Kit, following the manufacturer’s protocol. 3. Prepare first-strand cDNA from either total RNA or mRNA using a Pharmacia First-Strand cDNA Synthesis Kit, following the manufacturer’s protocol. Prime the reactions with primers supplied with the kit, using either 5 pg of a N&I-d(T)1 8 bifunctional primer or 0.04 pg pd(N)6 in the reaction.

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and Cloning of Fab Fragments

The procedures followed are summarized in Fig. 4. 1. Follow standard technrques for PCR using an annealing temperature of 52°C and 35 cycles. Each reaction should be performed with individual primer sets m reaction volumes of 50 uL 2. Test for the presence of amplified antibody DNA by running an aliquot (7%) of each individual PCR reaction on a 1.5% agarose gel. The expected size of the amplified antibody fragments is 660 bp. 3 Pool the PCR products with common 3’-primers, and extract once with phenol and once with phenol:chloroform:isoamyl prior to precipitating the DNA using standard procedures. 4. Run the recovered DNA on a preparative 1.5% agarose gel. 5. Excise the appropriate (660 bp) DNA bands (other bands may be present and should be ignored) from the gel, and extract the DNA by electroelution (see Note 2). 6. Quantify the purified DNA, and digest with the appropriate restriction enzymes for 3 h at 37°C. The hght chain should be incubated with J&z1 and Sac1 and the heavy chain with XhoI and SpeI. The number of enzyme units and buffers to use are summarized m Table 1. 7. Precipitate the restricted DNA and resuspend the DNA in 50 yL sterile water. 8. Purify the insert for hgation by passage through a Chromaspin 100 column, followmg the manufacturer’s instructions.

3.3. Preparation

of Vector DNA

1. Electroporate 1 pL of a 1:lOO dilution of supplied vector DNA into a 40-uL ahqout of electrocompetent XL 1-blue cells (see Section 3.5.), plate out dilutions onto LB agar plates (100 ug/mL carbenicillin), and incubate overnight at 37OC. 2. Pick a single colony, inoculate 10 mL SB (50 ug/mL carb), and incubate in a shaking incubator for 8 h. 3. Use this culture to inoculate 1 L of SB/carb and incubate, shaking (at 250 rpm) at 37OC overnight. 4. The following day, prepare phagemid DNA using the Qiagen method with a tip500 column. 5. In preparation for the hgation of the light chain into the vector (see Section 3.4.), digest the pComb3HSS vector DNA with XbaI and Sac1 for 3 h at 37’C (usmg the number of enzyme units and required buffers as summarized m Table 1). 6. Precipitate the digested DNA prior to running it out on a 0.8% Seakem GTG gel with appropriate mol-wt markers. 7. Excise the appropriate linearized vector band, and extract the DNA from the gel chip by electroelution (see Note 3). 8. Quantitate the DNA (see Note 5). 9. After ligation of the light chain into the vector (see Section 3.4.), repeat the above steps from 2, but restrict the DNA with XhoI and SpeI for 3 h at 37°C (using the number of enzyme units and required buffers as summarized in Table I), thus

399

Phage-Display Antibodies Purify and extract mRNA Spleen d

calls AAAAA

+

Syntheslse first strand cDNA

Amphfy of cDNA of Heavy and Light chain antibody CHl and V domains by PCR + Clone Light Cham mto pComb3H Vector + *

Competent Cells (XL1 - Blue)

Clone Heavy Cham mto Vector plus Light Cham e Package Phage e

Competent Cells (XL1 - Blue) Helper

Phage

t Pan (x4) with Antigen

Make Soluble Fab

Test by ELISA

Fig. 4. Cartoon representing the preparation and cloning of Fab fragments into pComb3H-SS.

preparing the vector containing the light-chain library for ligating in the heavy chain (see Section 3.4.).

3.4. Ligation of PCR Fragments info fhe Wecfor The prepared antibody PCR fragments are ligated in turn (first light chain, followed by heavy chain) into the restricted, quantitated vector. The insert

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Table 1 Equivalent Enzyme Amounts and Buffers for Vector and PCR Insert Digestion (Using Enzymes and Buffers Obtained from Boehringer Mannheim)

Enzyme

Buffer

SpeI

H H A A

XhoI sac1 XbaI

pComb3H-SS or pComb3H-H + L, U/l.tg DNA

PCR inserts U/ug DNA

3

17 70 35 70

9 5 9

ligation efficiencies and the extent of background (vector alone) hgation should be tested on a small scale prior to library construction. 1. For the test ligation, ligate 250 ng vector with or without 50 ng insert for 2 h at room temperature. These hgations should be performed in a 20-pL total volume with 1 pL T4 ligase. Electroporate 1 pL of each hgation mix into 40-uL aliquots of competent XLl-blue cells (see Section 3.5.), and plate out aliquots of 1, 10, and 100 yL. The background ligation should be ~20% of the ligation with insert, 2. For production of the library, incubate 1400 ng of vector with 450 ng of the prepared PCR product at room temperature overnight, in the presence of 10 pL T4 hgase and 40 pL 5X buffer in a total reaction volume of 200 pL. 3. Stop the reaction by heating at 65°C for 10 mm, ethanol-precipitate, and resuspend in 15 pL of water. 4. Microdialyze the DNA solution against water on a 0.025~pm Millipore membrane prior to transformation (this removes excess salt from the sample, which may interfere with electroporation).

3.5. Transformation of the Library Electrocompetent XLl-blue cells should be prepared and tested with 1 pL 0.01 pg/mL control pUC DNA before library preparation. Cells should transform with an efficiency of at least 2 x lo9 CFU/pg (see Note 6). I. Add all 15 pL of library DNA to two ahquots of 200 pL electrocompetent cells. 2. Transfer to 0.2-cm chilled cuvets and electroporate by pulsing at 2.5 kV, 2 pF and 200 Sz. 3. Flush the electroporation cuvets with a total volume of 5 mL SOC. 4. Immediately incubate the cells, shaking at 37°C for 1 h.

3.6. Recovery of the Library Initially the antibody light-chain fragments are cloned into the vector to prepare the light-chain library. The vector DNA is recovered from this and pre-

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pared for the insertion of the heavy-chain antibody fragments. The combined heavy- and light-chain (Fab) library is expressed, and recombinant phage-carrying Fab fragments are recovered. 1. After 1 h of incubation at 37”C, add 10 mL prewarmed (37’C) SB containing 20 yg/mL carb and 10 pg/mL tet to the transformed cells. 2. Titer the library by further diluting 10, 1, and 0.1 pL of the culture into 100 pL SB and plating out onto LB (carb) plates. Leave the plates to grow in a 37°C oven overnight. 3. Incubate the IO-mL culture for a further 1 h at 37°C with shaking. 4. Increase the carbenicillin concentration to 50 pg/mL, and incubate for another hour with shaking at 37°C. 5. Transfer the IO-mL culture to 100 mL prewarmed (37“C) SB containing 50 pg/mL carb and 10 pg/mL tet. 6. For recovery of the light-chain library, leave this culture incubating at 37°C while shaking, overnight (go to step 9 below). 7. For recovery of the final Fab (heavy- and light-chain) library, immediately add 1Or2 PFU of M13K07 helper phage to the culture, and then incubate at 37°C with shaking. 8. After 2 h, add 70 pg/mL of Kan, and then leave the culture to grow in a shaking incubator at 37°C overnight. 9. After construction of both the light and combined Fab libraries, prepare phagemid DNA from the overnight culture, using the Qiagen method. 10. After construction of the Fab (light and heavy chain) library, the phage-containing supernatant can be recovered and used for the selection of antigen-specific antibody fragments. Transfer the supematant after pelleting the cells to a clean bottle, and precipitate the phage by adding 4% (w/v) PEG-800 and 3% (w/v) NaCl. Il. Place the bottle on a shaker for 5 min to dissolve the PEG and NaCl, and then incubate on ice for 30 min. 12. Pellet the precipitate by centrifugation at 10,OOOg in a JAlO rotor at 4OC for 20 min.

13. Discard the supematantand allow the bottle to drain on paper towel for about 10 min to remove as much PEG solution as possible. 14. Resuspend the pellet in 2 mL PBS/l% BSA, and transfer to Eppendorf tubes prior to microcentrifugation for 10 min to pellet any residual cell debris. Recover the supematants to fresh Eppendorf tubes, and store at 4°C. Always reamplify library phage preparations prior to use in panning if they had been stored for more than 24 h, because the attachment of the Fab fragment

to the g3p phage coat protein is relatively unstable. Reamplification can be achieved by followmg the panning protocol from step 6 in Section 3.7. below.

15. Titer the phage suspensionby infecting 50-pL volumes of XLl-blue cells (OD A600 = 0.5) with 1 pL of 1W3, lOA, and lC@ dilutions of the phage suspension at

room temperaturefor 15min before plating out on LB carb platesand incubating overnight at 37°C.

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3.7. Panning Several rounds of panning of the Fab-carrying phage will be required to select for antigen-specific binding clones within a library. 1. Coat ELISA plate wells overnight at 4°C with 1-O. 1 pg antigen solution m coating buffer (see Note 7). 2. On the following day, empty the wells (the antigen may be retained for repeated use), wash them three times with water, fill them with 3% BSA in PBS blocking buffer, and incubate at 37°C in a humidified incubator. 3. After 1 h, replace the blocking buffer with 100 pL fresh phage suspension, and incubate for a further 2 h at 37’C m the humidified incubator. 4. Remove the phage from the wells (see Note 8), and wash by tilling them with PBST, pipeting vigorously up and down, and leaving for 5 min before removal. In the first round of panning, wash the wells once in this fashion; in the second round, wash five times; and in the thud and subsequent rounds, wash 10 times. 5. After washing the wells, elute the phage by adding 50 l.tL of elution buffer (O.lM HCl [adjusted with glycine to pH 2.2]/1 mg/mL BSA) to each well for exactly 10 min of incubation at room temperature. 6. Pipet up and down vigorously, remove the eluate, and neutralize with 3 uL neutralizing buffer. 7. Infect 2 mL (per well) log phase (OD &,,, = 1) XLl-blue cells with the eluted phage. Incubate the cells at room temperature for 15 mm to allow reinfection of the phage before proceeding with replication as described for the original Fab library in Section 3.6. (beginning with step 1, the addition of 10 mL prewarmed, antibiotic containing SB).

3.8. Conversion of pComb3H-SS to Soluble Fab-Producing Form 1. Prepare double-stranded DNA from the cellular pellet collected after the final round of panning, reinfection, and replication. 2. Digest 5 pg DNA with ,S’eI and MeI with the appropriate buffer (see Table 1) for 3 h at 37°C. This removes the g3p component from the vector, and the compatible “sticky” ends of vector sequence can be religated. 3. Precipitate the DNA and resuspend the pellet in 50 pL sterile water. 4. Run the digested product on a 0.8% GTG agarose gel. 5. Excise the 4136bp vector band, and electroelute the DNA. 6. Self-ligate 200 ng vector DNA in 20 uL total volume for at least 2 h at room temperature. 7. Transform 1 uL of the ligation mix into a 40-uL aliquot of electrocompetent XL1 -blue cells, and plate out dilutions. 8. Pick single colonies and inoculate 10 mL SB containing 50 pg/mL carb, and grow for 6 h at 37’C with shaking. 9. Streak or spot an LB carb plate with each culture prior to induction, because they may not be viable on the next day.

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10. Add IPTG to a final concentration of 1 n&f, and incubate overnight at 30°C (see Note 9). 11. Recover the cells from the overnight cultures by centrifugation. Although both supernatants and cell pellets will contain Fab fragments, a higher concentration will be obtained from the cell pellet. 12. Prepare periplasmic protein from the cells by freezing the pellets in a dry ice/ ethanol bath for 5 min. Once frozen, add 100 FL 10 mM Tris, pH 8.0, to the pellets, and allow them to defrost at room temperature, with occasional gentle mixing. As soon as the pellets have defrosted and resuspended, transfer the tubes to ice. After 1 h, pellet the cell debris by centrifugation and transfer the supernatants to fresh tubes ready to use in ELISA.

3.9. EL&A Analysis of Potential Antigen Recognizing Clones ELISA analysis should be performed following standard procedures (see also Chapters 5,6, 16, and 24). 1. Coat wells of an ELISA plate with 25 pL of 4.0 pg/mL antigen diluted in coating buffer. 2. Block the plates with 3% (w/v) BSA in PBS 3. Wash the plates four times with 3% (w/v) BSA in PBS. 4. For the first antibody, use 50 pL/well of Fab preparation directly (or diluted in PBS/3% BSA). 5. Wash the plates four times with 3% (w/v) BSA in PBS. 6. For the second antibody/enzyme conjugate, use anti-Fab antibody. 7. Wash the plates four times with 3% (w/v) BSA in PBS. 8. Identify positive clones by developing the ELISA with appropriate substrate solution (20).

3. IO. Fab Protein Purification The production of periplasmic proteins described in Section 3.8. can be scaled up. The cells should be induced with IPTG when their OD A600is about 1.0. Add 100 PM PMSF to the IO-nxl4 Tris, pH 8.0, solution during the periplasmic preparation stage. After removal of the cell debris by centrifugation, the Fab protein can be purified from the supernatant. Refer to ref. 20 for a variety of appropriate methods for antibody purification. Final analysis of the Fab activity and purity prior to epitope mapping can be achieved by ELISA titration and protein gel analysis.

4. Notes 1. I represents inosine, a base that will base-pair with T, A, and C. 2. Electroelution is the preferred method for obtaining efficient recovery of goodquality DNA (GeneClean will yield lower-quality inserts). If an electroelution apparatus is not available, the following protocol can be used: Transfer the gel chip to a length of preboiled dialysis tubing (e.g., Medicell size 5) with 400 pL

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3. 4. 5.

6.

7. 8. 9.

0.1X TAE buffer, All the air bubbles should be removed before clipping the tubing at both ends. Rest the “package” just submerged in an electrophoresis tank containing 0.2X TAE. Electrophoresis was carried out at 200 V for 30-60 min. The buffer containing DNA was illuminated over a UV transilluminator and removed to an Eppendorf tube. Chill the solution on ice for 2 min, prior to spinning in a microfuge for 10 min, to remove any residual gel material. Remove the supematant to a fresh tube. Extract first with an equal volume of phenol and then with an equal volume of phenol:chloroform:isoamyl alcohol (24:24: 1). Precipitate the DNA. The antibody response should be assessed by ELISA from a test bleed prior to recovery of the mouse spleens. All RNA work must be performed wearing gloves and with sterilized RNase-free pipets and solutions. The concentration of the DNA can be ascertained on a 0.7% agarose plate containing 0.7 pg/niL ethidium bromide. Dot 1 pL of the DNA to be assessed onto the plate along with 1 pL of dilutions of plasmid DNA of known concentration. Examine the intensity of fluorescence of the DNA dots by illumination with UV, and estimate the concentration of the sample from the fluorecence it gives relative to the fluorecence observed from the plasmid DNA dilutions. The preparation of highly electrocompetent cells is essential for the production of reasonably sized antibody libraries. Standard procedures should be followed to prepare the cells (22). It is imperative to keep the cells as cold as possible during the procedures. All solutions, pipets, and centrifugation rotors and bottles should be prechilled before use. Alternatively, ready-prepared electrocompetent XL1 -blue MRF’ cells can be obtained from Strategene (200158). Coating may also be performed at 37°C for 1 h. Other solid supports, such as Sepharose, may also be used for the immobilization of the anttgen. Bacteriophage should be removed to bleach solution to prevent it from contaminating future experiments. Optimum induction times will vary. It may be necessary to do a time-course analysis of the Fab production.

References 1. Pluckthun, A. and Skerra, A. (1990) Expression of functional antibody Fv and Fab. Methods Enzymol. 178,497-515. 2. Pluckthun, A. (1992) Biotechnological aspects of antibody production in E. coli. Immunol. Rev. 130,151-188. 3. Smith, G. P. (1985) Filamentous fusion phage; novel expression vectors that express cloned antigens on the virion surface. Science 228,13 15-l 3 17. 4. Cesareni, G. (1992) Peptide display on tilamentous phage capsids. FEBS Lett. 307,66-70. 5. Barbas, C. F., III, Kang, A. S., Lemer, R. A., and Benkovic, S. J. (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88,7978-7982.

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6. Clackson, T., Hoogenboom, H. R., Griffiths, A. D., and Winter, G. (1991) Making antibody fragments using phage display libraries. Nature 352, 624-628. 7. Garrard, J. G., Yang, M., O’Connell, M. P., Kelley, R. F., and Henner, D. J. (199 1) Fab assembly and enrichment in a monovalent phage display system. Bio/technology 9,1373-1377. 8. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M., and Gottesmann, K. S. (1991) Sequences of Proteins of Immunologtcal Interest, 5th ed. US Department of Health and Human Services, Public Health Service, Natl. Inst. of Health, Bethesda, MD. 9. Nisonhoff, A., Hooper, J. E., and Springs, S. B. (1975) The Antibody Molecule. Academic, New York. 10. Short, J. M., Fernandez, J. M., Sorge, J. A., and Huse, W. D. (1988) h ZAP: a bacteriophage 1expression vector with in vivo properties. Nucleic Acids Res. 16,7583-7600. 11. Bullock, W. O., Fenandez, J. M., and Short, J. M. (1987) Biotechniques 5,376-380. 12. Sambrook, J., Fritsch, E. F , and Maniatis, T. (1989) Molecular Cloning: A LaboratoryManual,2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 13. Burton, D. B., Barbas, C. F., III, Personn, M. A. A., Koenig, S., Chanock, R. M., and Lerner, R. A. (199 1) A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive donors. Proc. Natl. Acad. Sci. USA 88, 10,134-10,137. 14. Barbas III, C. F. (1993) Recent advances m phage display. Curr Opin. Biotechnol. 4,526-530. 15. Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., and Griffiths, A. D. (199 1) By-passing immunisation: human antibodies from V-gene libraries displayed on phage. J. Mol. Biol, 222,58 l-597. 16. McCafferty, J., Griffiths, A. D., Winter, G., and Chiswell, D. J. (1990) Phage antibodies: fllamentous phage displaying antibody variable domains. Nature 348, 552-554. 17. Hawkins, R. E., Russell, S. J., and Winter, G. (1992) Selection of phage antibodies by binding affinities: mimicking affinity maturation. J. Mol. Biol. 222,889-896. 18. Marks, J. D., Ouwehand, W. H., Bye, J. M., Finnern, R., Gorick, B. D., Voak, D., Thorpe, S. J., Hughes-Jones, N. C., and Winter, G. (1993) Human antibody fragments specific for human blood group antigens from a phage display library. Bio/ technology 11,1145-l 149. 19. Chiswell, D. J. and McCafferty, J. (1992) Phage antibodies: will new “coliclonal” antibodies replace monoclonal antibodies? Trends Biotechnol. 10,80-84. 20. Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 21. Kang, A. S., Burton, D. R., and Lerner, R. A. (1991) in Methods: A Companion to Methods in Enzymology, vol. 2 (Lerner, R. A. and Burton, D. R., eds.), Academic, Orlando, FL, pp. 11 l-l 18. 22. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., and Stuhl, K. (1994) Current Protocols in Molecular Biology, vol. 1, chapter 1, section 1.8.4. John Wiley.