Non-Radioactive Labelling: A Practical Introduction (Biological Techniques Series)

Non-radioactive Labelling A Practical Introduction

B IOLOGICAL TECHNIQUES A Series of Practical Guides to New Methods in Modern Biology

S e r i e s Editor

DAVID B S A T T E L L E Computer Analysis of Electrophysiological Signals J Dempster Fluorescent and Luminescent Probes for Biological Activity WT Mason (Editor) (published May 1993) Planar Lipid Bilayers W Hanke and W-R Schlue (published October 1993) In Situ Hybridization Protocols for the Brain W Wisden and BJ Morris (Editors) Manual of Techniques in Insect Pathology LA Lacey (Editor)

CLASSIC TITLES IN THE SERIES Microelectrode Methods for Intracellular Recording and Ionophoresis RD Purves Immunochemical Methods in Call and Molecular Biology RJ Mayer and JH Walker

BIOLOGICAL TECHNIQUES

Non-radioactive Labelling A Practical Introduction

A.J. GARMAN Zeneca Pharmaceuticals, Macclesfield, UK

Academic Press Limited 24-28 Oval Road, London NW1 7DX, UK http://www.hbuk.co.uk/ap/

United States Edition published by Academic Press, Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Copyright 9 1997 by ACADEMICPRESS

All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. The author and publisher have attempted to identify trademarks and trade names where applicable. However, the failure to do so does not indicate that such names or marks are not protected by applicable trademark laws. This book is printed on acid-free paper. Library of Congress Cataloging-in-Publication Data Garman, Andrew. Non-radioactive labelling: a practical introduction/Andrew Garman. p. cm.u(Biological techniques series) Includes index. ISBN 0-12-276045-X (alk. paper) 1. Biomolecules--Labeling. I. Title. II. Series. QP519.9.A37G37 1997 572.8--dc21 96-37104 CIP A catalogue record for this book is available from the British Library ISBN 0-12-276045-X Typeset by J&L Composition Ltd, Filey, North Yorkshire Printed in Great Britain by The Bath Press, Bath 97 98 99 00 01 02 EB 9 8 7 6 5 4 3 2 1

Series preface

The rate at which a particular aspect of modern biology is advancing can be gauged, to a large extent, by the range of techniques that can be applied successfully to its central questions. When a novel technique first emerges, it is only accessible to those involved in its development. As the new method starts to become more widely appreciated, and therefore adopted by scientists with a diversity of backgrounds, there is a demand for a clear, concise, authoritative volume to disseminate the essential practical details. Biological Techniques is a series of volumes aimed at introducing to a wide audience the latest advances in methodology. The pitfalls and problems of new techniques are given due consideration, as are those small but vital details that are not always explicit in the methods sections of journal papers. The books will be of value to advanced researchers and graduate students seeking to learn and apply new techniques, and will be useful to teachers of advanced undergraduate courses, especially those involving practical and/or project work. When the series first began under the editorship of Dr John E Treherne and Dr Philip H Rubery, many of the titles were in fields such as physiological monitoring, immunology, biochemistry and ecology. In recent years, most biological laboratories have been invaded by computers and a wealth of new DNA technology. This is reflected in the titles that will appear as the series is relaunched, with volumes covering topics such as computer analysis of electrophysiological signals, planar lipid bilayers, optical probes in cell and molecular biology, gene expression, and in situ hybridization. Titles will nevertheless continue to appear in more established fields as technical developments are made. As leading authorities in their chosen field, authors are often surprised on being approached to write about topics that to them are second nature. It is fortunate for the rest of us that they have been persuaded to do so. I am pleased to have this opportunity to thank all authors in the series for their contributions and their excellent co-operation. DAVID B SATTELLEScD

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Preface

It seems unsatisfactory to describe something by what it is not, but the terms 'non-isotopic' and 'non-radioactive' are here to stay. These terms, made popular by suppliers of research products, reflect, of course, the inherent disadvantages of radioactive labels: safety concerns, disposal costs, record keeping and (often) poor label stability. Non-radioactive alternatives have been slow to replace radiolabels but the trend is now firmly established and the use of non-radioactive labels is now the norm in many fields. For those considering use of such labels, perhaps for the first time, obtaining the desired labelled biomolecule can be perceived as a barrier. This book is aimed at overcoming this barrier. The impetus to write this book came when I had the need to train graduate staff to prepare labelled molecules of various kinds. I found no single book that I could recommend that would provide the necessary background, protocols and ancillary information. Rather this was distributed throughout various papers, notebooks and manufacturers' literature that I had accumulated over the years. Some multi-author books seemingly covering this field were too specialized for general use and did not cover the more introductory need that I faced. I felt that a single source of practical information and protocols, backed up by a modest amount of theory, would certainly be of value in my laboratory and therefore, I hoped, would also be useful in other laboratories. I also felt that such a book may be timely since, after nearly two decades of considerable development in non-radioactive labelling, there seemed to be a levelling off in the pace of development and it is now becoming clear which are the welltried and useful approaches. It also provided an opportunity to integrate methodologies that have been applied to labelling different types of biological molecules. All the major alternatives to radiolabels involve light measurement of some form or other, and the main categories to consider are absorbance, fluorescence and chemiluminescence. It is not the aim of this book to discuss the full range of possible labels in depth, though a general introduction is given in Chapter 3. Nor does it address how to use labelled molecules, i.e. detection protocols, instrumentation, etc., although nucleic acid detection is covered. Instead, this book addresses the crucial question of how best to link the label of choice to the biological molecule of interest. The methods described are predominantly chemical, but may be performed by non-chemists and do not in general require organic chemistry facilities. The chapter on oligonucleotide labelling, however, assumes knowledge of automated oligonucleotide synthesis using phosphoramidite chemistry.

viii

Preface Labelling is usually regarded as a matter of following recipes. This is successful when a well-described and relevant protocol can be found, but frequently one has to try to adopt a method which is either poorly described or far removed from the real need. A method that starts with 'dissolve 10 mg of the protein in 1 ml of 0.1 M sodium borate buffer, pH 8.5' is disheartening to someone starting with 500 ~tg of a valuable monoclonal antibody in 0.5 ml of an unspecified liquid. Often some understanding of the chemistry is needed in order to successfully adapt the procedure. This book seeks to describe the basic practical issues involved in labelling so that, whether using the protocols in this book, or others, the reader can make successful adaptations. Given the enormous range of biomolecules, labels and applications, the inclusion of protocols to suit every need is clearly not possible. The protocols included here are therefore intended to be representative and capable of adaptation. While a reasonably comprehensive review of the conjugation and labelling literature will be attempted, emphasis will be given to those systems and methodologies that have been tried and tested by myself and other groups and can be regarded as 'core' methods. However, realizing that diverse needs require diverse solutions, I have tried to indicate the potential of other methods and given references that will give further information. Keeping the number of references to manageable proportions has proved difficult: with the exception of some older landmark papers, I have often cited later rather than earlier references, since these show recent improvements. I apologize to any author whose work has been overlooked for this or any other reason. Few of the procedures described are novel: where possible original sources are credited, but many protocols have doubtless changed hands several times with modifications en route. Organization of this book Labelling biomolecules may be visualized as being in three-dimensional space, with the axes representing (a) the biomolecules, (b) the labels and (c) the coupling methods. This book does not attempt to explore this space systematically, but aims to give representative points in this space that may be used as departure points to develop a method for the particular molecule of interest. Much of the chemistry currently used for labelling nucleic acids and other biomolecules has its roots in protein labelling, and is largely based on linkages to amines and thiols. The book's layout reflects these developments, with Chapters 2-5 focusing on proteins. Thus, the remaining chapters build on many of these earlier themes, which, though they have been introduced in the context of proteins, are potentially of value in understanding and identifying the best options for labelling many other molecules that contain similar functional groups. In this way it is hoped that the cross-fertilization between nucleic acids and proteins, which has proved so successful to date, will be encouraged by this more unified approach, which is anyway more appropriate with so many laboratories becoming multi-disciplinary. Finally, the appendices contain some common procedures and basic data to allow labelling experiments to be planned with the minimum of recourse to other material. Safety Non-radioactive alternatives are not free from safety hazards, nor are the labelling and characterization methods. While the procedures in this book are believed to be safe, the investigator should take appropriate precautions to ensure the safety of the work undertaken. Neither the author nor the publisher accept any responsibility for any injury or other adverse outcomes caused by the carrying out of procedures described or referred to in this book.

Preface

ix

Request for information on developments Success with labelling depends to an extent on making the best use of the kind of anecdotal information and experiences which are often unpublished. Also, there may be errors and omissions in what is presented here. I would be interested to receive comments and suggestions of this nature which may be used to improve and keep up to date any future editions of this book. ANDREW J. GARMAN

Acknowledgements

Many of the procedures described here were adopted and/or developed in the context of core R&D groups at ICI Diagnostics, Cambridge Research Biochemicals and Zeneca Pharmaceuticals, and I am grateful to many colleagues who worked with me during those periods and whose efforts are reflected in one way or another in this book. In particular, I thank Maxine Campbell, Ian Hodgson, John Parker and Pirthipal Singh for contributing to the labelling know-how. The more specific contributions of others are acknowledged in the text. I also thank many colleagues, especially Pirthipal Singh, Ron Cotton, Dave Holland and David Carrick, for reading and commenting on the manuscript. Andrew J. Garman

Contents

Series Preface Preface Acknowledgements Abbreviations

v

vii x xiv

CHAPTER ONE

Introduction to non-radioactive signals and labels 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

Introduction Nomenclature Choice of signal and label" general considerations Fluorescence Time-resolved fluorescence Chemiluminescence and bioluminescence Enzyme labels Other labels Summary: which label?

1 1 2 4 5 6 9 14 15

CHAPTER TWO 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Labelling proteins and peptides" chemical background

20

Introduction Amino acid functional groups Reaction at amino groups Reaction at thiol groups Reaction at carboxylic acid groups Reaction at other amino acid side chains Reaction at carbohydrate groups Chemical modification of peptides Practical points

20 21 23 26 28 29 30 30 31

xii

Contents CHAPTER THREE Conjugation of proteins with enzymes and other proteins

33

Introduction Conjugation schemes Heterobifunctional approaches based on thiol chemistry Conjugation via carbohydrate groups

33 34 36 48

CHAPTER FOUR Fluorescent labelling of proteins and peptides

51

Introduction Labelling proteins with organic fluorophores Labelling with europium chelates Labelling of peptides

51 58 60 61

CHAPTER FIVE Use of tags in the labelling and detection of biomolecules

64

5.1 5.2 5.3 5.4 5.5 5.6

Introduction Which tag? Biotin and the alternatives Avidins and other tag binding agents Methods for tagging biomolecules Tagging of peptides Fusion protein tags

64 66 66 67 70 70

6.1 6.2 6.3 6.4 6.5 6.6

CHAPTER SIX Labelling of oligonucleotides Introduction On-column labelling using phosphoramidite reagents Labelling at introduced primary amino groups Labelling at introduced thiols Other chemical approaches Enzymic labelling

72 72 73 77 84 84 84

CHAPTER SEVEN Labelling of long nucleic acid probes

88

3.1 3.2 3.3 3.4

4.1 4.2 4.3 4.4

7.1 Introduction 7.2 Labelling by enzyme-mediated incorporation of modified nucleotides 7.3 Labelling by chemical modification CHAPTER EIGHT Labelling of small biomolecules 8.1 Introduction 8.2 Labelling with enzymes and other protein labels 8.3 Labelling with low molecular weight labels and tags

88 89 97

103 103 103 109

Contents

xiii

APPENDICES 1 2 3 4 5 6 7 8 9 10 11 12

111 115 117

List of suppliers Molecular weights Extinction coefficients for protein conjugation Excitation and emission wavelengths of chemilumophores and some fluorophores used for DNA labelling Determination of thiol groups using Ellman's reagent Use of disposable gel filtration columns Purity check for N-hydroxysuccinimide esters Values of pKa for amino acid functional groups Signal enzyme detection Reagents for changing functional groups Protein-protein conjugation: worked example HPLC of peptides and oligonucleotides

118 119 120 122 123 124 126 128 130

Index

131

Abbreviations

4CN ABTS AEC AMCA AMPGD AMPPD TM BCA BCIG BCIP BODIPY T M BSA CPG CPPCQ CPRG CSPD T M

DAB DELFIATM DEPC DIG DMF DMSO DNP dNTP DTNB DTTA ECL ECL EDC EDTA EMIT TM Fab' FACS

4-chloro- 1-naphthol 2,2'-azino-di-(3-ethylbenzthiazoline sulphonic acid) 3-amino-9-ethylcarbazole 7-amino-4-methylcoumarin-3-acetic acid disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-tricyclo [3.3.1.13'7]decan }-4-yl)phenyl ~-D-galactoside disodium 3-(4-methoxyspiro { 1,2-dioxetane-3,2'-tricyclo [3.3.1.13'7]decan }-4-yl)phenyl phosphate bicinchoninic acid 5-bromo-4-chloro-3-indoyl-13-D-galactoside 5-bromo-4-chloro-3-indoyl phosphate 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene bovine serum albumin controlled pore glass 2-(5 '-chloro-2 '-pho sphoryloxyphenyl)-6-chloro-4- (3H)- quinazolinone Chlorophenol red ~-D galactoside disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2 '-(5 '-chloro)tricyclo [3.3.1.13'7]decan }-4-yl)phenyl phosphate 3,3 '- diaminobenzidine dissociative enhanced lanthanide fluorescence immunoassay diethyl pyrocarbonate digoxigenin dimethyl formamide dimethyl sulphoxide dinitrophenyl deoxynucleoside triphosphate 5,5'-dithio-bis-(2-nitrobenzoic acid) diethylenetriamine-N1,N2.N3,N3-tetra-acetic acid enhanced chemiluminescence, or electrochemiluminescence 1-ethyl-3(3-di methyl aminopropyl)c arb odi-imide ethylenediaminetetra-acetic acid enzyme-monitored immunoassay technique fragment antigen binding fluorescence-activated cell sorting

Abbreviations FAM FISH FLPC FRET GFP HEPES HEX HPLC HPPA HRP JOE LC-SPDP MALDI MBS MES MHS MUP NBT NHS NIP NTA OD ONPG PBS PCR PMPI PNPG PNPP PVDF ROX SATA SDS SDS PAGE SIAB SMCC SMPB SMPT SPDP SSC TAMRA TE TET TFA TMB TNBS TRF Tris TSA X-gal

6-carboxyfluorescein fluorescence in situ hybridization fast protein liquid chromatography fluorescence resonance energy transfer green fluorescent protein 4-(2-hydroxyethyl)- 1-piperazine ethanesulphonic acid 4,7,2',4',5 ',7 '-hexachloro-6-carboxy fluorescein high performance (or pressure) liquid chromatography 3-(p-hydroxyphenyl)propionic acid horseradish peroxidase 6-c arboxy- 2' ,7 '- dimethoxy-4 ',5 '-dichlorofluore scein long chain SPDP matrix assisted laser desorption ionization m-maleimidobenzoyl-N-hydroxysuccinimide ester 4-morpholine-ethanesulphonic acid maleimidohexanoyl-N-hydroxysuccinimide 4-methylumbelliferyl phosphate nitroblue tetrazolium N-hydroxysuccinimide nitroiodophenol nitrilotriacetic acid optical density o-nitrophenyl- ~-D-galactoside phosphate buffered saline polymerase chain reaction p-maleimidophenyl isocyanate p-nitrophenyl-13-D-galactoside p-nitrophenyl phosphate polyvinylidene difluoride 6-carboxy-x-rhodamine N-succinimidyl-S-acetylthioacetate sodium dodecyl (or lauroyl) sulphate SDS polyacrylamide gel electrophoresis N-succinimidyl (4-iodoacetyl)aminobenzoate succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1-carboxylate succinimidyl 4-(p-maleimidophenyl)butyrate 4-succinimidyloxycarbonyl-methyl-cx-(2-pyridyldithio)toluene N-succinimidyl 3-(2-pyridyldithio)propionate salt/sodium citrate N,N,N' ,N'-tetrameth yl-6-carbo x yrhodamine Tris/EDTA buffer 4,7,2',7'-tetrachloro-6-carboxyfluorescein trifluoroacetic acid 3,3 ',5,5 '-tetramethylbenzidine trinitrobenzenesulphonic acid time-resolved fluorescence tris(hydroxymethyl)aminomethane tyramide signal amplification 5-bromo-4-chloro-3-indoyl-13-D-galactoside

xv

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CHAPTER

ONE

Introduction to non-radioactive signals and labels

1.1 INTRODUCTION There are many advantages to non-radioactive signals. The most immediate attractions are the freedom from safety issues, regulations, paperwork, disposal costs, etc. But there are also more positive reasons that sometimes apply: improved stability, reduced costs, improved spatial resolution, and greater scope for signal modulation, a feature which permits homogeneous assays to be designed. Radiolabels have an inherent sensitivity limit defined by the specific activity of the label and the time period over which one is prepared to measure. Many non-radioactive signals are potentially much more sensitive, capable of detecting down to a few hundred molecules. However there are some disadvantages. There are frequently sources of noise and background interferences with which to contend. One cannot always continuously monitor the label as one can with high energy radio-emitters. Nor should

it necessarily be assumed that non-isotopic labels have no safety issues: as with any other laboratory chemical, the hazard level needs to be understood and the appropriate precautions taken. Selection of the most appropriate signal for an application is important and the wide range of options available can be confusing. This chapter introduces the more important signal systems and is intended to offer some guidance in selecting the most appropriate for the application of interest.

1.2 NOMENCLATURE The literature and common usage is often inconsistent in its terminology. At this point, it is useful to define some terms. Signals and labels. The signal is the phenomenon that is measured; the label is the entity that

Non-radioactive labelling generates the signal. For example, fluorescence may be generated by a fluorescent label or by the action of an enzyme label on a fluorogenic substrate. The label can then be either a fluorophore, or an enzyme; the signal in both cases is fluorescence. To distinguish the two types of label, we can use the terms 'direct fluorescence' and 'enzyme-generated fluorescence'. Labels and tags. The literature (and this book) often speaks of 'labelling with biotin'. Of course, to locate or measure the biotin, it is necessary to use a biotin binding protein linked to a label (for example, fluorescein-labelled streptavidin). The role of the biotin is purely mediatory, and to call it a 'label', though this is well-established practice, is confusing. For the purposes of this discussion, we will call such systems (which also include haptens) 'tags', and reserve 'labels' for entities that actually give a measurable signal. The use of tags is described in Chapter 5.

Fluorescent labels and fluorescent probes. Labels passively report the quantity and/or the location of the molecule to which they are attached. Fluorescent probes (not nucleic acid probes) probe the chemical environment and can report conformational changes, cell membrane polarity, the presence of metal ions etc. Though there is a grey area in these definitions, fluorescent probes are not simply alternatives to

radiolabels and are therefore not covered in this book. Luminescent signals. Fluorescence, phosphorescence, luminescence, chemiluminescence and bioluminescence are terms that are often confused. All signals that involve the emission of light are called luminescence, and are caused by the transition of a molecule from an electronically excited state to a lower energy state. Luminescence may be classified by the stimulus that causes the initial excitation. Thus photoluminescence covers all processes where the stimulus is itself a photon, and includes fluorescence, timeresolved fluorescence and phosphorescence. Chemiluminescent systems are triggered by a chemical stimulus, whilst bioluminescence is a chemiluminescent phenomenon in, or originating from, living organisms. Chemifluorescence is an unnecessary and confusing term that may be found in certain product literature: it is enzyme-generated fluorescence. Table 1.1 summarizes the main types of luminescent processes that are relevant to bioanalysis.

1.3 CHOICE OF SIGNAL AND LABEL: GENERAL CONSIDERATIONS Which factors effect the choice of system for any particular application? Sensitivity, labelling

Table 1.1 Classification of luminescent signals

Types/comment

Timescaleof light output

Examples

Process

Stimulant

Photoluminescence

Light

(i) Fluorescence (ii) Time-resolved fluorescence (iii) Phosphorescence

Chemiluminescence

Chemical

(i) Direct seconds (ii) Enzyme generated hours

Acridinium ester Dioxetane substrates

Bioluminescence

Chemical

Chemiluminescence in living organisms, protein-mediated

Varies, may be complex

Firefly, jelly fish

Electrochemiluminescence

Electrical

Involves radical formation

Varies

Ruthenium complex

Radioluminescence

Radioactivity

nanoseconds

Scintillants

nanoseconds milliseconds > seconds

Fluorescein Europium chelates Erythrosin

Introduction convenience, track record and the availability of detection instrumentation are some of the many factors that are generally considered. But the application and institutional environment also generate a set of factors or pressures that will affect the choice of label. Some of these pressures, and where they typically apply, are given in Table 1.2. Given then this diversity of needs, and the strong commercial and academic interest in the development of non-radioactive alternatives, it is not surprising that there are a vast number of labels from which to choose. It is to the immunoassay field that most invention has

to n o n - r a d i o a c t i v e

signals and labels

3

been directed (for surveys, see e.g. Ngo, 1988; Gosling, 1990) but the benefits of this investment are now felt in many different fields. Attention is frequently focused upon the relative sensitivity of various labels. Here we need to distinguish between the sensitivity of the label measured in isolation and the sensitivity that is achievable in real assays using off-the-shelf instrumentation. There may be several orders of magnitude difference between these two measures. This has been considered in some detail for immunoassays (Kominami, 1994). Figure 1.1 gives an indication of the relative sensitivities of the major signals obtained in practice.

Table 1.2 Factors influencing choice of label

Factor/issue Cost constraints Ease of use Ease of labelling Compatibility with format/instrumentation Stability of label Freedom to operate with respect to patents Precision and robustness Throughput potential Low learning time available Time to read the signal Flexibility in reading the signal Dynamic range Requirement for homogeneous assays

Where typically applicable Most institutions but to varying extents Research; routine operation by less skilled staff Research; where many labelled species are required General Diagnostics, screening Commercial operations Diagnostics especially Diagnostics, screening operations Research, low-throughput needs On-site testing High-throughput situations Immunoassay: some analytes Diagnostics, high-throughput screens

Figure 1.1 Indication of the relative sensitivities of various signal systems obtainable with generally available instrumentation. Light grey area of the bars indicates dependency on format and instrumentation used.

Non-radioactive labelling The dynamic range of a signal is also an important factor for some applications. Here colour (absorbance) is usually at a disadvantage, being restricted to about two orders of magnitude, while fluorescence and chemiluminescence can typically cover four or more orders.

1.4 FLUORESCENCE Fluorescent labels have a long history of utility in bioanalysis. However, in the last decade or so, there has been an acceleration of interest in the application of fluorescent labels. To support this contention, one might cite the success of fluorescence DNA sequencing and other fluorescence gene analysis methods, the establishment of time-resolved fluorescence, the growth in fluorescence-activated cell sorting (FACS), the emergence of improved fluorophores, for example long-wavelength fluorophores, the increased interest in homogeneous fluorescence techniques and advances in measurement techniques such as fluorescence imaging, confocal fluorescence microscopy and fluorescence correlation spectroscopy. Further evidence is provided by the recent emergence of a much wider range of fluorescence instrumentation for bioanalytical applications. Fundamentally, this interest is because fluorescent labels are stable, easy to use and can be very sensitive. The Achilles' heel of fluorescence is that the environment fluoresces as well; as a result, progress in fluorescence has largely been about obtaining selectivity, i.e. improving the signal-to-noise ratio.

1.4.1 Fluorescent labels

At its limit, fluorescence can detect single molecules, either by fluorescence correlation spectroscopy (Eigen & Rigler, 1994) or other methods (Nguyen et al., 1987; Nie et al., 1994; Barnes et al., 1995). While chemiluminescence is often regarded as the most sensitive signal system (and often is, depending on the basis for the comparison), it should be borne in mind that a chemiluminescent molecule can decay only once, giving a single photon, while a fluorophore

is continuously excited, having the capacity to emit typically tens of thousands of photons (before chemical degradation, or photobleaching, occurs). In this respect, a fluorophore is analogous to an enzyme, having the capacity for converting photons of one wavelength to photons of a higher wavelength. This comparison is not altered if the fluorescence or chemiluminescence is generated by a (real) enzyme, since the enzyme adds an extra level of amplification for both systems. The realization of this extreme sensitivity potential is in practice limited by interfering signals. This may derive from biological components, plastics, reagent impurities, light scattering from particles or contamination from a variety of sources. The severity of these problems varies according to the particular application, but it is frequently the case for general biochemical and immunochemical assays that it is the noise, rather the instrumentation, that limits sensitivity, even with instrumentation of modest sensitivity (e.g. fluorescence plate readers). Several ploys have been developed to avoid the noise problem. Apart from time-resolved fluorescence, which is discussed below, there is a move towards the use of fluorophores that are excited and emit at longer wavelengths, where noise sources are less of an issue. Fluorophores with high Stokes' shifts (the difference between excitation and emission maxima) are more easily distinguished from noise. Fluorescence correlation spectroscopy and confocal microscopy also largely eliminate noise problems since only fluorophores in solution are detected. A brief survey of fluorescent labels that might be considered for labelling biomolecules is given in Chapter 4, which also describes protein labelling. These include protein fluorophores, namely the phycobiliprotein family. Mention should be made here of the so-called green fluorescent protein (GFP) of the jellyfish (Aequoria victoria) (Chalfie et al., 1994) and mutants thereof (Heim et al., 1995). As well as its use as a marker for gene expression, GFP fusion proteins (Wang & Hazelrigg, 1994) have considerable potential, both for intracellular studies and as a means of labelling proteins (Cubitt et al., 1995).

I n t r o d u c t i o n to n o n - r a d i o a c t i v e s i g n a l s a n d l a b e l s One further useful feature of fluorescence is that it forms the basis of a number of different homogeneous assays, or non-separation assays. These include fluorescence polarization (Jolley et al., 1981; Garman & Moore, 1990), energy transfer (Ullman et al., 1976; Cardullo et al., 1988; Morrison et al., 1989) and fluorescence correlation spectroscopy (Eigen, 1994).

1.4.2 Enzyme-generated fluorescence Enzyme-generated fluorescence is theoretically the most sensitive signal available, since, as discussed above, it has effectively two levels of amplification. Fluorescent substrates with the potential to measure sub-attomole quantities of enzyme are available for the two hydrolytic enzymes commonly employed as labels, alkaline phosphatase and [3-galactosidase. These substrates (see below) are stable and convenient to use, making this an attractive way forward for high sensitivity applications in both the immunochemical and molecular biology fields. In practice however, enzyme-generated fluorescence has achieved similar levels of sensitivity observed with enzyme generated chemiluminescence using dioxetane substrates (see below). Because the level of fluorescence is greater, enzyme-generated fluorescence is much less affected by noise issues, and can be at least as sensitive as radiolabels. In this case, sensitivity is more frequently limited by the quality (e.g. the affinity) of the assay components themselves, and the extent of non-specific binding. Enzyme labels are not as stable as other labels (though they are generally adequate) and labelling may be more involved. However, these are minor disadvantages.

1.5 TIME-RESOLVED FLUORESCENCE Although there are several examples of long-lived fluorescence, it is labels based on lanthanide chelates that have become established in immunoassay and, increasingly, other applications. The lanthanides that show useful fluorescence in certain chelates are europium (Eu3+), dysprosium

5

(Dy3+), samarium (Sm 3+) and terbium (Tb3+). Of these, europium has been the most exploited. The rationale of time-resolved fluorescence (TRF) is based on: (i) the long Stokes' shift, which distinguishes the label from many noise sources; (ii) the fact that the fluorescence is longer lived than most noise sources and therefore, with appropriate instrumentation, the (short-lived) background fluorescence noise may be filtered, or 'gated' out. These two factors give the approach considerably improved sensitivity compared with conventional fluorophores.

1.5.1 The DELFIA system The most widely used time-resolved fluorescence system is the Wallac DELFIA T M ('dissociative') system (Soini & Kojola, 1983; Hemmil~i et al., 1984; Hemmil~i, 1988). Here the label may be regarded as an indirect label in the sense that the europium label is bound to the antibody as a non-fluorescent chelate; at the end of the assay, the europium is dissociated from the conjugate by addition of an 'enhancement' reagent (containing acid, I]-diketone ligands and a detergent) which forms a micellar, multi-component fluorescent complex (see Figure 1.2A). This extra step is inconvenient, but nevertheless, this approach yields one of the most sensitive non-enzymic systems for microtitre plate applications. In a comparison with 125I and enzyme labelling (Madersbacher et al., 1993) it performed best in terms of sensitivity and dynamic range. A microtitre plate reader, labelling reagents and conjugates are available (Wallac). Imaging systems for time-resolved fluorescence are not yet widely available however. The dissociative method is effectively based on a free europium assay and, as such, is susceptible to environmental contamination by dust, etc. Cleanliness is therefore important and this approach may not be suitable for demanding environments, e.g. out-of-lab testing.

1.5.2 Stable fluorescent chelates Several europium chelate labels that are stable and fluorescent (i.e. do not require a dissociation

6

Non-radioactive labelling

N

N

N

enhancement

.

solution (H*)

"

CO0" COO" CO0" CO0EU3.

R

R

coo

N

R

COO-

Eu3*

N

~ N~ N. . NN ~

~

,N~~ -~

Figure 1.2 Three types of europium chelate time-resolved fluorophore: (A) the DELFIA system; (B) phenanthroline dicarboxylic acid chelates; (C) the tris-bipyridine europium cryptate. For details, see text. step) have been described. The most developed is probably the Cyberfluor system, which was developed initiallyforimmunochemicalapplications (Diamandis, 1988, 1991, 1992; Khosravi & Diamandis, 1987): the label is based on the phenathroline 2,6-dicarboxylic acid europium complex (Figure 1.2B). A disadvantage for some applications is that the sample (microtitre plate well, blot etc.) must first be dried. The system has been extended to DNA probes (Christopoulos et al., 1991), but remains relatively unexplored. Labelling reagents, conjugates and instrumentation are available (Cyberfluor). A europium cryptate label based on three bipyridyl units (see Figure 1.2C) has also been reported (Alpha et al., 1987) and is being exploited for DNA detection (Lopez et al., 1993), immunodiagnostics (Mathis, 1993)and protein interaction studies (Mathis, 1995). The attraction of this label is that it is being used in an energy transfer homo~geneous system. This has also been reported by Selvin & Hearst (1994) and Selvin et al. (1994). Environmental effects on a lanthanide chelate fluorescence have also been exploited to give a homogeneous

immunoassay (Mikola et al., 1995). Using yet another homogeneous principle, an assay for DNA based on europium fluorescence, has been described (Coates et al., 1994): this is based on target-dependent assembly of a sensitized europium complex. A number of other fluorescent lanthanide chelate labels have been described, but have yet to be widely used as labels (Remuifi~in et al., 1993; Saha et al., 1993).

1.6 CHEMILUMINESCENCE AND BIOLUMINESCENCE For most people there is an inherent fascination in organisms and chemical reactions that glow in the dark, and considerable effort has been devoted to exploiting such systems for bioanalytical purposes. At least 36 distinct chemiluminescent reactions have been described (Kricka, 1991); many of these have been studied intensively. The subject is well described, in both a historical and biological context, by Campbell (1988) and also reviewed by, for

I n t r o d u c t i o n to n o n - r a d i o a c t i v e signals a n d labels

nescent organisms, and is responsible for example for the excitation of GFP in Aequorea (Cubitt et al., 1995). Chemiluminescence energy transfer has been exploited to improve the efficiency of light output from chemiluminescent substrates (dioxetanes, see below) and also forms the basis of a proximity-based homogeneous assay (Williams & Campbell, 1986; Campbell, 1988).

example, Kricka (1991) and Stanley & Kricka (1991). Literature and product surveys appear regularly in the Journal of Bioluminescence and Chemiluminescence. The high sensitivity usually associated with chemiluminescent signals is related to: (i) the relative absence of noise sources which respond to the chemical stimulus (though this makes demands on the quality of the reagents), and (ii) the instrumental set-up which allows the detector to be placed close to the sample. However, direct chemiluminescent tags give out only one photon per label, after which the label is lost. It is for this reason that in the most successful chemiluminescent approaches, the label is not a chemilumophor per se, but a catalyst for a chemiluminescent reaction, particularly an enzyme (see below). The resulting generation of many photons per attached label is capable of giving extremely high sensitivity. Chemiluminescent energy may be used to excite fluorescence in a nearby fluorophore by a (FSrster) radiationless energy transfer mechanism. This phenomenon occurs in many biolumi-

1.6.1 Chemical systems Two of the best studied systems are shown in Figure 1.3, i.e. the luminol system (or isoluminol, as shown) and the acridinium esters. Of these, the acridinium ester (Weeks et al., 1983) is used in an immunodiagnostic system (MagicLite TM, Ciba Corning) while the luminol system, though it has been used as a direct tag (Kohen et al., 1985; De Boever et al., 1986), is much more widely used as a horseradish peroxidase substrate (see below). Labelling reagents have

O

0 H2021 OH" =,= NH

O-

peroxidase v

+ N2 + H20 + light

NH2 0

NH2 0 isoluminol

CHa

~Hs

~Ha H2021 OH"

(~-~N

~

O

R acridinium ester

R CH3 I

,k

CH3 I

+ light

0

Figure 1.3 Two chemiluminescence reactions.

7

0

Non-radioactive labelling been designed to introduce these direct labels to proteins and other biomolecules. The light emission with direct chemilumophores is fast and is complete in a matter of seconds ('flash kinetics'). This does present instrumentation difficulties, requiting in situ injection of the triggering agent. Although this hurdle has been overcome for tubes and microtitre plates, it is a barrier to using this system in other formats and in very high throughput systems. More seriously for the general researcher, there is no possibility of repeat measurements; once oxidized, the label is gone. Sensitivity is instrumentation dependent but can reach attomole levels. Acridinium ester chemiluminescence forms the basis of one of the most sensitive homogeneous assays for DNA (Arnold et al., 1989); this is based on the protection against hydrolysis afforded by intercalation of an acridinium ester attached to an oligonucleotide probe when hybridized to target DNA.

1.6.2 Bioluminescent proteins The jellyfish protein aequorin, formed from the apo protein and the cofactor coelenterazine, may be stimulated to emit light by calcium ions, and may be used either to measure calcium as a label for, e.g., immunoassay. Recombinant aequorin is available (Stults et al., 1992; SeaLite Sciences) which may be chemically conjugated. Fusion proteins offer an alternative way of labelling with aequorin (Casadei et al., 1989).

1.6.3 Enzyme-generated chemiluminescence Enzyme-generated chemiluminescence is very sensitive and of growing importance in many research and diagnostics applications, with alkaline phosphatase and horseradish peroxidase dominating the field. The kinetics of light output is prolonged ('glow kinetics') which simplifies instrumentation, and allows a wider range of assay formats and film detection to be used. Enzyme-stimulated chemiluminescence is discussed in more detail below (Section 1.7). Of the bioluminescent enzymes, the best studied is the luciferase family. Luciferases are

widely used to detect or measure ATP (and hence microbial life), and have been investigated for immunoassay (Baldwin et al., 1986). Chemically conjugating luciferases has proved difficult and they have not been used as labels to any great extent.

1.6.4 Electrochemiluminescence In this system, the label is a ruthenium complex which is excited on the surface of an electrode, giving off light at 620 nm (Blackburn et al., 1991; Yang et al., 1994). The process requires tripropylamine radicals that are generated on the surface of the electrode, but the label is able to cycle (Figure 1.4) which permits very high sensitivities to be achieved. Signal-to-noise ratios and the linear dynamic range are also excellent, and non-separation formats are possible. Active ester (NHS) labelling reagents are available (IGEN). The main drawback for general research use is the specialist nature of the instrumentation which is required to bring the assay complex, electrode and photomultiplier together. It has been used for immunoassay

TPA+" .~,~H+ TPA J(bpy),+~

TPA"

Ru(bpy) 2+* ,(bpy)2+~

~ Photon (620 nm)

Figure 1.4 Mechanism of electrochemiluminescence. Reproduced with permission from Blackburn et al. (1991).

I n t r o d u c t i o n to n o n - r a d i o a c t i v e s i g n a l s a n d l a b e l s and polymerase chain reaction (PCR) product detection (DiCesare et al., 1993).

1.7 ENZYME LABELS Any reasonably stable enzyme that gives an easily detectable product can in principle be used as a signal enzyme, and many have. However, three enzymes have dominated this type of labelling: horseradish peroxidase (HRP), alkaline phosphatase and ~-galactosidase. Apart from special applications, such as homogeneous immunoassays (see Section 1.7.4), it is not necessary to look outside of these three. HRP is the most widely used signal enzyme in research (small-scale) immunoassays, but for larger immunodiagnostic systems and nucleic acid detection, alkaline phosphatase dominates. 13-Galactosidase is less frequently used now as a signal enzyme, though as a cell reporter gene it is very well established. An overview of substrates for these enzymes is given in Table 1.3. It is important to distinguish between substrates that give soluble products (used for example in microtitre plate assays) and substrates that give products that immediately precipitate or otherwise localize on the solid phase (used for example for membrane-based assays or histochemical analysis). Methods for using some of the more important substrates are given in Appendix 9.

9

dize a variety of compounds, giving coloured, fluorescent or chemiluminescent products. It is inexpensive and can be conjugated easily by a variety of routes. The mechanism has been well studied and is more complicated than the hydrolase signal enzymes (Walker et al., 1992). The enzyme is slowly activated during the reaction (Tijssen, 1985), which means that prolonged incubation does not give proportionally higher signals. HRP is not a robust enzyme, and is adversely affected by azide, oxygen and several aromatic compounds. It has good storage stability, but relatively poor heat stability, an issue for nucleic acid hybridizations. There are numerous chromogenic substrates that have been developed for HRP; convenient tablet and proprietary formulations are available (e.g. from Boehringer, Sigma, Pierce). Opinion on the relative safety of HRP substrates changes from time to time and the reader is advised to obtain up-todate safety information.

1.7.1.1 H R P detection: colour (solution)

For immunoassay and other microwell assays, the two main substrates used are ABTS (2,2 '- azino- di- (3-ethylbenzthiazoline- sulphonic acid)), which gives a green reaction product, and TMB (3,3 ',5,5 '-tetramethylbenzidine) which gives a blue product. Both of these reactions become yellow after addition of an (optional) acidic stop reagent.

1.7.1.2 H R P detection: colour (precipitating)

1.7.1 Horseradish peroxidase

HRP is a haem-containing protein, molecular weight 44 000, that is able to use H202 to oxi-

Three main substrates have been employed for localizing HRP activity: DAB (3,3'-diaminobenzidine), AEC (3-amino-9-ethylcarbazole) and

Table 1.3 Summary of principle substrates for common signal enzymes Enzyme

Chromo g enic (soluble product)

Chromo g enic (precipitating product)

Fluo ro g enic

Chemiluminescent

HRP

TMB, ABTS

DAB, AEC, 4CN

PHHA

Luminol (ECL)

Alkaline phosphatase

PNPP

BCIP (_+ NBT)

4MUP, FDP, Attophos

AMPPD, CDP etc. LumiPhos T M

13-Galactosidase

PNPG, ONPG, CPRG

BCIG (_+ NBT)

4MUG, FDG

AMPGD, LumiGal T M

10

Non-radioactive labelling

4CN (4-chloro-l-naphthol). Of these, DAB and AEC are the most sensitive but DAB is a known carcinogen. DAB may be enhanced by heavy metals such as nickel (Adams, 1981). Formulations of TMB that localize the product are also available. A good review of HRP substrates in the context of nucleic acid detection is provided by Verlander (1995).

1.7.1.3 H R P detection: fluorescence

Various dyes fluoresce when oxidized by HRP. Of these HPPA (3-(p-hydroxyphenyl)propionic acid) has been used to assay and detect the enzyme (Zaitsu & Ohkura, 1980).

1.7.1.4 H R P detection: chemiluminescence

HRP is able to catalyse the oxidation of luminol, giving off light (Figure 1.3). This reaction was of minor analytical importance until the discovery that the duration and intensity of light output could be increased greatly in the presence of firefly luciferin (Whitehead et al., 1983) and certain organic compounds, notably iodophenol (Thorpe & Kricka, 1986). The mechanism of this reaction is complicated (Vlasenko et al., 1989) but need not concern the user; yet again it is important to note that the time course of light emission is relatively short lived, peaking within minutes and decaying over just a few hours. This system, known as 'enhanced chemiluminescence' (ECL; but not to be confused with electrochemiluminescence which also gives rise to this acronym), is the most sensitive detection system for HRP. It has been used in clinical immunodiagnostics (the Amerlite system) and is also popular for Western blotting (Vachereau, 1989; for a discussion on background noise, see Pampori et al., 1995). Reagents are available from Amersham and Pierce. Greater sensitivity may be obtained from the Pierce proprietary formulation in Western blotting (SuperSignal; R. Forder, personal communication, 1995). One major downside of ECL is that there is only one opportunity to develop the signal, since HRP is effectively inactivated during the reac-

tion. The practical consequences can be severe: any problem, mistake or interruption and the experiment is lost.

1.7.1.5. H R P amplification systems

Catalysed reporter deposition is an interesting approach to increasing enzyme signals. In this technique (Bobrow et al., 1989, 1991), the primary label is HRP which acts on biotinyl tyramide to deposit biotin on the assay solid phase. This biotin is then detected conventionally with a streptavidin-enzyme conjugate (the enzyme may be alkaline phosphatase, HRP or any other label). The amplification factor is therefore due to the increased amount of biotin deposited, compared to a conventional single biotin tag, and may be one to two orders of magnitude. The system, known as TSA TM, is available from NEN and Dako.

1.7.2 Alkaline phosphatase Although many alkaline phosphatases have been extensively studied, it is the c a l f intestinal enzyme that dominates the field. This enzyme is robust, easy to conjugate and is capable of greater sensitivity than HRP. Its turnover of substrate is continuous, such that the signal obtained is roughly proportional to the incubation period; this gives valuable flexibility. A dimeric protein of molecular weight ca. 150000, alkaline phosphatase is a metalloenzyme, requiring both magnesium and zinc for activity. However, provided the enzyme is not allowed to become deficient in these metals, it may be handled in metal-free buffers, provided also that chelates are avoided. Phosphate is inhibitory and should be diluted or washed away before measurement (Tijssen, 1985), but it may be present during labelling and storage. Furthermore, phosphate does appear to enhance storage stability. Alkaline phosphatase is capable of hydrolysing many of phosphate esters and this has aided the development of a wide variety of substrates (see Figures 1.5 and 1.6). This diversity, and the capability of very high sensitivity, make alkaline phosphatase a good enzyme in which to invest.

I n t r o d u c t i o n to n o n - r a d i o a c t i v e signals a n d labels O

O

II

HO-P-OH I

II

el

O2N

H O - P -OH I

N H PNPP

BCIP

O

O

HO - P - OH I

HO - P - OH !

II

~

11

CH3

H O - P - OH ~ O MUPP

II

,9 O--P - O H

bH

AttophosTM

fluorescein

diphosphate

Figure 1.5 Some important chromogenic and fluorogenic substrates for alkaline phosphatase. For details, see text. 1.7.2.1 Alkaline phosphatase detection: colour (solution) PNPP (p-nitrophenyl phosphate) is a simple but very effective substrate, releasing the yellow (405 nm)p-nitrophenolate ion. Though not by any means the most sensitive substrate for this enzyme, it is nevertheless adequate for the majority of routine immunoassays, particularly if prolonged enzyme incubations are acceptable.

1.7.2.2 Alkaline phosphatase detection: colour (precipitating) The standard method for localizing alkaline phosphatase activity is the BCIP/NBT system. BCIP (5-bromo-4-chloro-3-indoyl phosphate) is dephosphorylated to an active intermediate that oxidatively dimerizes to give an insoluble indigo dye (McGadey, 1970). The intensity of the signal is greatly increased in the presence of NBT (nitroblue tetrazolium) which gives an intense red/purple formazan product, again insoluble. Related substrates are the salmon and magenta phosphates (Avivi et al., 1994). Azo dyes can be formed using naphthol AS phosphate derivatives in the presence of diazonium salts (Kunz & West, 1992); though less sensitive than BCIP/

NBT, different colours may be generated with different derivatives.

1.7.2.3 Alkaline phosphatase detection: fluorescence 4-Methylumbelliferyl phosphate is an excellent fiuorogenic substrate for alkaline phosphatase and has long been used in immunodiagnostics (e.g. Giegel et al., 1982). It is about 10-fold more sensitive than PNPP and has high stability to hydrolysis. The (soluble) product, 4-methylumbelliferone, is excited at 360 nm and measured at 450 nm and can be read in conventional fluorescence plate readers. Difficulties in preparing fluorescein diphosphate in pure form (Rotman et al., 1963) have hitherto prevented the general use of this substrate, which is otherwise attractive given the ease with which the product fluorescein may be measured. It is now available commercially (Molecular Probes, USA; Haugland, 1992). A newer fluorogenic substrate, Attophos, shows promise as a very sensitive detection system (Cano et al., 1992), matching in sensitivity the chemiluminescent dioxetane substrates described below (Kerkhof, 1992). The reaction product is soluble but appears to localize on membranes; it may be excited at 440 nm and

12

Non-radioactive labelling R

O- 0 O- CH3

alkaline phosphatase

R . ~ j OO-O-ell3 O"

0 I

O-P-OH I OH

O

R~

0 cH3 "

+

0~~'~

9 light O-

Figure 1.6 Mechanism of light generation from the dioxetane chemiluminescent alkaline phosphatase substrates. R = H: AMPPD, Lumiphos; R = CI: CSPD. detected at 560 nm. The system fits well with fluorescent imaging instrumentation such as the FluorImager T M and Storm T M (Molecular Dynamics). A precipitating fluorogenic substrate, CPPCQ, has been developed by workers at Molecular Probes for histochemical applications (Huang et al., 1992, 1993). The product emits at 520 nm when excited at 320-420 nm. A time-resolved fluorescence detection system for alkaline phosphatase (EALL, enzymeamplified lanthanide luminescence) has been described (Kronem Sytems; Evangelista et al., 1991; Christopoulos & Diamandis, 1992). The substrate is a phosphate ester of salicylic acid; the salicylic acid product then forms a fluorescent complex with terbium and ethylenediaminetetraacetic acid (EDTA).

1.7.2.4 Alkaline phosphatase detection: chemiluminescence

The development of dioxetane-based chemiluminescent substrates for various hydrolytic enzymes, but particularly alkaline phosphatase (Schaap et al., 1987; Bronstein & McGrath, 1989; Bronstein et al., 1989a), has been one of the most significant steps forward in the field of non-isotopic signalling in recent years. The main feature is very high sensitivity, but there are also a host of other useful features that make the reagents attractive to use. Nucleic acid detection has been the focus of attention (Clyne et al.,

1989; Bronstein et al., 1990), but other applications such as Western blotting (Bronstein et al., 1992a) and immunodiagnostics (Bronstein et al., 1990) are equally important. The enzyme reaction is given in Figure 1.6. Enzymic removal of the phosphate group generates an unstable intermediate which decomposes to give two ketones, one of which, the methyl meta-oxybenzoate anion, is in an excited state that collapses to emit light at 477 nm. The kinetics of light emission is therefore a product of the linear generation of the unstable intermediate and the exponential decay of this intermediate to give light. After a build-up phase, the light is then produced in a steady 'glow' that can last for up to several days. The CSPD T M substrate has faster kinetics, but more importantly gives a lower non-enzymic background. The time to plateau is also influenced by the chemical environment. The prolonged light output permits multiple exposures to be made and avoids the time constraints associated with HRP chemiluminescence. Filter spots and streaks were an occasional disappointing feature of Lumiphos and AMPPD, which, even with very careful technique, could not always be eliminated. CSPD T M and, more recently, 'CDP-Star xM', are better in this respect; also, detection protocols have improved. The quantum yield for this process is low (0.13%); however, in solution, the quantum

I n t r o d u c t i o n to n o n - r a d i o a c t i v e signals a n d labels yield may be increased with 'enhancers', which are essentially fluors that trap the energy by a more efficient process and re-emit also with high quantum yield, giving an overall quantum yield of 48% (Schaap et al., 1989). On nylon membranes, this enhancement is not necessary since the hydrophobic environment substantially increases the quantum yield. Nitrocellulose and PVDF membranes give poor results, though the treatments that make them nylon-like are available (Bronstein et al., 1992b). An unexpected feature of these substrates is the minimal product diffusion rate on nylon membranes giving rise, for example, to tight bands on Southern blots. Sensitivity is the main feature of dioxetane substrates, exceeding that of 32p (Giles et al., 1990), enhanced HRP chemiluminescence (Sandhu et al., 1991) and BCIP/NBT (Bronstein et al., 1989b). Generally, the sensitivity is traded in for shorter exposure times, so that typically an exposure of a few hours gives equivalent results to an exposure of several days with 32p. Other attractive features of this system are the high storage stability of the substrate in ready-to-use form, the ease of stripping and reprobing with Southern blots, and the ability to use X-ray film detection, giving results in the same way as autoradiography. Chemiluminescence plate readers may also be used. One drawback of dioxetane substrates is the price, which is high compared with other substrates. Whether the concomitant convenience justifies this will depend on the application and the situation, but typically the extra cost is insignificant compared with the alternatives when the system as a whole is costed. Dioxetane substrates, enhancer formulations and related products are available from Tropix, Lumigen and other suppliers.

13

1.7.2.6 Alkaline phosphatase amplification systems

Very high sensitivities may be achieved with a colour end-point using an ingenious 'enzyme amplification' scheme (Self, 1985; Stanley et al., 1985; Johannsson et al., 1986). In this scheme, alkaline phosphatase converts NADP to NAD +, which feeds into a redox cycle, each 'turn' of which generates a molecule of the red/ purple dye formazan. This extra level of amplification extends the sensitivity of alkaline phosphatase down to 0.01 attomole. It is important to note that the generation of colour is not linear with time and that therefore there is not a linear trade-off of sensitivity with signal development time. Reagents for this system (AMPAK TM) are available from Dako. A fluorescence analogue of this approach has also been reported (Cook and Self, 1993).

1.7.3 [~-Galactosidase The [3-galactosidase from Escherichia coli is a large (molecular weight 465 000), tetrameric enzyme which has been popular for immuneassay, though its use appears to be declining (Gosling, 1990). Its size is a disadvantage, especially in immunohistochemistry, and it is relatively expensive. In other respects it is a perfectly adequate signal enzyme; easy to conjugate and reasonably robust. It contains free cysteines which can be employed for conjugation. An elegant homogeneous immunoassay, termed CEDIA (cloned enzyme donor immunoassay), has been developed; this is based on the inhibition of the formation of (active) tetramers of [3galactosidase (Henderson et al., 1986). 1.7.3.1 ~-Galactosidase substrates

1.7.2.5 Bioluminescence systems

The firefly luciferase may be coupled with alkaline phosphatase; here alkaline phosphatase acts on D-luciferin-O-phosphate and the luciferin product feeds into the luciferase reaction (Hauberet al., 1989). Very high sensitivity, down to 0.03 attomole, is claimed with a photon-counting camera.

Like alkaline phosphatase, [3-galactosidase is a hydrolytic enzyme which is relatively tolerant to the nature of the leaving group. As a result, nearly all the substrates developed for alkaline phosphatase (see above) have their ~-galactosidase analogues, where phosphate is replaced by 13-D-galactopyranoside. Thus we have, for example, ONPG and PNPG (o- and p-nitrophenyl

14

Non-radioactive labelling

galactopyranoside), BCIG (5-bromo-4-chloro-3indoyl galactopyranoside; also known as X-gal), MUG (4-methylumbelliferyl galactopyranoside) and FDG (FACS-GAL, fluorescein digalactoside). FDG may be detected with the Fluorlmager (Molecular Dynamics). The chlorophenol red derivative (CPRG) is often preferred to the nitrophenyl substrates. Chemiluminescent dioxetane substrates AMPGD, Galacto Light T M (Tropix) and Lumigal T M (Lumigen) have been developed (Beale et al., 1992). It should be noted however that dioxetane light emission is strongest at alkaline pH whereas I]-galactosidase operates optimally near neutrality; hence a compromise pH is necessary or, alternatively, one can raise the pH to measure the signal.

devised. The power of the PCR has been exploited for the detection of antibody-antigen complexes (Sano et al., 1992; Zhou et al., 1993; Joerger et al., 1995). In this extremely sensitive technique, 'immuno-PCR', the antibody is linked to a DNA tag which is amplified by PCR and detected conventionally by electrophoresis (although less manipulative detection could be devised). As little as 580 antigen molecules have been detected, though in principle, single molecule detection is possible. Few conventional analytical applications need anything like this sensitivity, but this approach is attracting interest for virus detection and suggests new horizons for bioanalysis. 'Midivariant' RNA may be amplified by QI]replicase to give up to 10 ~2 copies. This system, with fluorescence detection, has been proposed as general reporter system (Burg et al., 1995).

1.7.4 Homogeneous enzyme immunoassays

Those prepared to invest in a homogeneous immunoassay will need to consider other enzymes that have been exploited for this purpose. Here the objective is to construct a conjugate of the analyte (the analyte is usually of low molecular weight) and an enzyme, such that when it binds antibody the enzyme activity is modulated (Bastiani et al., 1973). The most easily understood modulation is steric interference with substrate binding by the attached antibody, but allosteric effects can also be exploited. The system (known as 'EMIT') can then be used in competition mode, where free analyte competes with enzyme-analyte for antibody, the level of analyte thereby affecting the level of enzyme activity. Glucose-6-phosphate dehydrogenase, malate dehydrogenase and lysozyme are examples of enzymes that have been used for this purpose (Tijssen, 1985).

1.8 OTHER LABELS

1.8.2 Gold

Molecules labelled with colloidal gold particles have been widely used in electron microscopy, but have also been employed in molecular biology (Van de Plas and Leunissen, 1992) and immunoassay (Moeremans et al., 1984; Rocks et al., 1991; Urdal et al., 1992). Gold labelling gives a permanent and very visual signal and, not needing any complex instrumentation, is suited (for example) to off-site testing. The ultra-small gold particles (Leunissen et al., 1989) have an average diameter of ca. 1 nm, i.e. small compared with the size of the protein to which they are attached. The gold-labelled complex, once formed, may be considerably enhanced by silver staining. The primary labelling of proteins with gold is not straightforward and it is therefore best to approach this label first by means of prelabelled streptavidin, protein A, protein G and secondary antibodies, which are commercially available form several sources (e.g. Amersham, Biocell, Aurion, Janssen).

1.8.1 Nucleic acid labels

1.8.3 Surface plasmon resonance

Though not in widespread use, several interesting labels based on nucleic acids have been

In this technique (J6nsson et al., 1991), the label is the mass of the molecule itself. This biosensor

I n t r o d u c t i o n to n o n - r a d i o a c t i v e s i g n a l s a n d l a b e l s technique (BIAcore TM, Pharmacia) exploits the change in refractive index at the surface of a gold-coated glass plate that occurs when one protein binds to another that is bound to the surface. The signal is a function of the total mass of the protein complex, hence only proteins and large peptides give a sufficient change in mass. It is of considerable value for characterizing protein-protein interactions, particularly antigens and antibodies.

1.8.4 Particles/beads as labels

Immunoassays can be configured in a variety of ways to bring about the cross-linking of particles which may be detected by nephelometry, turbidometry or particle counting. This is generally a latex particle which may be passively coated with protein, or, alternatively, a variety of chemically functionalized particles may be obtained for covalent attachment. Generally, specialized equipment is required for quantitation, although microtitre plate readers have been used for turbidometry (Collet-Cassart et al., 1989). Semi-quantitative results may be obtained by visual inspection and such an approach is suitable for low-sensitivity, out-oflab tests. Derivatization of latex particles has been described by Bangs (1984).

1.9 SUMMARY: WHICH LABEL? Given the breadth of possible applications and the variety of environments, it is clearly hard to make firm recommendations that would suit every situation; but here are some guidelines. It is probably useful to consider what degree of sensitivity is required: adjectives such as super-sensitive and ultra-sensitive have seductive appeal but many applications are not very demanding in this respect and conventional (and cheaper) technology can be used. One must also think about the format and the instrumentation to be used: signals and labels cannot be considered in isolation and one must consider the sensitivity of the analytical system as a whole. Fluorescent labels are stable, easy to label

15

with and give an instant signal. For this reason they should be considered first, at least for lower sensitivity applications. Signal-to-noise may be an issue, but fluorescence imaging and use of long-wavelength dyes may surmount this. Timeresolved fluorescence should be considered for more demanding applications, but this is presently limited mainly to microplate applications. If fluorescent labels seem impractical, the second main option is an enzyme label. Alkaline phosphatase is suggested as the first all-round choice, although for some applications (e.g. immunoassay, Western blotting) or where the ultimate in sensitivity is not required, HRP is also an option. Introducing new labels can involve considerable investment of time and money: overall, the best advice is to keep it simple and, wherever possible, follow good precedents.

REFERENCES Adams, J.C. (1981). Heavy metal intensification of DAB based HRP reaction product. J. Histochem. Cytochem. 29, 775. Alpha, B., Lehn, J.M. & Mathis, G. (1987). Energy transfer luminescence of europium (III) and terbium (III) cryptates of macrobicyclic polypyridine ligands. Angew. Chem. 26, 266-267. Arnold, L.J., Hammond, P.W., Wiese, W.A. & Nelson, N.C. (1989). Assay formats involving acridinium-ester-labeled DNA probes. Clin. Chem. 35, 1588-1594. Avivi, C., Rosen, O. & Goldstein, R.S. (1994). New chromogens for alkaline phosphatase histochemistry: salmon and magenta phosphate are useful for single- and double-label immunohistochemistry. J. Histochem. Cytochem. 42, 551-554. Baldwin, T.O., Holzman, T.F. & Holtzman, R.B. (1986). Active center-based immunoassay approach using bacterial luciferase. Meth. Enzymol. 133, 248-264. Bangs, L.B. (1987). Uniform Latex Particles. Seradyn, Inc. Barnes, M.D., Whitten, W.B. & Ramsey, J.M. (1995). Detecting single molecules in solution. Anal. Chem. 67, 418A-423A. Bastiani, R.J., Phillips, R.C., Schneider, R.S. & Ullman, E.F. (1973). Homogeneous immunochemical drug assays. Am. J. Med. Technol. 39, 211-216.

16

Non-radioactive

labelling

Beale, E.G., Deeb, E.A., Handley, R.S., AkhavanTafti, H. & Schaap, A.P. (1992). A rapid and simple chemiluminescent assay for Escherichia coli ~-galactosidase. Biotechniques 12, 320-323. Blackburn, G.F., Shah, H.P., Kenten, J.H., Leland, J., Kamin, R.A., Link, J., Peterman, J., Powell, M.J., Shah, A., Talley, D.B., Tyagi, S.K., Wilkins, E., Wu, T-G. & Massey, R.J. (1991). Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem. 37, 1534-1539. Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. & Litt, G.J. (1989). Catalysed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. lmmunol. Meth. 125, 279-285. Bobrow, M.N., Shaughnessy, K.J. & Litt, G.J. (1991). Catalysed reporter deposition, a novel method of signal amplification. Application to membrane immunoassays. J. Immunol. Meth. 137, 103-112. Bronstein, I. & McGrath, P. (1989). Chemiluminescence lights up. Nature 333, 599-600. Bronstein, I., Edwards, B. & Voyta, J.C. (1989a). 1,2Dioxetanes: novel chemiluminescent enzyme substrates. Applications to immunoassays. J. Biolumin. Chemilumin. 4, 99-111 (1989). Bronstein, I., Voyta, J.C. & Edwards, B. (1989b). A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. Anal. Biochem. 180, 95-98. Bronstein, I., Thorpe, G.H.G., Kricka, L.J., Edwards, B. & Voyta, J.C. (1990). Chemiluminescent enzyme immunoassay for alpha-fetoprotein. Clin. Chem. 36, 1087-1088. Bronstein, I., Voyta, J.C., Murphy, O.J., Bresnick, L. & Kricka, L.J. (1992a). Improved chemiluminescent Western blotting procedure. Biotechniques 12, 748-753. Bronstein, I., Fortin, J. & Voyta, J.C. (1992b). Nitroblock enhancement of AMPPD chemiluminescent signal in the detection of DNA. Biotechniques 12, 500-502. Burg, J.L., Cahill, P.B., Kutter, M., Stefano, J.E. & Mahan, D.E. (1995). Real-time fluorescence detection of RNA amplified by Q[3 replicase. Anal. Biochem. 230, 263-272. Campbell, A.K. (1988) Chemiluminescence. VCH, Weinheim. Cano, R.J., Torres, M.J., Klem, R.E. & Palomares, J.C. (1992). DNA hybridization assay using Attophos, a fluorescent substrate for alkalinephosphatase. Biotechniques 12, 264-267. Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C. & Wolf, D.E. (1988). Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85, 8790-8794. Casadei, J., Powell, M.J. & Kenten, J.H. (1989) Characterization of a chimeric aequorin molecule

expressed

in myeloma cells.

J.

Biolumin.

Chemilumin. 4, 246-350.

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I n t r o d u c t i o n to n o n - r a d i o a c t i v e s i g n a l s a n d l a b e l s E.F. (1991). Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays. Anal. Biochem. 197, 213-224. Garman, A.J. & Moore, R. (1990). Detection of nucleic acid sequences using fluorescence polarisation. Eur. Patent 382433. Giegel, J.L., Brotherton, M.M., Cronin, P., D' Aquino, M., Evans, S., Heller, Z.H., Knight, W.S., Krishnan, K. & Sheiman, M. (1982). Radial partition immunoassay. Clin. Chem. 28, 1894-1898. Giles, A.F., Booth, K.J., Parker, J.R., Garman, A.J., Carrick, D.T., Akhavan, H. & Schaap, A.P. (1990). Rapid, simple, non-isotopic probing of Southern blots for DNA fingerprinting. Adv. Forens. Haemogen. 3, 40. Gosling, J.P. (1990). A decade of development in immunoassay methodology. Clin. Chem. 36, 1408-1427. Hauber, R., Miska, W., Schleinkofer, L. & Geiger, R. (1989). New, sensitive, radioactive-free, bioluminescence-enhanced detection system in protein blotting and nucleic acid detection. J. Biolumin. Chemilumin. 4, 367-372. Haugland, R.P. (1992). Handbook of fluorescent probes and research chemicals. Molecular Probes, Eugene, OR. Heim, R., Cubitt, A.B. and Tsien, R.Y. (1995). Improved green fluorescence. Nature 373, 663664. Hemmil~i, I. (1988). Lanthanides as probes for timeresolved fluorometric immunoassays. Scand. J. Clin. Lab. Invest. 48, 389-400. Hemmil~i, I., Dakubu, S., Mukkala, V.-M., Siitari, H. & L6vgren, T. (1984). Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 335-343. Henderson, D.R., Friedman, S.B., Harris, J.D., Manning, W.B. & Zoccoli, M.A. (1986). CEDIA, a new homogeneous immunoassay system. Clin. Chem. 32, 1637-1641. Huang, Z., Terpetschnig, E., You, W. & Haugland, R.P. (1992). 2-(2'-Phosphorylphenyl)-4(3H)quinazolinone derivatives as fluorogenic precipitating substrates of phosphatase. Anal. Biochem. 207, 32-39. Huang, Z., You, W., Haugland, R.P., Paragas, V.B., Olson, N.A. & Haugland, R.P. (1993). A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ. J. Histochem. Cytochem. 41, 313-317. Joerger, R.D., Truby, T.M., Hendrickson, E.R., Young, R.M. & Ebersole, R.C. (1995). Analyte detection with DNA-labelled antibodies and polymerase chain reaction. Clin. Chem. 41, 1371-1377. Johannsson, A., Ellis, D.H., Bates, D.L., Plumb, A.M. & Stanley, C.J. (1986). Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole. J. Immunol. Meth. 87, 7-11. Jolley, M.E., Stroupe, S.D., Schwenzer, K.S., Wang,

17

C.J., Lu-Steffes, M., Hill, H.D., Popelka, S.R., Holen, J.T. and Kelso, D.M. (1981 ). Fluorescence polarization immunoassay. III. An automated system for therapeutic drug determination. Clin. Chem. 27, 1575-1579. J6nsson, U. et al. (1991). Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 11, 620-627. Kerkhof, L. (1992). A comparison of substrates for quantifying the signal from a nonradiolabeled DNA probe. Anal. Biochem. 205, 359-364. Khosravi, M. and Diamandis, E.P. (1987). Immunofluorometry of choriogonadotrophin by timeresolved fluorescence spectroscopy, with a new europium chelate as label. Clin. Chem. 33, 1994-1999. Kohen, F., Pazzagli, M., Serio, M., de Boever, J. & Vandekerckhove, D. (1985). Chemiluminescence and bioluminescence immunoassay. In Alternative Immunoassays (ed. W.P. Collins) John Wiley, Chichester. Kominami, G. (1994). Sensitivity of immunoenzymometric assay and detection method of enzyme. J. Immunoassay 15, 79-92. Kricka, L.J. (1991). Chemiluminescent and bioluminescent techniques. Clin. Chem. 37, 1472-1481. Kunz, W. & West, S. (1992). Azo dyes. In Nonradioactive labeling and detection of biomolecules

(ed. C. Kessler), pp. 161-164. Springer, New York. Leunissen, J.L.M., Van de Plas, P.F.E.M. & Borghgraef, P.E.J. (1989). Auroprobe One: a new and universal ultra small gold particle based (immuno)detection system for high sensitivity and improved penetration. Aurofile 2, 1-2, Janssen Life Sciences. Lopez, E., Chypre, C., Alpha, B. & Mathis, G. (1993). Europium (III) trisbipyridine cryptate label for time-resolved fluorescence detection of polymerase chain reaction products fixed on a solid support. Clin. Chem. 39, 196-201. Madersbacher, M., Shu-Chen, T., Schwarz, S., Dirnhofer, S., Wick, G & Berger, P. (1993). Timeresolved immunofluorometry and other frequently used immunoassay types for folliclestimulating hormone compared by using identical monoclonal antibodies. Clin. Chem. 39, 1435-1439. Mathis, G. (1993) Rare earth cryptates and homogeneous fluoroimmunoassays with human sera. Clin. Chem. 39, 1953-1959. Mathis, G. (1995). Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin. Chem. 41, 1391-1397. McGadey, J. (1970). A tetrazolium method for nonspecific alkaline phosphatase. Histochemie 23, 180-184. Mikola, H., Takalo, H. & Hemmil~i, I. (1995).

18

Non-radioactive labelling

Syntheses and properties of luminescent lanthanide chelate labels and haptenic antigens for homogeneous immunoassays. Bioconj. Chem. 6, 235-241. Moeremans, M., Daneels, G., Van Dijck, A., Langanger, G. & De Mey J. (1984). Sensitive visualisation of antigen-antibody reactions in dot and blot immuno overlay assays with immunogold and immunogold/silver staining. J. Immunol. Meth. 74, 353-360. Morrison, L.E., Halder, T.C. & Stols, L.M. (1989). Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. Anal. Biochem. 183, 231-244. Ngo, T.T. (1988). Non-isotopic immunoassay. Plenum, New York. Nguyen, D.C., Keller, R.A., Jett, J.H. & Martin, J.C. (1987). Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser-induced fluorescence. Anal. Chem. 59, 2158-2161. Nie, S., Chiu, D.T. & Zare, R.N. (1994). Probing individual molecules with confocal fluorescence microscopy. Science 266, 1018-1021. Pampori, N.A., Pampori, M.K. & Shapiro, B.H. (1995). Dilution of the chemiluminescence reagents reduces background noise on Western blots. Biotechniques 18, 588-590. Remuifi~in, M.J., Rom~in, H., Alonso, M.T. & Rodriguez-Ubis, J.C. (1993). Synthesis and luminescence properties of europium (III) and terbium (III) complexes with polyacid chelates derived from 2,6-bis(N-pyrazolyl)pyridine. J. Chem. Soc. Perkin Trans 2, 1099-1102. Rocks, B.F., Patel, N. & Bailey, M.P. (1991). Use of a silver-enhanced gold-labelled immunoassay for detection of antibodies to the human immunodeficiency virus in whole blood samples. Ann. Clin. Biochem. 28, 155-159. Rotman, B., Zderic, J.A. & Edelstein, M. (1963). Fluorogenic substrates for 13-o-galactosidases and phosphatases derived from fluorescein (3,6-dihydroxyfluoran and its monomethyl ether). Proc. Natl. Acad. Sci. USA 50, 1-6. Saha, A.K., Kross, K., Kloszewski, E.D., Upson, D.A., Toner, J.L., Snow, R.A., Black, C.D.V. & Desai, V.C. (1993). Time-resolved fluorescence of a new europium chelate complex: demonstration of highly sensitive detection of protein and DNA samples. J. Am. Chem. Soc. 115, 11032-11033. Sandhu, G.S., Eckloff, B.W. & Kline, B.C. (1991). Chemiluminescent substrates increase sensitivity of antigen detection in Western blots. Biotechniques 11, 14-16.

Sano, T., Smith, C.L. & Castor, C.R. (1992). Immuno-PCR: very sensitive antigen detection by means of specific antibody DNA conjugates. Science 258, 120-122. Schaap, A.P., Sandison, M.D. & Handley, R.S.

(1987). Chemical and enzymatic triggering of 1,2-dioxetanes. 3: alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane. Tetrahedron Lett. 28, 1159-1162. Schaap, A.P., Akhavan, H. & Romano, L.J. (1989). Chemiluminescent substrates for alkaline phosphatase: application to ultra-sensitive immunoassays and DNA probes. Clin. Chem. 35, 18631864. Self, C.H. (1985). Enzyme amplification- a general method applied to provide an immunoassisted assay for placental alkaline phosphatase. J. Immunol. Meth. 76, 389-393. Selvin, P.R. & Hearst, J.E. (1994). Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. Proc. Natl. Acad. Sci. USA 91, 10024-10028. Selvin, P.R., Rana, T.M. & Hearst, J.E. (1994). Luminescence resonance energy transfer. J. Am. Chem. Soc. 116, 6029-6030. Soini, E. & Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates. A new generation of non-isotopic immunoassays. Clin. Chem. 29, 65-68. Stanley, C.J., Johannsson, A. & Self, C.H. (1985). Enzyme amplification can enhance both the speed and the sensitivity of immunoassays. J. Immunol. Meth. 83, 89-95. Stanley, P.E. & Kricka, L.J. (eds) (1991). Bioluminescence and chemiluminescence: current status.

John Wiley, Chichester. Stults, N.L., Stocks, N.F., Rivera, H., Gray, J., McCann, R.O., O'Kane, D., Cummings, R.D., Cormier, M.J. & Smith, D.F. (1992). Use of recombinant aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli. Biochemistry 31, 1433-1442. Thorpe, G.H.G. & Kricka, L.J. (1986). Enhanced chemiluminescent reactions catalysed by horseradish peroxidase. Meth. Enzymol. 133, 331-353. Tijssen, P. (1985). Practice and theory of enzyme immunoassays. Lab. Tech. Biochem. Mol. Biol. 15. Ullman, E.F., Schwarzberg, M. & Rubenstein, K.E. (1976). Fluorescence excitation transfer immunoassay. J. Biol. Chem. 251, 4172-4178. Urdal, P., Borch, S.M., Landaas, S., Krutnes, M.B., Gogstad, G. & Hjortdal, P. (1992). Rapid immunometric measurement of C-reactive protein in whole blood. Clin. Chem. 38, 580-584. Vachereau, A. (1989). Luminescent immunodetection of Western blotted proteins from Coomassiestained polyacrylamide gel. Anal. Biochem. 179, 206-208. Van de Plas, P.F.E.M. and Leunissen, J.L.M. (1992). Colloidal gold as a marker in molecular biology: the use of ultra-small gold particles. In Non-

I n t r o d u c t i o n to n o n - r a d i o a c t i v e s i g n a l s a n d l a b e l s radioactive labeling and detection of biomolecules (ed. C. Kessler). Springer, Berlin. Verlander, P.C. (1992). Detection of horseradish peroxidase by colorimetry. In Nonisotopic probing blotting and sequencing (ed. L.J. Kricka). Academic Press, London. Vlasenko, S.B., Arefyev, A.A., Klimov, A.D., Kim, B.B., Gorovits, E.L., Osipov, A.P., Gavrilova, E.M. & Yegorov, A.M. (1989). An investigation on the catalytic mechanism of enhanced chemiluminescence. J. Biolumin. Chemilumin. 4, 164-176. Walker, M.R., Stott, R.A. & Thorpe, G.H.G. (1992). Enzyme-labeled antibodies in bioassays. Bioanal. Appl. Enzymes 36, 179-208. Wang, S. & Hazelrigg, T. (1994). Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369, 400-403. Weeks, I., Beheshti, I., McCapra, F., Campbell, A.K. & Woodhead, J.S. (1983). Acridinium esters as high-specific-activity labels in immunoassay. Clin. Chem. 29, 1474-1479. Whitehead, T.P., Thorpe, G.H.G., Carter, T.J.N., Groucott, C. & Kricka, L.J. (1983). Enhanced luminescence procedure for sensitive determination of peroxidase-labelled conjugates in immunoassay. Nature 305, 158-159. Williams, E.J. & Campbell, A.K. (1986). A homogeneous assay for biotin based on chemiluminescence energy transfer. Anal. Biochem. 155, 249-255. Yang, H., ~Leland, J.K., Yost, D. & Massey, R.J.

19

(1994). Electrochemiluminescence: a new diagnostic and research tool. Biotechnology 12, 193-194. Zaitsu, K. & Ohkura, Y. (1980). New fluorogenic substrates for horseradish peroxidase: rapid and sensitive assays for hydrogen peroxide and the peroxidase. Anal. Biochem. 109, 109-113. Zhou, H., Fisher, R.J. & Papas, T.S. (1993). Universal immuno-PCR for ultra-sensitive target protein detection. Nucl. Acids Res. 21, 6038-6039.

FURTHER READING Campbell, A.K. (1988). Chemiluminescence. VCH Ellis Horwood, Chichester. Collins, W.P. (Ed.) (1985). Alternative immunoassays. John Wiley, Chichester. De Luca, M.A. & McElroy, W.D. (1986) B ioluminescence and chemiluminescence. Part B. Meth. Enzymol. 133. Hemmil~i, I.A. (1991). Applications of fluorescence in immunoassays. John Wiley, Chichester. Kricka, L.J. (1990). Selected strategies for improving sensitivity and reliability of immunoassays. Clin. Chem. 40, 347-357. McComb, R.B., Bowers, G.N. & Posen, S. (1979). Alkaline phosphatase. Plenum Press, New York. Tijssen, P. (1985). Practice and theory of enzyme immunoassays. Elsevier, Amsterdam.

CHAPTER

TWO

Labelling proteins and peptides: chemical background

2.1 INTRODUCTION Being large, polyfunctional molecules, proteins offer a wide variety of possibilities for chemical modification for labelling purposes. Arguably, this diversity has been a cause for difficulties in the past, since for many schemes there is plenty of scope for overmodification, polymerization and poor reproducibility. This may be avoided however by careful selection and control of the most appropriate coupling chemistry. Regardless of the label selected, be it a low molecular weight label introduced by use of specially designed reagents, or protein (for example, enzyme) label introduced by one of numerous conjugation schemes, the type of chemistry used is similar. Nucleic acid labelling by chemical modification also draws on many of

the schemes originally employed for protein labelling. This chapter is intended to introduce some of the more commonly used chemistries and the reaction conditions that are required. This is intended to help understand what features of a labelling protocol are important and why; this is of importance when modifying a literature protocol (including protocols in this book), as well as helping to derive protocols from first principles, where necessary. The chemistry of peptide labelling and conjugation is broadly similar to proteins except that peptides tend to be more idiosyncratic in terms of solubility and general handling conditions and, of course, not all the amino acids generally present in proteins will be represented. Aspects peculiar to peptides will be discussed in Section 2.8.

Proteins and peptides: chemical background

and Arg have also been designed, but these have not been extensively used. Lys, Cys, Asp and Glu bear functional groups that under moderate conditions are quite reactive and it is to these residues that the majority of conjugation and labelling reagents are directed. The N-terminal a-amino group and the C-terminal carboxyl group are also sufficiently reactive, provided that they are not blocked by a posttranslational modification; amidated C-termini, for example, are essentially unreactive. In addition, the carbohydrate groups of glycoproteins are generally reactive to periodate oxidation, affording a useful route for labelling.

2.2 AMINO ACID FUNCTIONAL GROUPS

2.2.1 Choice of attachment site A diagrammatic view of a protein, seen from a chemical modification perspective, is given in Figure 2.1. Of the 20 amino acids found in proteins, the side chains of only a small number are useful for the attachment of labels. That is not to say that the others are totally unreactive, but simply that reaction under moderate, aqueous conditions is either very difficult, inconvenient or difficult to control. In this class we have Gly, the hydrophobic side chains of Ala, Val, Leu, Ile, Pro and Phe and the amides of Asn and Gln. The hydroxyls of Ser and Thr are in general unreactive under moderate aqueous conditions, though reaction at hyper-reactive serines of, for example, serine proteases is possible and useful. N-terminal serine is also modifiable (Geoghegan et al., 1993). Trp and Met are capable of modification but are not useful for conjugation purposes. Labelling reagents that react at Tyr, His

H2N ~

21

2.2.2 Reactivity of side chains The protein modification reactions used in labelling are invariably under kinetic control; i.e. it is the most reactive side chains in a given class that are preferentially modified. The reactivity of any particular functional group varies greatly depending on, inter alia, the local chemical environment and the degree to which the

. SH

COOH

H2 O

/ ~ Asn

Phe

Met

Trp

Pro

COOH /

H2

§ H2N

NH2

Figure 2.1 Schematic showing a protein from a chemical modification perspective. Residues in the top half of the diagram are highly suitable for labelling purposes becoming (clockwise) progressively less suitable. Unreactive amino acids or those usually found unexposed to solvent are shown in the centre.

22

Non-radioactive labelling

group is exposed. The latter factor depends also on the size of the modifying reagent, smaller reagents being able to penetrate the crevices of a protein's surface better than larger reagents. The ionization state of the group to be modified is a critical factor for most reactions. Appendix 8 shows the pKa of ionizable groups found in proteins.

2.2.3 Minimal modification The obvious concern when chemically modifying any protein is that the modification introduced will diminish or destroy its biological activity. It is not difficult to do this with many chemical reagents. It must make sense therefore to ensure that the degree of modification is no

greater than is required to achieve the desired labelling. A look at an antibody molecule may be instructive here: let us suppose we wish to make a 1:1 antibody-enzyme conjugate (i.e. one antibody linked to one enzyme). A typical IgG antibody contains about 120 lysine side chains which provide suitable attachment points (see Figure 2.2). Yet, in principle, only one of these needs to be modified to provide a link with an enzyme. It is possible to tune the reaction conditions such that, on average, only one or two lysine residues per molecule are modified. This means that there will be a small number of the most reactive lysine amino groups (but probably greater than one or two) which will be partially modified. Unless the most reactive lysine residue happens to be in the antigen binding site, the antibody should retain its activity.

Figure 2.2 The primary amino groups of immunoglobulin G (human). Composite structure of 2 Fab domains (top) and an Fc domain (bottom), showing the a carbon chain and primary amines (black circles). Diagram kindly provided by A. Slater, based on structures by Deisenhofer (1981) and M. Marquat & R. Huber (structure deposited in Brookhaven protein structure database, code 2IG2, 1989).

Proteins and peptides: chemical background The success of amino-directed conjugation schemes, particularly those that do not lead to extensive modification, shows that the most reactive lysines are rarely involved in antigen binding. Minimal modification (< 3 mol mol -~) of both polyclonal and monoclonal antibodies rarely gives rise to any significant loss of activity. A similar viewpoint can be adopted for other proteins. To a rough approximation, the probability of success is related to the proportion of the protein's surface area that is not required for activity. With an antibody (and many enzymes), this is generally high. At the other extreme, with peptides the probability is much lower (although, even so, there are many successful examples). The key then is minimal modification. For many applications, a single label (a single attachment point) will suffice. Other applications, for example fluorescent labelling, may require a higher degree of modification. Chemical labelling is therefore often an empirical activity; conditions are chosen, the product made and characterized and the desired activity assessed. Depending on the outcome, a repeat preparation with adjusted reaction conditions is undertaken, until an acceptable or optimal product results. With proteins at least, the window of conditions that will give a satisfactory conjugate is generally wide. However, it is usually worthwhile employing whatever tools are available to characterize labelled species and intermediates, i.e. to determine the degree of modification.

2.3 REACTION AT AMINO GROUPS The most convenient and useful functional group for labelling and conjugation is the primary amino group, provided by the e-amino group of lysine and the N-terminus (N-terminal proline provides a secondary amino group but this is useful also). A very large number of reagents have been devised for reaction at protein amino groups and it is usually the case that there is no need to consider any other attachment point for labels or cross-linking agents. Because of the importance of the primary amino group, it is worthwhile considering the relevant chemistry in some depth.

23

2.3.1 Acylation Of the many reactions that may be performed at protein amino groups, the most useful for labelling purposes is acylation, or reactions that may be considered analogous to acylation. Acylation reactions may be described by the following general scheme: -NH2 + X-CO-R -NHCO-R where ( ~

>

+ HX

is the protein, X is

a

leaving group

and R is the function being introduced. The active reagent X - C O - R may be produced in situ by the action of an activating agent, such as a carbodi-imide, on the free carboxylic acid, but much more preferable is the use of stable active esters that may be stored as solid reagents. Of the many investigated, the ester N-hydroxysuccinimide (NHS), together with the more water-soluble sulphonated form (NHSS), have proved to be the most enduring: o

O

O

o

II

II

R-C-O-N

R-C-O-N

O NHS ester

O NHSS ester

Their success is due to their stability as reagents, convenient reaction times due to their reactivity with protein amino groups (typically 0.5-2 h), and relative ease of synthesis. Cyclic anhydrides of dicarboxylic acids have also been used widely (though less often i n cross-linking agent design). For example, succinic anhydride is ring opened by amino groups, effectively 'converting' an amino group to a carboxyl: O

-NH2+O~ 0 -N-C

"

0

II

0 II C-OH

L__/

24

Non-radioactive labelling

2.3.2 Reactions analogous to acylation

the products formed are not stable and readily hydrolyse.

Reactions which may be considered analogous to acylation are:

2.3.3 Extent of reaction (i)

Reaction with sulphonyl halides, usually chlorides, to yield sulphonamides: - N H 2 + R-SO2C1 - N H S O 2 - R + HC1

(ii) Reaction imidates"

with

imidoesters

to

yield

NH~II -NH2 + R-C-O-R' +

NH 2 II - N H C - R + R'OH where R' is usually methyl or ethyl. The link formed retains the originally positive charge on the lysine: this is often important for structural investigations. It is also no bad thing for labelling and conjugation, but neutral (e.g. amide) links have generally not been a problem. (iii) Reaction with isothiocyanates to yield thioureas" - N H 2 + R - SCN

>

-NH-C-NH-R The reactions (i)-(iii) have certain features in common that affect the choice of reaction conditions. These will be discussed below. All the links introduced by the above schemes are stable to all the conditions likely to be encountered in the detection procedures, including elevated temperatures at neutral pH, and are also stable to prolonged storage. The specificity of these amino group-modifying reactions is high. Guanidino groups have a higher pKa, > 12, and are unreactive. Free thiols, if present, and the imidazole ring of histidines may be modified, but

For any labelling or conjugation reaction with proteins, it is essential to be able to control the degree of modification, i.e to ensure that the protein is neither under- nor over-modified. This is at best a semi-empirical activity which, in principle, may be achieved in a number of ways, for example by varying the pH, the reaction time, the temperature or the concentration of the reacting species. Of these, varying the concentration of the reagent is the most usual and is recommended. Two approaches can be employed: the choice of these depends on local needs and, in particular, the quantities of protein available. Firstly, one can carry out a series of reactions (say four or five reactions) with varying reagent concentrations. It is best to vary the concentrations logarithmically, e.g. 3-fold differences between concentrations. After analysis, the desired degree of modification may be selected (interpolating between concentrations if necessary), and the reaction repeated on a bigger scale, if required. Secondly, one can make a 'best guess', and carry out a small-scale trial reaction. The degree of modification obtained can then be used as a guide to the best concentration to use in the fullscale reaction to get the desired degree of modification. The dependence of the degree of modification on reagent concentration varies between proteins, but is always curved to some extent (an example may be found in Figure 4.4). This is because, as we have discussed, amino groups are modified in the order of their reactivity, thus progressively greater and greater concentrations of reagent are required to modify successive lysines as the degree of modification is increased. A rule of thumb that applies to NHS esters at least, is that to increase or decrease the degree of modification 2-fold, the reagent concentration needs to be adjusted up or down by about 2.5-fold (but this does depend on the protein). A 5-fold change may require a 10fold change in reagent concentration (or more).

Proteins and peptides: chemical background 2.3.4 Choice of reaction conditions 2.3.4.1 Choice of buffer Because these reactions require the amino group to be in its neutral, unprotonated form, the rate of reaction is very dependent upon pH. The pKa of the epsilon amino group of lysine is in the range 9.5-10.5. However, lower pH values, ca 7.0-9.5, are generally selected because: (i) a sufficient fraction is unprotonated to provide adequate reaction rates, and (ii) there is a competing hydrolysis reaction which increases markedly with pH. Since this means that reagents have to be used in excess, for economical and other reasons it is generally desirable to keep this excess to a minimum. NHS esters, for example, are frequently used at pH as low as 7.0-7.5, where reaction with amino groups is sufficiently fast and the competing hydrolysis reaction is slow. As a general rule, NHS esters are best reacted in the pH range 7.5-8.0, while isothiocyanates, which are less reactive but more water-stable, are reacted in the range pH 8.5-9.5. Most buffer systems appropriate for the adopted pH are acceptable, providing that they do not contain a reactive amine. Thus phosphate, borate, triethanolamine, bicarbonate and HEPES are all suitable. Tris and glycine contain amino groups, however Tris is relatively slow to react and, though not ideal, may be used if compensating higher concentrations of reagent are used. The strength of the buffer needs to be in excess of the reagent concentration used. In most cases the reagent concentration will not exceed a few millimolar and therefore 10 mM buffer is sufficient; however, 50 mM or 100 mM is normal. Sodium chloride often appears in protocols, usually for historical reasons; it has no chemical role and generally it may be omitted, although with some proteins it can enhance protein solubility. Phosphate-buffered saline (PBS) can be used for NHS reactions provided that the reagent is used at low concentrations (1 mM or less). Buffer powders are convenient for PBS (e.g. Difco haemagglutination buffer powder). A free choice of buffer is possible when the protein is supplied lyophilized. If it is supplied in an unsuitable buffer, there is the option of

25

transferring it to the buffer of choice by dialysis or using gel filtration. This may involve unacceptable losses, and is in any case inconvenient; so it is best if possible simply to add the desired components to the solution. This may simply involve pH adjustment (though this may require some care to avoid extremes of pH): this is best achieved by adding concentrated buffer solutions, rather than addition of inorganic acids or bases which would cause local pH extremes upon addition. It is often preferable first to experiment at the pH meter with larger volumes of the solutions without the protein present, and then scale down pro rata with the protein solution in question. Some antibodies are supplied by manufacturers in unbuffered saline; addition of a 10• concentrate of the buffer of choice is convenient in this case. Mention should also be made of the importance of ensuring that the protein is free from ammonia (or other nucleophiles). Most commonly, this arises when ammonium sulphate precipitates have been inadequately dialysed or gel filtered. Even micromolar quantities of ammonium ions will compete for the reagent and give rise to reduced modification. Bearing in mind that ammonium sulphate precipitation uses molar quantities of this salt, this clearly calls for an efficient de-salting process. Water-miscible solvents, commonly used to help less soluble reagents into solution, frequently have an effect on reaction rates. Generally they suppress the ionization of the amino group, making it more reactive, although this effect is negligible when the solvent is present at just a few per cent. 2.3.4.2 Protein concentration This should be as high as possible, provided that the solution is not noticeably viscous. It may be that the modified protein is less soluble: if modification causes cloudiness or overt precipitation, then it may be sensible to reduce the protein concentration. Overall, 5-10 mg m1-1 is ideal for most modifications, but 1-5 mg m1-1 is also fine. Below 0.5 mg m1-1, concentration of the protein should be considered (Centricon ultrafiltration devices (Amicon) are convenient). Protein losses due to sticking to surfaces can be

26

Non-radioactive labelling

significant, particularly if the protein is diluted further upon purifying the product. However, concentration may also cause losses, so discretion is called for. 2.3.4.3 Reagent concentration

This is the most convenient parameter to adjust to achieve the desired degree of modification. An excess over protein concentration will always be necessary, even if a substitution of 1:1 is desired. The magnitude of the excess will depend on the extent of the modification required, the sensitivity of the reagent to the competing hydrolysis reaction, the protein concentration and many other variables. The protocols in subsequent chapters will give a guide as to suitable concentrations in different situations. As a rule of thumb, a suitable starting concentration for minimal modification of proteins with many NHS esters and other amine-directed reagents is ca 0.5-1.0 mM; this should at least give a measurable modification which can then be optimized.

of the molar ratio, care needs to be exercised in using it as a tool for adopting and changing protocols; secondly, one should seek to work with as high a concentration of protein as is practical and convenient. It also follows that experimental descriptions that state the molar ratio with no mention of any absolute concentration are not adequate. 2.3.4.5 Reaction temperature

Room temperature is normal since (as well as being convenient) it is the usual result of the compromise between achieving an acceptable rate of reaction and not denaturing the protein. In laboratories where the temperature can fluctuate it is helpful, for the sake of consistency, to use an incubator or water bath at 25~ Elevated temperatures (e.g. 37~ have been used for the slower reactions; conversely, where protein stability is an issue, many reactions are quite effective at 4~ Lower temperatures are also appropriate for the more reactive reagents such as the sulphonyl chlorides.

2.3.4.4 Molar ratios

2.3.4.6 Reaction time

The reagent excess required for a protein modification reaction is often expressed in the literature as a molar ratio. This can be a source of difficulties, since absolute as well as relative concentrations are important. The reason for this may be seen by an example. Let us consider a reaction that gives an acceptable degree of modification at 2.5 mg ml-1 protein and a molar ratio (reagent: protein) of 5:1. If the method is subsequently performed with the protein at 0.5 mg ml-1 and the same molar ratio and reaction conditions, the extent of reaction will be much reduced since both reactants are present at 5 times lower concentration and hence the initial rate of reaction will be 25 times slower. This may be compensated for to some extent by increasing the time of reaction, but the competing reagent hydrolysis reaction, which obeys pseudo-first order kinetics, is unaffected by the dilution and hence always dominates at lower protein concentrations. The result is inevitably a poor degree of modification. Two lessons emerge: firstly, though it is useful to be aware

One might expect that the longer the reaction, the greater the extent of reaction. This is in general true. However, with reagents that are hydrolysed, there is little point in prolonging the reaction beyond about two hydrolysis halflives. That aside, and given the other range of variables that are available to control the degree of modification, the main parameter to consider is convenience, i.e. it should be as short as the speed of reaction and the economics of reagent usage will allow. Reaction times suggested in the protocols in this book tend to err on the short side for convenience; however, where the reagent is valuable or in short supply, longer reactions with lower reagent concentrations may be appropriate.

2.4 REACTION AT THIOL GROUPS Relatively few of the proteins that are labelled or used for labelling have free, reactive thiol

Proteins and peptides" chemical background (sulphydryl) groups, the cysteine residues more often existing as disulphides. Nevertheless, given that thiols may be introduced into proteins using a variety of chemical modification (and protein engineering) approaches, thiol chemistry has proved to be very useful for labelling and conjugating proteins, due to the high reactivity of thiols and the high specificity of reagents that have been designed to react with them. Schemes for introducing thiols by means of such reagents will be discussed in Section 3.3.1. However, we can consider the chemistry regardless of the origin of the thiol.

2.4.1 Reaction types

2.4.1.1 Disulphide exchange -SH + R-SS-X -SS-R

>

+ X-SH

In this scheme, the function R is introduced to the protein via a disulphide. The disulphide may be symmetrical (X = R), but more typically, asymmetrical disulphides are employed, where X is chosen to be a good thiol leaving group. This means that fast reaction rates can be achieved without a large excess of reagent. The leaving group most widely used in the design of labelling and cross-linking agents is the 2-pyridyl group; as well as enhancing the reactivity of the disulphide, this group has the added advantage that the released group, which tautomerizes to pyridine 2-thione, may be readily quantified by its absorbance at 343 nm (c = 8080 M-~ cm-~). - S - S ~

H The above reaction scheme is also exploited in the well-known reaction for estimating thiol concentration, employing the symmetrical disulphide, DTNB (5,5'-dithiobis(2-nitrobenzoic acid); Figure 2.3; Ellman, 1959). The liberated anion

~

27

--SH COO" CO0"

S

\

/

NO2

+

S-

O2N

COO"

COO" yellow (412 nm)

Figure 2.3 Thiol determination using DTNB (Ellman's reagent). absorbs at 412 nm (e = 14 150 M- 1 c m - 1). This reagent is of great analytical importance in any conjugation work involving thiols, and its use is described in Appendix 5. The mixed disulphide that results from reaction of protein thiols with Ellman's reagent, or symmetrical disulphides such as 2-pyridyl disulphide, are in themselves thiol-reactive compounds, and may be used preparatively to link to other thiols:

(~-S-S-~~

+ HS-R

9

- S - S- R +S = ~ , ~ I'~I--

H

Although this approach gives a stable protein intermediate, and a conjugation stepthat may be monitored spectrophotometrically, this scheme is not often used for preparing conjugates. Compared with other links described in this book, disulphide links are the easiestto reverse. Though of interest in cross-linking studies, this reversibility is sometimes associated with concerns about the stability of conjugates made by using disulphides. This aspect is discussed further in Section 3.3.2.5.

2.4.1.2 Alkylation with haloacetyl reagents Protein thiols react very readily with haloacetyl reagents to give a thioether: -SH + XCHzCO-R - S--CHzCOR + HX

>

28

Non-radioactive labelling

This reaction is a bimolecular nucleophilic substitution reaction and is fast. The role of the carbonyl group is to activate the methylene carbon to nucleophilic attack, the attacking species being the thiol anion. The reaction rate is fastest with X = I and consequently iodoacetyl reagents are preferred.

2.4.1.3 Alkylation with maleimido reagents Carbonyls also have an activating role in the reaction of the maleimide double bond with protein thiols: O

O

| sH+ N R O

N-FI O

The specificity of these thiol reagents depends to a large degree on the higher reactivity of thiol nucleophiles compared with amino groups. The thiol anion is the reactive species, and has a pKa of ca 9.0. Iodoacetyl and maleimido groups will react, albeit more slowly, with amino groups. However, by limiting the reaction time, the reagent concentration and the pH, a high degree of specificity for thiols can be achieved. Since all the commonly used reagents are quite reactive to thiols, and side reactions with water much less significant, molar ratios close to unity may be employed. Both maleimido and iodoacetyl reagents do hydrolyse in water however, and reagents and protein derivatives cannot be maintained in water for extended periods (days), or for shorter periods at higher pH. The thioether bonds formed by these reactions are very stable, affording excellent stability to labelled proteins prepared by this route.

2.4.2 Choice of reaction conditions

2.4.2.1 Choice of buffer Thiol-containing proteins are prone to oxidation and disulphide formation and the use of de-gassed buffers is recommended. Oxidation is promoted by certain metals, and for that reason EDTA is

often included in the buffer (for example, 2 mu). Use of good quality buffer components is however usually sufficient to avoid these problems, but EDTA is suggested if the thiol-containing protein has to be kept in solution for any length of time. EDTA should not be used with alkaline phosphatase which it will inactivate. The pH may be in the range 6-9; usually 7.5 or 8.0 is used. A wide variety of buffers may be employed: 0.1 M Tris is satisfactory.

2.4.2.2 Protein concentration There are few demands here, and reactions down to 0.1 mg m1-1 are successful. High concentrations (>10 mg ml-1) may encourage undesirable intermolecular disulphide formation before and during the reaction. A concentration of 1-5 mg ml- ~ is ideal.

2.4.2.3 Reagent concentration Generally, because of the high reaction rates possible, equimolar ratios may be used, though at typical protein concentrations it is usual to employ a small excess of reagent (e.g. 5- to 10-fold).

2.4.2.4 Reaction temperature and time As discussed above, thiol groups may be assayed readily using Ellman's reagent (see Appendix 5) and this may be used to monitor the loss of thiols in a reaction. Provided the protein concentration is greater than 1 mg m1-1, most reactions are complete in ca 1 h at room temperature. More labile proteins may be modified overnight at 4~

2.5 REACTION AT CARBOXYLIC ACID GROUPS The carboxyl functions of glutamic and aspartic acid, and of the C-terminus, may be reacted with amines and converted to amides. In this case the protein carboxyl needs first to be activated by use of a water-soluble carbodi-imide, such as EDC (1-ethyl-3-(dimethylaminopropyl) carbodi-imide): C H 3 C H 2 - N = C = N - CH2CH2CH2N (CH3)2

Proteins and peptides: chemical background In principle, protein amino groups can also react, giving rise to cross-linked products. This can be minimized however by using only minimal quantities of carbodi-imide and by using an excess of the amine component. This reaction is carried out typically at pH 4.5-5.0: acetate buffers cannot be used (for obvious reasons) and as a result automatic or manual pH-statting has been employed. MES buffers (0.1 M, pH 4.7) can also be used for this reaction. The reaction yield can be markedly improved by the addition of N-hydroxysuccinimide which forms an active ester in situ (Staros et al., 1986). An analogous reaction occurs with hydrazides, which being more nucleophilic, can deliver greater specificity compared with protein amino groups, allowing the use of lower reagent concentrations. Water-soluble carbodi-imides are not totally specific and modification at tyrosine and (if present) sulphydryl groups may be expected to occur (Carraway & Koshland, 1968; Carraway & Triplett, 1970). The former may be reversed by hydroxylamine treatment whereas the latter is irreversible. From the protein-labelling point of view, such modifications may lead to loss of activity, but likewise, so could the intended carboxyl modification. Again an empirical approach to find a suitable 'window' is called for. Carboxyl modification has had a greater role in the synthesis of hapten carrier conjugates than for labelling proteins; the carrier activity of a protein is hard to destroy. For labelling proteins with labels that have functional amines or hydrazides, this is a useful option, though for glycoproteins, coupling to periodate-oxidized carbohydrates should also be considered (see below). Generally, however, carboxyl modification is not the best way of modifying proteins for labelling purposes and there are usually better alternatives.

29

istry has been developed over many decades that is targeted at other residues. The driving force for this has been the quest (a) to understand the role of those residues in the biological function of the protein, and (b) to understand the structure of protein-containing complexes by means of chemical cross-linking methodologies. While the range of commercially available specialist chemical reagents for these chemistries is more limited, nevertheless this chemistry may be exploited for labelling and conjugation, and may have a role in certain circumstances where amino or thiol chemistry is inappropriate or has failed.

2.6.1 Reaction at tyrosine

The tyrosine phenol may be acylated with active esters, isothiocyanates and other amine-reactive reagents; however, they are unstable, hydrolysing readily under physiological conditions. Reaction at tyrosine is a side reaction that is sometimes observed with lysine-directed reagents. Precautionary treatment of the reaction mix with hydroxylamine (1.5 M, pH 8.0, 1 h) has been suggested (Brinkley, 1992) and should be considered if loss of label is observed on storage. Diazotization of the phenol has been used for labelling purposes, but this reaction is more often used for structural studies.

2.6.2 Reaction at histidine

Acylation of the histidine imidazole is also possible but such adducts hydrolyse spontaneously. Alkylation with iodoacetates is possible, and given the widespread availability of iodoacetate-containing reagents, this is an interesting route for labelling cysteine-free proteins and peptides. Specificity for histidines rather than amino groups may be achieved by maintaining the pH at ca 6.5.

2.6 REACTION AT OTHER AMINO ACID SIDE CHAINS 2.6.3 Reaction at arginine

For reasons given above, we have given amino, thiol and carboxyl chemistry special attention for labelling and conjugation. However, chem-

The high pKa of arginine (12-13) means that high pH (e.g. > pH 10) is required for reaction: this

30

Non-radioactive labelling

makes it unsuitable for general use. Glyoxal and diketones have been used for structural studies, but few reagents have been described which are suitable for general labelling purposes.

ever, there are some differences that should be borne in m i n d .

2.8.1 Solubility

2.7 REACTION AT CARBOHYDRATE GROUPS Many protein molecules of interest bear carbohydrate groups and are useful for labelling purposes (see Figure 2.4). Mild oxidation with periodate yields a bis-aldehyde, which may be reacted with an amine component to give a Schiff's base. This, being rather unstable, is then reduced using sodium cyanoborohydride to give either a secondary or a tertiary amine. The exposure of the protein to both an oxidizing and a reducing environment, with the risks that this may involve, make this a less attractive scheme except for those well-established procedures (e.g. for horseradish peroxidase antibody conjugates) which have been shown to be useful. Hydrazide reagents react similarly, and the hydrazone linkage formed is more stable than the Schiff's base and does not require a further reduction step.

2.8 CHEMICAL MODIFICATION OF PEPTIDES The chemistry appropriate to proteins is, as would be expected, applicable to peptides. HowRO\

~'---;~,' OR periodate OHOH

RO\

"o '.'.|

While most proteins are soluble in most neutral and moderately alkaline buffers, the solubility of peptides and labelled peptides is difficult to predict, and handling peptides which are poorly soluble in water can be problematic. If solubility problems are encountered, the following is suggested. Inspect the sequence to see if the insolubility is likely to be related to hydrophobicity. A predominance of hydrophobic groups would suggest this. A high ionic strength in the buffer is unhelpful in this case and reducing it to the minimum required (1-5 mM) or even the use of water alone may be successful. In addition, the use of water-miscible organic solvents in the buffer is indicated. Dimethyl sulphoxide (DMSO) is a good choice, but N,N-dimethylformamide (DMF), n-butanol, ethanol, dioxan and acetone have also been used for this purpose. These solvents may be used at least up to 50% by volume, though 10% often suffices. (ii) Alternatively, the charges on the peptide may be the cause. As with any chemical, acidic peptides are more soluble in basic buffers, and vice versa. The charge on the peptide at the pH of choice may be estimated with knowledge of the pKa of the

(i)

i~--- O1~OR (~NH2 OO

RO\

)

RO\

o...|

Figure 2.4 Oxidation of vic-diols with periodate and subsequent reaction with primary amines. Both aldehyde functions may in principle react which would give after reduction a tertiary (cyclic) amine.

Proteins and peptides: chemical background

(iii)

(iv)

(v)

(vi)

amino acid side chains (see Appendix 8). If the overall charge is zero, then adjustment of the pH up or down may have a profound effect on solubility. This may take the pH outside the range appropriate for the intended reaction, though occasionally, where the insolubility is kinetic rather than thermodynamic, adjustment of the pH back to the desired pH will not result in precipitation. As alternative approaches to dissolving difficult peptides, sonication or prolonged heating with stirring may be attempted, but in the author's experience these ploys are rarely successful, precisely because insolubility is generally thermodynamic rather than kinetic. Despite indications in the sequence, solubility properties are difficult to predict, and experimentation with small quantities of peptide and different solvents is useful. Occasionally, though the starting peptide is insoluble, the modified product of the reaction is soluble, due to a change in the overall charge on the molecule, or because the introduced group is more hydrophilic. In this case, the reaction may be started with a suspension of the peptide, and solubilization becomes an indicator of the reaction taking place successfully. However, reaction rates may be drastically reduced with this approach. If the reverse of (iv) takes place (namely, a solution reaction gives an insoluble product), this may provide a useful way of purifying product, but clearly one then has to resort to efforts such as described above to solubilize and use the product subsequently. When passing the reaction through a chromatography column, precipitation may occur if the column buffer is different from the reaction buffer. For simple gel filtration columns, there is no strong need for a buffer, and water is suggested as the eluent. For high-performance liquid chromatography (HPLC) purification, common purification schemes (Appendix 12) rarely cause difficulties except for very hydrophobic peptides.

31

2.8.2 Reactivity of amino acid side chains Amino acid functional groups (e.g. amino groups) vary greatly in their reactivity depending on the chemical environment and labelling reactions, select the most reactive. In a peptide, there may only be one or two amino groups and there is no easy way of knowing in advance what their reactivity may be. Thus while proteins are relatively invariant in the conditions required for modification, peptides are much more idiosyncratic, and may require much more forcing conditions. An empirical approach is required.

2.8.3 Reduced range of amino acids to modify The discussion on protein modification above emphasized the utility of amino and thiol groups. If neither of these groups is present in the peptide of interest, then alternative ploys may be considered. The peptide may be synthesized with an extra lysine or cysteine present. To minimize the effect on biological activity, the natural position is at the N- or C-terminus. Because of the attractions of thiol-based labelling chemistry, cysteine is particularly convenient and this approach is commonly adopted in the synthesis of conjugates with carrier proteins for immunization. (ii) Other amino acid side chains may be considered for conjugation, for example carboxyls or histidines.

(i)

2.9 PRACTICAL POINTS

2.9.1 Reagents Most reagents for the chemical modification of biological molecules are water-sensitive to a greater or lesser extent. Manufacturers tend to recommend storage conditions, but if in doubt storage with desiccation at -20~ is suggested. A plastic sealable box containing self-indicating

32

Non-radioactive labelling

silica gel is suitable for this. Ensure that solid reagents are equilibrated to room temperature before opening the bottle in order to avoid condensation forming on the reagent. Many reagents are only poorly soluble in aqueous solutions. Preparation of a stock solution at for example 100 times final in an organic solvent is a common ploy, followed by dilution into the reaction. DMSO and DMF are good for this purpose; dioxan, acetonitrile and methanol are alternatives. DMSO should not be used with sulphonyl chlorides. Very hydrophobic reagents may precipitate out upon dilution, depending on the concentration; in these cases the reaction needs to be carried out in the presence of an organic solvent, for example higher concentrations of DMSO, DMF, acetonitrile or other solvents. Separate experiments to test the ability of the protein to withstand such conditions need to be carried out.

2.9.2 Reactions 2.9.2.1 Reaction vessels Plastic is preferable to glass for most proteins. Polypropylene is preferred over polystyrene since it is less 'sticky' to proteins. However, being opaque it is less easy to see the reaction and check for signs of precipitation or nonhomogeneity. For small volumes (<1 ml), either small polystyrene tubes or Eppendorf tubes may be used. For 1-5 ml, polystyrene bijou bottles are convenient. 2.9.2.2 Adding reagents It is advisable to add all reagents slowly over the course of at least 20 seconds, with stirring; this avoids high local concentration of reagents. A convenient technique is to stir with the pipette tip as the reagent is expelled. 2.9.2.3 Stirring Once a homogeneous solution is obtained, there is no need to stir the reaction. Indeed, in some

cases stirring may even be harmful, promoting denaturation or oxidation. Stirring-induced precipitation has even been observed (Saunders, 1979).

REFERENCES Brinkley, M. (1992). A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2-13. Carraway, K.L. & Koshland, D.E. (1968). Reaction of tyrosine residues in proteins with carbodiimide reagents. Biochim. Biophys. Acta 160, 272-274. Carraway, K.L. & Triplett, R.B. (1970). Reaction of carbodiimides with protein sulphydryl groups. Biochim. Biophys. Acta 200, 564-566. Deisenhofer (1981). Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of Protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20, 2361-2370. Ellman, G.L. (1959). Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 70-77. Geoghegan, K.F., Emery, M.J., Martin, W.H., McColl, A.S. & Daumy, G.O. (1993). Site-directed double fluorescent tagging of human renin and collagenase (MMP-1). Substrate peptides using the periodate oxidation N-terminal serine. An apparently general strategy for provision of energy-transfer substrates for proteases. Bioconjugate Chem. 4, 537-544. Saunders, G.C. (1979). In Immunoassay in the Clinical Laboratory (eds R.M. Nakamura, W.R. Dito & E.S. Tucker III), p. 99. Liss, New York. Staros, J.V., Wright, R.W. & Swingle, D.M. (1986). Enhancement by N-hydroxysuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156, 220-222.

FURTHER READING Glazer, A.N., DeLange, R.J. & Sigman, D.S. (1975). Chemical modification of proteins: selected methods and analytical procedures. Elsevier, Amsterdam. Means, G.E. & Feeney, R.E. (1971). Chemical modification of proteins. Holden-Day, San Francisco.

CHAPTER

3

Conjugation of proteins with enzymes and other proteins

3.1 INTRODUCTION Here we deal with the attachment of a protein label or an anti-tag protein to a protein of choice. Enzymes constitute by far the most important class, but other examples include fluorescent proteins such as the phycobiliproteins, bioluminescent proteins such as aequorin, and anti-tag proteins such as streptavidin. Photoaffinity labelling is not considered here; this subject has been reviewed recently (Kotzyba-Hibert et al., 1995). The protein-protein conjugation field has been driven by the very widespread need to prepare enzyme labelled antibodies, and this area been reviewed as the technology developed (O'Sullivan & Marks, 1981; Ishikawa et al., 1983; Tijssen, 1985). Much of this chapter revolves around enzyme-antibody conjugates and the material is presented largely in these terms. However, the approaches employed

have also been successfully applied to other protein-protein conjugates and the conjugation methods presented here, especially those involving thiol-reactive heterobifunctional agents, can certainly be adapted to suit other proteins. The term conjugation is to be preferred here over cross-linking: the latter term is best reserved for (e.g. structural) studies where a cross-link is introduced in a preformed complex, i.e. where there is already an affinity between the components of the complex. High cross-linking efficiencies can be achieved in these situations, essentially because the protein components do not have to find each other in free solution. Having made that distinction, there is an overlap in the repertoire of reagents used for both conjugation and cross-linking, and all such reagents are generally referred to as cross-linking reagents. When surveying commercially available cross-linking agents, it is important to bear in mind the purpose for which the agent was designed. For example, the large class of

34

Non-radioactive labelling

photo-activatable cross-linking agents are quite unsuited for conjugate preparation. The field of protein-protein conjugation does not suffer from a shortage of possible conjugation schemes, with a large variety of reagents available and an even larger variety of ways of using them. There is a shortage, however, of studies that compare the advantages and disadvantages of the various methods in a systematic way, and where key factors such as the degree of modification of each protein are controlled for. As a result, conjugation is often considered to be a somewhat empirical activity, where luck plays a part. This should not be the case. Let us start by considering what we want to achieve. The ideal conjugate should retain the full biological activity of both the protein components, and should not generate high background signals. Often, more attention is given to maximizing or enhancing of activity than reducing background noise, but it is generally the case, with enzymes at least, that the sensitivity of any application is ultimately limited by non-specific binding rather than the signal. Ideally also the preparation method should be simple and reproducible. As is often the case, there is no method that fulfils all of these requirements, and there is generally a trade-off between conjugate quality and ease of preparation. Choosing between the two is not hard however, when one considers the downstream work that can be involved squeezing acceptable performance out of a suboptimal conjugate. It is well worth investing time and effort in advance to ensure that the conjugate that is prepared is, as far as one can reasonably be certain, of good quality. This underlies the emphasis that will be given in this chapter to three aspects of conjugation: (i) good chemical control, (ii) characterizing intermediates and (iii) the principle of minimal chemical modification. A further parameter that needs to be considered is the composition of the conjugate. It is possible to adjust the degree of substitution of the antibody, or use polyfunctional linker groups to achieve several enzymes per antibody. Where there is a need to extract maximum sensitivity out of a system, this makes sense. However for most immunoassays with good quality antibodies, there is no need to aim for high ratios, and 1:1 conjugates are perfectly acceptable.

They have the added advantages that they are easier to make reproducibly, they are easier to characterize and they tend to give lower assay backgrounds than more polymeric conjugates. For immunohistochemical applications, where tissue penetration is perceived to be an issue, 1:1 conjugates are also to be preferred.

3.2 CONJUGATION SCHEMES Many approaches to conjugation have been devised. We can distinguish two main types, designated by the nature of the coupling reagents used: homobifunctional and heterobifunctional. It is convenient now to discuss homo- and heterobifunctional schemes in general terms, using 'A' to denote the protein to be labelled, and 'S' to denote the signal protein.

3.2.1 Homobifunctional reagents These are reagents of general structure: X - R - X , where X is a protein-reactive group and R is a linker group. The construction of the desired conjugate, 'A-S', is simple: just mix A and S together, and add the cross-linking reagent (Figure 3.1A). X is able to react with functional groups on both proteins, and AS will form. Of course, AA and SS will also form. There is also nothing to prevent the formation of AAS, ASA, SSA, SAS and a very large number of higher oligomers. In short, the outcome is a high degree of heterogeneity associated with a number of disadvantages: (i) the more polymerized species tend to lead to higher non-specific binding in many assay situations; (ii) since AA and SS species, and the like, are unproductive, the method is wasteful; and (iii) the method is difficult to control and it is difficult to guarantee that the same spectrum of species will be formed on a repeat preparation. The classic homobifunctional reagent is glutaraldehyde, which may be used in a one-step method, as described above, or a two-step method, where one species is activated before contacting the second (reviewed in Ishikawa et al., 1983). Both methods give poor yields of

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d o t h e r p r o t e i n s

Q

~ X-R-X

@

that for the above reasons homobifunctional reagents, particularly glutaraldehyde, should no longer be recommended for conjugation purposes. Though they have a long history, the disadvantages of these approaches are too serious to ignore, particularly in the light of much better heterobifunctional reagents that have become well established over the past decade or more. As noted above, these comments do not necessarily apply to the cross-linking of (preformed) complexes.

@

3.2.2 Heterobifunctional reagents

~ X-R-Y

~

35

R-Y

1o B~

Figure 3.1 Protein-protein conjugation schemes based on (A) homobifunctional, heterobifunctional cross-linking agents.

and

(B)

conjugate (Tijssen, 1985) and are frequently associated with reduced activity of one or both of the protein components. High background signals are often a problem. The chemistry is more complicated than simple Schiff's base formation (Hardy et al., 1976) and different batches of glutaraldehyde can give very different conjugation outcomes. Several other symmetrical cross-linking agents are available commercially, for example based on dicarboxylic acids (Abdella et al., 1979). When the reactive group X is hydrolysable by water, for example when X = NHS, then one end of the cross-linking agent may react with protein and the other hydrolyse. Not only is this inefficient, but it increases the possibility that the protein may be inactivated by this unnecessary modification. Many practitioners, including the author, feel

These may be represented by the structure X-R-Y, where X and Y are two different reactive groups, with a degree of specificity for a particular type of amino acid side chain. One protein component (say, S) is reacted with the reagent to give S-R-Y; this is then reacted with protein A to give S - R - A (Figure 3.1B). In this way, species such as A-A and S-S cannot form. In practice, this scheme is an oversimplification since Y has to be specific for a reactive group that is present on A but not on S: generally this need is met by ensuring that A, but not S, has a thiol group, and that Y bears a thiol-reactive group (A and S may of course be interchanged). This provides the principle conjugation scheme presented in this chapter (Section 3.3). Overall, the heterobifunctional approach offers a higher degree of chemical control, particularly since the intermediates may be characterized and the degree of modification kept to a minimum.

3.2.3 Zero-length cross-linking agents This term has been used to described agents that directly join two proteins together without the introduction of any extra atoms (i.e. a spacer group). An example would be the direct activation of carboxyl groups with water-soluble carbodi-imide, followed by reaction with the amino groups of the second protein. Clearly, as with homobifunctional reagents, a large spread of products will result, and with carbodi-imide extensive precipitation of protein may occur. In general, zero-length cross-linking is not

36

N o n - r a d i o a c t i v e labelling

appropriate for conjugation needs. The one notable exception to this is the frequently used scheme for the conjugation of glycoproteins via their carbohydrate groups using periodate oxidation (see Section 3.4). Because only one protein is oxidized, and the carbohydrate acts in effect as a spacer, the general problems of zero-length linkers do not apply.

3.2.4 Gene fusion conjugates The construction of protein-protein conjugates by the expression of fused genes is immediately attractive, offering the possibility of a limitless supply of well-defined conjugate, with a good prospect of full retention of the biological activity of both components. The approach, which has been reviewed (Lindbladh et al., 1993), has been used to prepare conjugates of antigens with alkaline phosphatase and ~-galactosidase and also conjugates of Protein A with several different enzyme labels and bioluminescent proteins. Preparation of antibody-enzyme conO

enzyme}--NH

jugates is more difficult due to the complex chain structure of the immunoglobulin molecule, though antibody-alkaline phosphatase fusion proteins have been reported (Weiss & Orfanoudakis, 1994; Carrier et al., 1995). Fusion conjugates of ligands with alkaline phosphatase have been used in receptor identification studies (Tartaglia et al., 1995). Clearly however, the investment required for a single conjugate is significant, with design, expression, purification and stability issues to address. For that reason, the approach is not well suited for general research purposes, but also, significantly, it has not yet been widely adopted for the commercial production of conjugates.

3.3 HETEROBIFUNCTIONAL APPROACHES BASED ON THIOL CHEMISTRY In this approach, one of the proteins must contain a free thiol. This is not the limitation that it

O O +

NH2 O O

O

NH;

[enzyme]--N

HS" - ~ ~ N

N H

antibody]

O

0 / [enzyme].--N ~ O N

S ~

NH~ N H~r-antibody]

O Figure 3.2 Typical conjugation scheme involving thiolation of one component (antibody), reaction of the second component (an enzyme label) with a heterobifunctional cross-linking agent (SMCC), followed by the conjugation step.

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d o t h e r p r o t e i n s may seem since thiols may readily be either generated by limited disulphide reduction or added by means of a thiolating reagent. Figure 3.2 shows an illustrative scheme where an antibody is thiolated by means of 2-iminothiolane and an enzyme derivatized by means of succimidyl 4-(N-maleimidomethyl)cyclohexane1-carboxylate (SMCC). In this scheme, the thiolating agent is effectively also a cross-linking agent. One may regard the whole operation as putting a 'hook' on one protein and an 'eye' on the other, followed by bringing the two together. Note that cross-linking agents are reacted at their amino-reactive groups first, otherwise reaction of the 'other end' would expose the active ester portions to water and these groups would substantially hydrolyse before being used (they would also react with any protein). The advantages of this approach are as follows. (i)

Chemical control. Formation of homodimers or homomultimers is minimized. The desired reaction of thiol with the thiol-reactive group (e.g. maleimido) is much faster than the side reactions that would lead to homo-species (disulphide formation, reaction of thiol-reactive group with amino groups on the same protein). (ii) Analysis. The extent of reaction of each component may be readily measured. (iii) Efficiency. Every linking group introduced is relatively chemically stable and able to make a productive link with the other protein component. There is little 'nonproductive' modification. The last point should mean that there is no need to introduce more than one 'hook' or 'eye' per protein component. In practice, a degree of modification in the range 1-4 moles linking group per mole protein is required to achieve reasonable yields of conjugate (this may be due to modification in clefts that are 'inaccessible' to the other protein component). However, the propensity for the high, non-productive modification found with many homobifunctional reagents is nevertheless avoided. Although the scheme may appear to be relatively complex, it is not unduly time-consuming to carry out. Those readers unfamiliar with the

37

various steps involved may find it helpful to read the worked example given in Appendix 11. Alternatively, to simplify the procedure, kits for conjugating alkaline phosphatase and HRP, based on lyophilized, pre-activated enzymes (Garman & Parker, 1991) are available commercially (Immunolink TM, Genosys).

3.3.1 Reagents for introducing thiols into proteins Thiols may be generated by disulphide reduction or by reaction with thiolating agents. A scheme describing the principle thiolating options is given in Figure 3.3.

3.3.1.1 Reduction of protein disulphides For most proteins, total reduction of disulphides is not an option, but partial reduction may be considered. Partial reduction of whole antibodies and F(ab')2 fragments has been used extensively for the preparation of enzyme conjugates. Conjugation of enzymes to the thiol of Fab' antibody fragments has the advantage that the site of attachment is known and it is remote from the antigen binding site. Furthermore the conjugate produced is believed to show lower non-specific binding, better penetration for immunohistochemistry and reduced serum interferences in immunoassay. For these and other reasons, Fab' conjugates are preferred to whole antibody conjugates by many workers (e.g. Ishikawa et al., 1983; Rugg et al., 1986). From an economic perspective, it is useful to note that the inevitable losses in preparation are compensated by the fact that two moles of Fab' are produced per mole of antibody. A general method for the generation of Fab' fragments is given in Protocol 3.1 (based on Ishikawa et al. (1983) and Lamoyi (1986)). For other proteins where there is less information on the effect of partial reduction, the use of thiolating agents, as described below, is likely to be more fruitful.

3.3.1.2 2-Iminothiolane (Traut's reagent) This cyclic reagent (Jue et al., 1978) is ringopened by protein amino groups to generate a

38

Non-radioactive labelling

SH I

I

I

r'-

(~NH 2 disulphide reduction

DTT/ l +

! I I

2-iminothiolane ~ N H

2

o

o o

o

o

SPDP

0

0

O-

o

o

o

0 SATA

F i g u r e 3.3 Some options for introducing thiols to a protein. For details, see text.

Protocol 3.1 Preparation of antibody (IgG) Fab' fragments, a 1. Prepare IgG in 0.1 M sodium acetate pH 4.2 at 10 mg ml-~.b 2. Add porcine pepsin (Sigma) to a final concentration of 0.5 mg m l - ' . Pepsin may be added as the solid or as a 2 mg ml-~ stock in the pH 4.2 buffer. Incubate overnight at 37~ c 3. Add 1/40th volume of 2 M Tris-HC1, pH 8.0 and adjust pH to 8.0 with 0.5 M NaOH. 4. Apply to a column of Ultrogel T M AcA-44 (30 times sample volume) equilibrated in 0.1 M sodium borate buffer, pH 8.0. Collect the peak corresponding to the Fab' fragment. 5. Dialyse or de-salt the Fab' into 0.1 M sodium phosphate buffer, pH 6.0 and add 0.1 M 2-mercaptoethylamine in the same buffer to give a final concentration of 15 mM, followed by 0.1 M EDTA to give a final concentration of 1 mM. 6. Incubate at 37~ for 1 h. 7. Desalt the reaction mixture into 0.1 M sodium phosphate pH 7.4 (Appendix 6). Place on ice and use as soon as possible. 8. Analyse for thiol content using Ellman's reagent (Appendix 5). a Whole antibodies may be partially reduced also: start at step 5. b If dialysis is used for buffer exchange, dialyse against 0.1 M sodium acetate (pH 7.0) and then adjust pH to 4.2 with 2 Macetic acid dropwise with thorough stirring. c Different antibody preparations and pepsin preparations may mean that digestion conditions need to be modified. IgG may be digested at lower concentrations. Mouse monoclonal antibodies vary in their susceptibility to pepsin both between and within subclasses (Lamoyi, 1986). In all cases, it is advisable to follow the course of the digestion for example by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) or analytical gel filtration HPLC.

Conjugation

of proteins with enzymes

free thiol, thereby introducing a thiol in a single step. A further advantage compared with amideforming reagents is that the positive charge on the lysine is retained, which in some circumstances may be beneficial with respect to the retention of biological activity. 2-Iminothiolane was believed originally to be in the ring-opened form and was known as methyl 4-mercaptobutyrimidate (Traut et al., 1973). Protocol 3.2 describes the use of 2iminothiolane for a typical protein (for alkaline phosphatase thiolation, see steps 4 - 6 of Protocol 8.3).

3.3.1.3 SPDP and related reagents SPDP was one of the first heterobifunctional reagents to be described (Carlsson et al., 1978). It is a versatile reagent since it can be used either to introduce thiols groups to a protein, or it may be used to provide a thiol-reactive group (see Section 3.3.2.3). A further advantage of this reagent is that the pyridine-2-thione released upon reduction is easily measurable by means of its absorption at 343 nm (e =

Protocol

3.2

and other proteins

39

8080 M-1 cm-1); this may be exploited to determine the degree of thiolation. Variations on the SPDP theme include watersoluble analogues (sulpho-SPDP), long-chain versions (LC-SPDP and sulpho-LC-SPDP) and SMPT. SMPT gives rise to a disulphide with improved stability, at least in vivo (Thorpe et al., 1987).

3.3.1.4 SATA SATA (N-succinimidyl S-acetylthioacetate) reacts with amines via the active ester group to give a protein derivative with thiols protected with the acetyl group (Duncan et al., 1982). Subsequent deprotection is achieved with hydroxylamine. A method for the use of S A T A is given in Protocol 3.3.

3.3.1.5 Other cyclic reagents N-Acetylhomocysteine thiolactone is ringopened by amino groups to generate a thiol and an amide linkage (Benesch & Benesch,

Introduction

of thiols with

2-iminothiolane. 1. Obtain protein at 1-5 mg ml-~ in 50 mM sodium phosphate buffer pH 7.4 (1.0 ml). 2. Prepare a 2 mg ml-~ solution of 2-iminothiolane in the same buffer (use immediately). 3. Add 50 lal of 2-iminothiolane solution to the protein and incubate at 37~ for 30 min. Final concentration of 2-iminothiolane is 0.7 mM.a'b 4. Equilibrate a de-salting column (e.g. Pharmacia PD-10, see Appendix 6) in a suitable buffer, e.g. PBS (preferably de-gassed). 5. Apply reaction mixture to column and isolate the modified protein. Keep on ice and use as soon as possible after analysis, c 6. Take a sample of the product and measure protein concentration (e.g. by absorbance) and thiol concentration (Appendix 5). 7. Calculate the molar concentration of both total protein and thiol groups in the product, and thereby determine the degree of modification (moles thiol per mole protein). a This protocol will give a degree of substitution close to 2 mol mol -~ for most antibodies modified at 1 mg ml -~. Other proteins may differ substantially; for 1 example, a preparation of streptavidin at 1 mg ml- required 3.5 mM final 2iminothiolane concentration. b For conjugation, a degree of modification of 1.5-2.5 mol mol-~ is ideal for a good yield of 1:1 conjugate. When attempting this for the first time or with a new protein a small-scale trial reaction is advised. c The thiolated protein should be used immediately in the next conjugation step since the thiol groups will oxidize. Alternatively use 1 mM EDTA in the column buffer.

40

Non-radioactive labelling

Protocol 3.3 Introduction of thiol groups using SATA. 1. Obtain protein preferably at 5-10 mg m1-1 in 50 mM sodium phosphate buffer, pH 7.5. 2. Prepare a 50 mM solution of SATA in DMSO or DMF. 3. Add SATA to the protein to give a final reagent concentration of 0.5 mM and allow to react at room temperature for 30 min. 4. Isolate product by de-salting on a Sephadex T M G25 (PD-10) column, or equivalent (Appendix 6). The product should be retained in this form until just before it is required, when it should be de-protected. To measure the degree of modification, a portion may be de-protected as follows, and an Ellman's assay performed (Appendix 5). 5. Add 1/10th volume of deacetylation solution: 0.5 u hydroxylamine, 50 mM sodium phosphate, 25 mM EDTA, pH 7.5. Incubate at room temperature for 2 h. 6. The de-acetylated product may be used for conjugation without further purification.

1958). S-Acetylmercaptosuccinic acid anhydride is similar to SATA, and may be de-protected likewise with hydroxylamine (Klotz & Heiney, 1962). Both these reagents are important historically, but are now less preferred because of the redundant pendant functions (N-acetylamino and carboxymethyl, respectively) that are introduced into the linkage.

3.3.1.6 Thiolation at the Fc protein of immunoglobulin Though random modification of immunoglobulin molecules does not usually interfere with antigen binding, a site-specific approach where linkage could be directed to the Fc portion would be useful. This is one advantage of carbohydrate modification, which is exclusively located in the Fc portion (see Section 3.4). Husain & Bieniarz (1994) have reported an interesting hybrid approach where thiols are introduced by reacting periodate-oxidized antibody with cysteamine (H2N(CH2)2SS(CH2)2 NH2), followed by reduction. This is then reacted with alkaline phosphatase derivatized with an (extra long) analogue of SMCC.

3.3.2 Reagents for introducing thiol-reactive groups into proteins Of the chemical functions which are reactive to thiol groups, there are three that have been

widely used for conjugate synthesis, namely maleimido, iodoacetyl and pyridyldithio (see Section 2.4). Most of the reagents used to introduce these groups are amino-reactive NHS esters (or sulpho-NHS analogues). Some of more widely used reagents are shown in Figure 3.4; a larger list has been compiled by Wong (1991).

3.3.2.1 Active ester reagents for introducing maleimido groups Several reagents are available for introducing the maleimido function onto protein amino groups (see Figure 3.4). Of these, SMCC (Yoshitake et al., 1979) and MBS (Kitagawa & Aikawa, 1976) have been widely used. SulphoNHS versions of many of these reagents are also available, including SMCC, MBS and SMPB. The modification of various proteins with SMCC is described in Protocols 3.4, 3.5 and 3.6. Reagents with straight-chain linkers of various lengths have been described (Keller & Rudinger, 1975; Ji, 1979; Kitagawa et al., 1981): the shorter linkers are directed towards cross-linking studies; the longer ones, for example MHS (C5 spacer), are better suited for conjugation needs. Adverse interactions of the cyclohexane group of SMCC with serum components has been observed and some workers prefer to use MHS, available from Boehringer (C. Lockhart, personal communication, 1995).

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d other p r o t e i n s

0 N

0 N

0 O--N 0 0 SMCC

0

0 oN~

0 H ICH2/ ~

0~ N 0

0

SMPB 0

0 O--N 0 0 MHS 0

~ N S"s

y O-N 0

O--N 0 0 MBS

0

O,,~j O_N/~

O-N

0 0

0

SlAB

~ N S'S

41

SPDP

0

o

N

H LC-SPDP

0

~

0

Figure 3.4 Reagents for introducing thiol-reactive groups to a protein. SMCC, succinimidyl 4-(Nmaleimidomethyl)cyclohexane- 1-carboxylate; MBS, M-maleimidobenzoyl-N-hydroxysuccinimide; MHS, maleimidohexanoyl-N-hydroxysuccinimide; SMPB, succinimidyl 4-(p-maleimidophenyl)butyrate; SLAB, Nsuccinimidyl (4-iodoacetyl)aminobenzoate; SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate; LC-SPDP, long-chain version of SPDP.

3.3.2.2 NHS reagents for introducing iodoacetyl groups The NHS and sulpho-NHS reagent SIAB, based on 4-iodoacetylaminobenzoic acid (Figure 3.4), has been used extensively for conjugate synthesis (Weltman et al., 1983).

3.3.2.3 NHS reagents for introducing pyridyldithio groups SPDP is in many ways the archetypal heterobifunctional agent (Carlsson et al., 1978). It is

more versatile than most in that, as we have already seen, it may also be used to introduce thiols onto a protein. Protocol 3.7, based on M~rtensson et al. (1984), describes its use. Activated proteins are also stable for prolonged periods at 4~ or frozen. Pyridyldithio-activated proteins may also be prepared from a thiolcontaining protein by treatment with 2,2'-dipyridyl disulphide, while treatment with Ellman's reagent also gives a mixed disulphide that is thiol reactive (and analogous to pyridyldithio activation). As noted in Section 3.3.1.3, several variants on the SPDP theme are available.

42

Non-radioactive labelling

Protocol 3.4 Introduction of maleimido groups with SMCC. 1. Obtain protein at 1-10 mg ml-1 in 0.1 M triethanolamine HC1, pH 7.4. a 2. Prepare a stock solution of SMCC in dry DMF at 20 mM (6.7 mg ml-1). 3. Add SMCC stock to protein solution to give a final concentration of 0.5 mM. Allow to react at ambient temperature for 30 min. 4. Meanwhile, equilibrate and prime a disposable de-salting column (e.g. Pharmacia PD-10) in a suitable buffer, e.g. PBS. 5. Apply reaction mixture to the column and purify the modified protein (Appendix 6). For PD-10: apply reaction mixture (1 ml), allow to soak onto column, apply 1.7 ml buffer and run to waste, apply 1.6 ml and collect the product. Place on ice. Determine protein concentration by absorbance or other convenient method. 6. Analyse the product as follows: take 100 ktl product and add 20 ktl 0.5 mM 2-mercaptoethanol. As a control, take 100 ktl column buffer and add mercaptoethanol likewise. Seal and incubate at 37~ for 20 min. For each dilute to 1.0 ml (or similar volume) with PBS in a cuvette, place in spectrophotometer and zero at 412 nm. Perform a thiol estimation by addition of 25 ktl 1 mM Ellman's reagent (DTNB), recording the increase in absorbance, b 7. Calculate maleimido content as follows: M=

(OD (control) - OD (product)) X overall dilution factor 14 150

8. Calculate the degree of substitution: M/P (mol maleimido/mol protein). The optimum degree of modification will vary from protein to protein, but generally 2-4 is satisfactory, c If outside the range 1-5, a repeat preparation is advised using an appropriately altered SMCC concentration. Or 50-100 mM sodium phosphate buffer, pH 7.4. b The increase for the product should be lower than for the control, due to consumption of some of the thiols by the maleimido groups. For a reliable estimation, the absorbance increase for the product should be between 20% and 70% of that for the control. If above 70%, repeat with a higher concentration of protein or a lower concentration of 2-mercaptoethanol; if lower than 20%, repeat with a higher concentration of 2-mercaptoethanol. c Though measuring the degree of modification is to be recommended, it may be omitted once a procedure for a particular protein is defined and consistency obtained.

a

Protocol 3.5 Introduction of maleimido groups into alkaline phosphatase using SMCC. 1. To 0.25 ml alkaline phosphatase (calf intestinal, 10 mg ml-~ immunoassay grade, Boehringer), add 0.75 ml 0.1 M triethanolamine, pH 7.4, containing 1 mM MgCI2 and 1 mM ZnSO4. 2. Add 37 ~1 of 6.7 mg ml-1 (20 mM) SMCC (Pierce) in dry DMF. React for 30 min at room temperature, a 3. Recover product and analyse as described in Protocol 3.4. a These conditions should give a degree of modification of ca 3.5 mol mo1-1.

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d o t h e r p r o t e i n s

43

Protocol 3.6 Introduction of maleimido groups into HRP peroxidase using SMCC. 1. Dissolve 5 mg HRP (Biozyme) in 0.1 M triethanolamine, pH 7.4 (0.9 ml). 2. Add 100 gl of 16.7 mg m1-1 (50 mM) SMCC (Pierce) in dry DMF. React for 30 min at room temperature, a 3. Recover product and analyse as described in Protocol 3.4. a

These conditions should give a degree of modification of ca 2.0 mol mol-1

3.3.2.4 Thiols as thiol-reactive groups

Formally, thiols themselves should be included in this list, since gentle air oxidation of two thiol-containing proteins will give rise to disulphide-linked conjugates. However, this approach gives symmetrical products, is slow and gives poor yields. It is not a good approach to general conjugation needs.

3.3.2.5 Factors to consider in choosing a thiol-reactive group

For most purposes, any of the reagents in Figure 3.4 are perfectly adequate for conjugation purposes, and the advantages and disadvantages are relatively minor. The following points may be worth considering. Stability of the conjugate. Of the bonds that are formed when reacted with thiol groups, the alkyl thioether formed by iodoacetyl compounds is the most stable, while the disulphide formed by the SPDP group of reagents is the most labile. For demanding situations and long-term storage, iodoacetyl may therefore be best. For example, conjugates made with SIAB have been used in automated immunoassay systems where longterm storage is critical, and improved stability compared with maleimido links has been claimed (Rugg et al., 1986). However, many other workers have found the thioether link produced by maleimido groups to have adequate storage stability for immunoconjugates (also see Ishikawa et al., 1983). Specificity. The iodoacetyl group, though fast, tends to be less specific for thiols. Pyridyldisul-

phide is the most specific. In general, however, the specificity for thiols is high for all these groups and is not an issue for conjugations. Characterization. Assessing the degree of modification is easy for the pyridyldithio group, while maleimido and iodoacetyl have to be determined by back-titration with mercaptoethanol which is slightly less convenient. Light sensitivity. Iodoacetyl compounds are labile to light and frequently contaminated with elemental iodine (they are yellow rather than pure white) which may damage the protein. Reactions should be performed in the dark. Spacer groups. Heterobifunctional schemes such as shown in Figure 3.2 give a long overall spacing between the proteins, and the difference between a C2 and a C6 spacer is not significant. In general, spacers of length equivalent to C6 are preferred; if greater, they should contain hydrophilic functions, rather than (CH2)n. NHS or sulphonyl NHS. Avoidance of DMSO or other solvents is an attractive reason for using water-soluble reagents, particularly where the protein's resistance to such solvents is unknown. Most proteins used for labelling are resistant to low concentrations of DMSO and DMF however. The ability to add solid reagent to a reaction is also attractive, though in some cases the amount added is so small that the preparation of a stock solution at higher concentration is not avoided. The ability to use an aqueous stock is useful, though it has to be used immediately.

44

Non-radioactive

labelling

Protocol 3.7 Introduction of pyridyldithio groups to

IgG with SPDP. 1. 2. 3. 4. 5.

Prepare antibody at 5.0 mg ml-1 in 0.1 M sodium phosphate, pH 7.1. Prepare a 3.2 mM (1 mg ml-1) solution of SPDP in ethanol. Add SPDP to the antibody to give a final concentration of 180 laM. React for 60 min at 25~ De-salt the reaction by passage through a disposable PD-10 column, or equivalent, equilibrated in 0.2 M sodium phosphate, pH 7.1 containing 2 mM MgC12. 6. A portion may be analysed as follows. Dilute by 1/10 or 1/20 in 0.2 M sodium phosphate pH 7.1 and measure OD at 280 nm. Zero the cuvette at 343 nm and add 50 mM dithiothreitol to give a final concentration of 1 mM. Record the increase in OD. Calculate the pyridine-2-thione concentration using the extinction 8080 M-1 cm-l: this gives the concentration of pyridyldithio groups. The antibody concentration may be estimated from the OD at 280 nm using an extinction of 1.4 for 1 mg m1-1 solution (210 000 M-1 cm-1). However, since part of the OD at 280 nm is due to the pyridyldithio group, it is necessary to apply a correction. Using the pyridyldithio concentration determined above, calculate the OD 280 nm contribution using an extinction of 5100 M-1 cm-1. Deduct this figure from the observed OD 280 nm to obtain the OD 280 nm contribution of the protein and thereby derive the true protein concentration. Calculate the degree of substitution. Preparative reduction (if desired) may be carried out as follows. Transfer the modified protein into 0.1 Msodium acetate, 0.1 MNaC1, pH 4.5, by dialysis or gel filtration. Add dithiothreitol to a final concentration of 25 mM and incubate for 45 min at room temperature. De-salt into the buffer of choice (see also Protocol 3.2). The lower pH gives improved specificity of the desired reaction over protein disulphide reduction. (Carlsson et al., 1978).

Note:

3.3.3 Characterization of intermediates The principle of minimal modification implies that the active intermediates should be characterized (at least when a protein is derivatized for the first time). Characterization is quite straightforward and gives the experimenter some confidence that the activated components are worth proceeding with. It is less important for repeat batches, provided that nothing has changed. Often a new batch of reagent, a different protein preparation or a different operator introduces undetected variables that significantly alter the outcome and this is a further reason why an element of characterization can be valuable. It is important to note that (with the exception of pyridyldithio groups) thiol-reactive groups, and thiols in particular, are not stable for long periods, and characterization and use should be completed without delay. The thiol-containing component may easily be quantified using Ellman's reagent (Appendix 5), and given that thiolated proteins are prone to

oxidation, the thiol content should be checked before conjugation. Thiol-reactive groups may be measured by back-titration by reacting with a small excess of mercaptoethanol, followed by determination of the unreacted mercaptoethanol with Ellman's reagent (see e.g. Protocol 3.4). This procedure has adequate sensitivity for most conjugation needs, though a more sensitive procedure based on papain activation has been described (Singh, 1994). Pyridyldithio groups may be measured by measuring the pyridine-2thione upon reduction with dithiothreitol (Protocol 3.7). For thiol-based linkage chemistry, a degree of modification greater than 5 mol mol -~ may be considered excessive. However, 1 mol mol -~ is likely to give low yields of conjugate. In the absence of any other information, 2-3 mol mol -~ is a suitable window to aim for, but this should be 'fine-tuned' if there is evidence (for example, from gel filtration profiles) of under- or over-conjugation. See comments on optimizing the degree of modification in Section 2.3.3.

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d o t h e r p r o t e i n s

3.3.4 The protein-protein conjugation step It is advisable to prepare the thiol-reactive component first, since this will be more stable than the thiolated protein. The thiolated component should be conjugated immediately since there is a risk of thiol oxidation. Having appropriately derivatized the two proteins, the conjugation step per se is straightforward, requiring simply that the two components are mixed together and incubated (Protocol 3.8). The following is intended to help the choice of reaction conditions.

3.3.4.1 Molar ratio

To obtain a 1:1 conjugate, the components may be mixed in equimolar quantities. However, it is often beneficial to have one component in a small excess, say 2- to 4-fold, over the other (normally one would arrange that the less valuable component was in an excess over the more valuable component): this can drive the reaction further to completion and ensure that all of the more valuable component is conjugated. For the construction of enzyme-antibody conjugates, an excess (2- to 4-fold) of enzyme ensures good yields of 1:1 conjugate, while the unconjugated enzyme is easily removed upon purification. 3.3.4.2 Time and temperature

This is highly dependent on the protein concentration and the reactivity of the thiol-reactive group. Maleimido couplings are typically car-

Protocol 3 . 8 C o n j u g a t i o n

45

ried out at 4~ for 16 h providing that the overall protein concentration is greater than 1 mg ml-1. One to two hours at room temperature is however usually sufficient, especially for reactions with iodoacetyl reagents. Disulphide formation with pyridyldithio reagents may be followed in the spectrophotometer, monitoring pyridine-2thione release at 343 nm.

3.3.4.3 Buffer

This is determined by the buffers that have been used in the preparation of the intermediates. There are no major demands on the buffer provided that the pH is in the range 7-8. Tris or phosphate are suitable. Metals should be avoided (Mg 2+ and Zn 2+, commonly used with calf intestinal alkaline phosphatase, are unnecessary at this stage). 3.3.4.4 Blocking reactive groups

It might be thought that a conjugate that has unreacted groups (a thiol, or a thiol-reactive group) may give rise to storage instability or higher non-specific binding. In practice, provided that the conjugation reaction is sufficiently prolonged, e.g. overnight, there are unlikely to be any reactive groups remaining and, in general, it is not necessary to add blocking reagents. If problems are encountered, then blocking with mercaptoethanol and N-ethyl maleimide should be considered. These should be added just prior to conjugate purification. A suitable scheme is as follows.

of thiolated and maleimido

proteins. 1. Calculate the molar quantities of each protein that are available. 2. Assuming both components are in appropriate buffers (see text), mix the components, preferably with the maleimido component in a 2- to 4fold excess over the thiolated component. For coupling maleimido enzymes to thiolated antibody, an enzyme:antibody ratio of 2-4 should be used. 3. Cap vial and incubate overnight at 4~ (or 2-4 h at room temperature), a a Conjugation times will depend on protein concentrations: the above is suitable for proteins in the range 1-2 mg ml-1

46

Non-radioactive labelling

1. Add mercaptoethanol to a concentration 5 times that of the maleimido component in the conjugation reaction. Allow to react for 15 min. at room temperature. 2. Add N-ethyl maleimide in a small excess (50%) to the mercaptoethanol. Allow to react for 15 min at room temperature. Purify by gel filtration immediately.

3.3.5 Conjugate purification Many practitioners use the conjugate without any purification, but this is not to be recommended. Simple passage down a gel filtration column not only permits a useful degree of purification (which may lead to better signalto-noise ratios), it also affords an opportunity to inspect the product mix (since there will certainly be more than one species present). This will hopefully give evidence that some conjugate has been produced, but also it characterizes the conjugate in the sense that the elution profiles of any subsequent batches may be usefully compared to assess the extent of con-

jugation. Given the molecular weight changes that conjugation produces, gel filtration is the natural purification method for protein conjugates. See Protocol 3.9 for a general method. Any gel filtration method fractionating over an appropriate molecular weight range can be used but HPLC or FPLC methods are preferred: these offer a useful degree of resolution with run times of an hour or less. As an example, the purification of an alkaline phosphatase-antibody conjugate on Superose 6HR T M is given in Figure 3.5. As discussed above (Section 3.3.4.1), use of an excess of one component over the other should ensure that all of the less abundant protein should be conjugated. In this way, for antibody-enzyme conjugates, one can ensure that the free antibody content of the conjugation mix is low. Other procedures may give more unconjugated antibody, and to obtain conjugates of maximum activity further purification will be required. Affinity techniques are suggested, for example the use of Con-A Sepharose T M for the purification of HRP conjugates (Tijssen, 1985). Removal of unconjugated enzyme will not be necessary in most cases since gel filtration gives

Protocol 3 . 9 C o n j u g a t e p u r i f i c a t i o n b y F P L C . 1. Equilibrate a Superose 6 HRT M column (24 ml, FPLC, Pharmacia) with the conjugate storage buffer at 0.5 ml min-i. PBS (Difco haemagglutination buffer is convenient) containing 0.1% sodium azide is recommended. It is useful to have the column calibrated with molecular weight markers and with the proteins that are to be conjugated. Set UV detector at 280 nm and set the fraction collector to collect 0.5 ml fractions. 2. Apply the sample (volume should not exceed 5% of the column volume, i.e. ca 1.2 ml. Larger sample volumes may be concentrated before use, for example, using a disposable Centricon filter device (Amicon). Collect 0.5 ml fractions. 3. Inspect the elution profile. Identify the 1:1 peak and select fractions to pool. a,b " Addition of BSA (preferably heat-treated) to 0.1% improves storage stability. b Variations: Use of Superdex-200 (Pharmacia) will give better resolution since this matrix fractionates over a narrower range. The upper molecular weight cut-off is ca 500 kDa which does not allow analysis of the higher molecular weight components which will elute as a single peak. Conjugates of other proteins may require the use of other gel filtration matrices, depending on their molecular weight. Fractions corresponding to 2:1, 1:2 and 2:2 conjugates are generally usable and may be included in the pool. Highly polymerized species should be excluded.

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d o t h e r p r o t e i n s 1'8 F

47

AP

1.6

1.4

1.2

1.0 o oo oJ

a 0

0.8 1.2 0.6 0.8 0.4

o

173 O 0.4

0.2

01

0

I

4

,,,,-l-V'

8

1

12

1

16

20

Volume (ml)

Figure 3.5 Purification of an alkaline phosphatase sheep anti-mouse polyclonal antibody conjugate, prepared according to the scheme shown in Figure 3.2. Dotted line shows a conjugate activity profile determined on a coated plate ELISA. AP designates unconjugated alkaline phosphatase. Solid bar shows the recommended conjugate pool, containing predominately 1:1 conjugates, with some higher molecular weight species. an adequate purification, and furthermore, even quite large amounts of unconjugated enzyme (if not over-modified) do not usually interfere with immunoassay procedures. However, where necessary, an affinity chromatography approach based on an immobilized ligand that binds the protein to be conjugated may prove useful. For antibodies, the use of nickel chelate affinity columns, which binds antibody but not HRP or alkaline phosphatase, may be considered (FreeZyme TM, Pierce). The use of this approach may be considered as an alternative to gel filtration, rather than as an addition. However, as discussed above, the ability to inspect the range of conjugate species and pool accordingly is an attractive advantage of gel filtration.

3.3.6 Conjugate storage Conjugates of both alkaline phosphatase and HRP are stable at 4~ for many months, up to a year or more. For alkaline phosphatase conjugates, phosphate appears to aid stability. A suitable buffer is 0.05 M sodium phosphate, pH 7.4 containing 0.1% bovine serum albumin (BSA; heat-treated) and 0.1% sodium azide. Freezing can cause alkaline phosphatase to lose much of its activity; if storage at - 2 0 ~ is desired, use of phosphate buffers and 20% ethylene glycol is successful. Azide inactivates HRP, and other anti-bacterial agents (e.g. 0.02% thiomersal) must be used, or alternatively the conjugate should be stored sterile. Preferably,

48

Non-radioactive labelling

HRP conjugates are stored frozen, though stability of concentrated solutions at 4~ is also good. For room temperature storage, polyethylene glycol has been recommended as a stabilizing agent (Boyd & Yamazaki, 1993). Flash freezing in liquid nitrogen is a better option for more unstable proteins than slow freezing. Repeated freeze-thaw cycles should be avoided with all proteins.

3.4 CONJUGATION VIA CARBOHYDRATE GROUPS

3.4.1 Principles Attachment of protein labels at the carbohydrate portion of glycoproteins has been widely used in the preparation of HRP conjugates (Nakane &

Protocol

3.10

Kawaoi, 1974), but may be used for most glycoproteins where the carbohydrate group is not required for activity. The chemistry is introduced in Section 2.7 and Figure 2.4. Vicinal diols are oxidized by periodate to give a dialdehyde which is then reacted with amino groups on a second protein to give a Schiff's base-linked conjugate. This linkage is not itself stable, but is then reduced to give either a secondary or a tertiary amine, which is very stable. As well as HRP, this chemistry has been used for attaching labels to immunoglobulins. Since the carbohydrates on IgG are located on the Fc portion, one can be confident that antigen binding is unaffected (see also Section 3.3.1.6). Polymerization can, in principle, result from this conjugation scheme since protein amino groups can react with aldehydes on the same protein. Self-condensation of HRP is however minimized by the naturally lower amino group content of this enzyme, and this is a further

Preparation of HRP-antibody conjugates by the periodate method.

1. Prepare a solution of HRP (5-20 mg ml-l) in sodium acetate buffer, pH 5.0. 2. To this add 1/10th volume of 0.1 Msolution of sodium periodate (fresh) in water. Allow to react for 30 min at room temperature in the dark. 3. Quench the unreacted periodate by the addition of 1/10th volume of ethylene glycol, a 4. Isolate the activated enzyme by passage through a disposable gel filtration column (see Appendix 6) or by dialysis. 5. Prepare the antibody (or other protein to be labelled) at 2.5 mg ml-1 in PBS. 6. To this solution add sufficient HRP to give a 10-fold excess (by weight) over the antibody (the concentration of HRP may be estimated by the dilution factors and complete recovery from the column assumed). 7. Incubate at room temperature for 2 h. 8. Towards the end of the incubation period, prepare a 300 mg m1-1 solution of sodium cyanoborohydride in water (caution: toxic, cyanide released on exposure to acids). Use immediately. 9. Add 2.5 gl of the sodium cyanoborohydride solution per ml of reaction. Incubate for a further 2 h. 10. Isolate the conjugate by passage through a disposable gel filtration column ~Appendix 6) or by dialysis. Store the conjugate aliquotted at -20 ~ a Certain grades of ethylene gycol contain reactive impurities: use grades for this purpose (e.g. from Pierce Chemical Co.) b Azide inactivates HRP: do not use this as a preservative. Use 0.02% thiomersal.

C o n j u g a t i o n of p r o t e i n s w i t h e n z y m e s a n d o t h e r p r o t e i n s reason for this scheme working well with HRP. Overall, periodate oxidation is a relatively simple procedure, but requires some care to get consistent and optimal results. It is useful as a quick way to get a conjugate for less demanding uses. For high quality conjugates, heterobifunctional approaches are recommended (Section 3.3), even for HRP.

3.4.2 Methodology The method has undergone some evolution with time and there are several variations on the common theme (Tijssen & Kurstak, 1984; Tijssen, 1985; Madersbacher et al., 1992). It is now accepted that blocking of HRP amino groups, a feature of the original method, is unnecessary. Protocol 3.10 gives a basic method. The extent of conjugation may be modulated by the periodate concentration, the periodate treatment time and the coupling pH (amongst other factors). A high ratio of HRP to antibody helps to prevent formation of oligomers. As is generally the case for protein-protein conjugates, 1:1 conjugates give the best results. Increasing the ratio of enzyme to antibody in the product may give an increase in sensitivity, but usually at the expense of higher backgrounds, especially in immunohistochemical applications. Sodium cyanoborohydride is the preferred reducing agent since it is milder and more specific (note this reagent is toxic and will generate cyanide on exposure to acids). Pre-activated HRP is available from Pierce. Often conjugates prepared by this route are used without purification but this is not ideal. Optionally, with HRP IgG conjugates, free enzyme may be removed by precipitating the conjugate with ammonium sulphate at 70-80% saturation, while free IgG may be removed using Con-A Sepharose TM, which has an affinity for HRP, provided it is not over-oxidized (Tijssen, 1985). However, passage of the reaction mixture down a gel filtration column (e.g. Protocol 3.9) not only purifies the conjugate, it gives an impression of the extent of conjugation. Storage conditions for HRP conjugates are given in Section 3.3.6 above.

49

REFERENCES Abdella, P.M., Smith, P.K. & Royer, G.P. (1979). A new cleavable reagent for cross-linking and reversible immobilization of proteins. Biochem. Biophys. Res. Comm. 87, 734-742. Benesch, R. & Benesch, R.E. (1958). Thiolation of proteins. Proc. Natl. Acad. Sci. 44, 848. Boyd, S. & Yamazaki, H. (1993). Stabilization of peroxidase-antibody conjugate in solution by polyethylene oxide. Biotechnol. Techniques 7, 671-676. Carrier, A., Ducancel, F., Settiawan, N.B., Cattolico, L., Maill6re, B., L6onetti, M., Drevet, P., M6nez, A. & Boulain, J-C. (1995). Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human IgGs: application to anti-HBsAg detection. J. Immunol. Meth. 181, 177-186. Carlsson, J., Drevin, H. & Axen, R. (1978). Protein thiolation and reversible protein-protein conjugation. Biochem. J. 173, 723-727. Duncan, R.J.S., Weston, P.D. & Wrigglesworth, R. (1983). A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132, 68-73. Garman, A.J. & Parker, J.R. (1991). Pre-activated proteins for labelling oligonucleotide probes. European Patent Application 431882. Hardy, P.M., Nicholls, A.C. & Rydon, H.N. (1976). The nature of the cross-linking of proteins by glutaraldehyde. Part I. Interaction of glutaraldehyde with the amino-groups of 6-aminohexanoic acid and of o~-N-acetyl lysine. J. Chem. Soc. Perkin. Trans. 1 9, 958-962. Husain, M. & Bieniarz, C. (1994). Fc site-specific labeling of immunoglobulins with calf intestinal alkaline phosphatase. Bioconjugate Chem. 5, 482-490. Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y. & Ueno, T. (1983). Enzymelabeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J. lmmunoassay 4, 209-327. Ji, T.H. (1979). The application of chemical crosslinking for studies on cell membranes and the identification of surface reporters. Biochim. Biophys. Acta 559, 39. Jue, R., Lambert, J.M., Pierce, L.R. & Traut, R.R. (1978). Addition of sulphydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate). Biochemistry 17, 5399-5405. Keller, O. & Rudinger, J. (1975). Preparation and some properties of maleimido acids and malcoyl derivatives of peptides. Helv. Chim. Acta 58, 531. Kitagawa, T. & Aikawa, T. (1976). Enzyme coupled

50

Non-radioactive labelling

immunoassay of insulin using a novel coupling agent. J. Biochem. 79, 233-236. Kitagawa, T., Shimozono, T., Aikawa, T., Yoshida, T. & Nishimura, H. (1981). Preparation and characterization of hetero-bifunctional cross-linking agents for protein modifications. Chem. Pharm. Bull. 29, 1130-1135. Klotz, I.M. & Heiney, R.E. (1962). Introduction of sulphydryl groups into proteins using acetylmercaptosuccinic anhydride. Arch. Biochem. Biophys. 96, 605. Kotzyba-Hibert, F., Kapfer, I. and Goeldner, M. (1995). Recent trends in photo-affinity labeling. Angew. Chem. Int. Ed. 34, 1296-1312. Lamoyi, E. (1986). Preparation of F(ab')2 fragments from mouse IgG of various subclasses. Methods Enzymol. 121, 652-663. Lindbladh, C., Mosbach, K. & Btilow, L. (1993). Use of genetically prepared enzyme conjugates in enzyme immunoassay. Trends Biochem. Sci. 18, 279-283. Madersbacher, S., Wolf, H., Gerth, R. & Berger, P. (1992). Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies. J. Immunol. Meth. 152, 9-13. M~rtensson, A.M., Gustafsson, J.-G. & Ljunggren, H.D. (1984). A modified, highly sensitive enzyme-linked immunosorbent assay (ELISA) for Rhizobium meliloti strain identification. J. Gen. Microbiol. 130, 247-253. Nakane, P.K. & Kawaoi, A. (1974). Peroxidaselabelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22, 1084-1091. O' Sullivan, M.J. & Marks, V. (1981). Methods for the preparation of enzyme-antibody conjugates for use in enzyme immunoassay. Meth. Enzymol. 73, 147-166. Rugg, J.A., Rigl, C.T., Leung, K., Lamar, S.L., Welsh, M., LeBlanc, M. & Evans, S.A. (1986). Radial partition immunoassay applied to automated quantification of human choriogonadotrophin with use of two monoclonal antibodies. Clin. Chem. 32, 1844-1848. Singh, R. (1994). A sensitive assay for maleimide groups. Bioconjugate Chem. 5, 348-351. Tartaglia, L.A. et al. (1995). Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263-1271.

Thorpe, P.E., Wallace, P.M., Knowles, P.P., Relf, M.G., Brown, A.N.F., Watson, G.J., Knyba, R.E., Wawrzynczak, E.J. & Blakey, D.C. (1987). New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Res. 47, 5924-5931. Tijssen, P. (1985). Practice and theory of enzyme immunoassays. Elsevier, Amsterdam. Tijssen, P. & Kurstak, E. (1984). Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays. Anal. Biochem. 136, 451-457. Traut, R.R., Bollen, A., Sun, T-T., Hershey, J.W.B., Sundberg, J. & Pierce, L.R. (1973). Methyl 4mercaptobutyrimidate as a cleavable cross-linking agent and its application to the Escherichia coli 30S ribosome. Biochemistry 12, 3266-3273. Weiss, E. & Orfanoudakis, G. (1994). Application of an alkaline phosphatase fusion protein system suitable for efficient screening and production of Fab-enzyme conjugates in E. coli. J. Biotechnol. 33, 43-53. Weltman, J.K., Johnson, S.A., Langevin, J. & Riester, E.F. (1983). N-succinimidyl-(4-iodoacetyl)aminobenzoate: a new heterobifunctional crosslinker. Biotechniques 1, 148-152. Wong, S.S. (1991). Chemistry of protein conjugation and cross-linking. CRC Press. Yoshitake, S., Yamada, Y., Ishikawa, E. and Masseyeff, R. (1979). Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)maleimide. Eur. J. Biochem. 101, 395-399.

FURTHER READING Hermanson, G.T. (1996). Bioconjugate techniques, Academic Press, San Diego. Tijssen, P. (1985) Practice and theory of enzyme immunoassays. Elsevier, Amsterdam. Wong, S.S. (1991 ). Chemistry of protein conjugation and cross-linking. CRC Press.

CHAPTER

FOUR

Fluorescent labelling of proteins and peptides

4.1 INTRODUCTION The list of fluorophores that are commercially available as protein-reactive reagents is now enormous, with a choice of chemistries available for the most popular. As a result, the labelling of proteins is usually a very straightforward matter, and the protocols can be summarized as: m i x r e a c t - separate. Those who have not fluorescently labelled proteins before are often surprised at how easy it is. One needs to consider the following questions: (i) which fluorophore?; (ii) which chemistry?; (iii) what degree of substitution?; and (iv) how should the product be characterized?

4.1.1 Choice of fluorophore Though the choice of fluorophore will depend on the application (immunoassay, immunohisto-

chemistry, protein-protein interaction studies, fluorescence-activated cell sorting, and many others), there are certain features that are almost universally desirable. (i)

Sensitivity. The brightness of a fluorophore is proportional to the product of the extinction coefficient and the quantum yield. However, when comparing fluorophores, other factors need to be taken into account, particularly instrumentation. One needs to be aware of the wavelength dependence of the power of the light source, the sensitivity of the detector and the transmittance of the optics. For example, many fluorescence plate readers are relatively insensitive towards UV (< 360 nm). Also, fluorophores are often selected because they can be excited by particular lasers, e.g. the argon ion laser (488 nm). (ii) Stability. The label should have both good chemical stability and good photochemical

52

N o n - r a d i o a c t i v e labelling

stability. The latter is particularly important where prolonged illumination of the sample is required, e.g. in histochemical applications. (iii) Physicochemical properties. The label should not change significantly the physicochemical properties of the protein to be modified. This means that the label should be reasonably water soluble and not make the protein 'sticky'. Introduction of charges, however, is often inevitable and usually tolerated. (iv) Stokes' shift. This should be as large as possible to distinguish the label from noise and minimize interference from scattering. (v) Wavelength. Biological components usually have a level of background fluorescence (autofluorescence), as do plastics and other materials. For higher sensitivity applications in particular, the fluorophore should be selected to be distinguishable from the prevalent noise. In general, the higher the wavelength, the better; ideally > 600 nm.

4.1.1.1 Organic fluorophores Thoughts about fluorescent labels usually start with fluorescein, a very popular choice for many years and in many ways the gold standard. Fluorescein is an excellent fluorophore: bright, stable, with good water solubility and low nonspecific binding. Its excitation is well matched to the argon ion laser (488 nm). But it is not perfect: it is not fluorescent at acid pH, its fluorescence declining rapidly below pH 7. Sensitivity is limited by its short Stokes' shift and because there are biological interferences in this wavelength range. The short Stokes' shift also gives rise to inner filter quenching (see Section 4.1.3) and its fluorescence is decreased somewhat when attached to proteins. Photobleaching can be a problem at high light intensities (e.g. immunohistochemical applications). Nevertheless, these drawbacks are not an issue in many applications and the availability of a wide range of fluorescein reagents, together with the suitability of much of the fluorescence instrumentation available, makes fluorescein a good first choice. The availability of anti-fluorescein anti-

bodies and conjugates may also be an advantage in some situations (e.g. where the option of signal amplification is desirable). Useful derivatives of fluorescein include erythrocin (a tetraiodo-derivative), eosin (a tetrabromo-derivative) and dimethoxyfluorescein (Khanna & Ullman, 1980). These have reduced fluorescence but the latter two in particular have been used as acceptors in energy transfer assays using fluorescein as donor. BODIPY FL is a newer fluorophore with similar excitation and emission to fluorescein but with some advantages: it is brighter, the emission peak is sharper and it is insensitive to pH. Significant quenching is seen with protein labelling however, and it is currently best employed labelling oligonucleotides or small molecules (Haugland, 1992). Longer wavelength BODIPY derivatives are also available (Haugland, 1992). Structurally related to the fluoresceins is the rhodamine family. These are less bright than fluorescein but, operating at longer wavelengths, often avoid background noise sources better. Several rhodamines are available (see Table 4.1); of these tetramethylrhodamine is the most widely used. Rhodamines have a reputation, often justified, of being 'sticky' and care is needed in developing applications. Texas Red T M is a more hydrophilic, longer wavelength rhodamine with broad emission above 600 nm, and is widely used in flow cytometry and other immunochemical applications. More recently, the quest for better longer wavelength fluorophores has led to the development of the cyanine (Cy) dye family (Southwick et al., 1990; Mujumdar et al., 1993). These are very bright dyes (Wessendorf & Brelje, 1992), compensating for their modest quantum yields (0.1-0.3) with high extinction coefficients (> 150 000). The Cy dyes are distinguished by a suffix where the first digit refers to the number of carbons in the central connecting chain (though usage is not always consistent). The second digit is an arbitary listing. The length of the central conjugated chain is related to the wavelength maxima; thus, the Cy3 dyes emit in the mid 500 nm range, the Cy5 in the mid 600 nm range and the Cy7 in the mid 700 nm range. Although the Stokes' shifts are small (resulting in self-quenching at higher substitu-

F l u o r e s c e n t l a b e l l i n g of p r o t e i n s a n d p e p t i d e s

53

Table 4.1 Fluorescence properties of some fluorophores Name

Excitation max. (nm)

Emmission max. (nm)

Extinction coefficient a

354 495 525 540 556 535 595 430 374-403 550 581 649 675 743

411 520 550 570 576 604 615 540 422-430 570 596 670 694 767

17 000 66 000 101 000 100 000 93 000 80 000 85 000 13 000 19 000-30 000 150 000 150 000 250 000 250 000 ca 250 000

AMCA Fluorescein Eosin Tetramethyl rhodamine Lissamine TM rhodamine B X-rhodamine Texas RedTM Lucifer Yellow TM Cascade Blue TM dyes Cy3 Cy3.5 Cy5 Cy5.5 Cy7 a

Of unreacted reagent (M- 1 cm- ~).

tions) and the absorption and emission peaks are broad, this dye family shows great promise and is the focus of much current interest. Cy5 is well suited for use with the Storm TM imager (Molecular Dynamics; Figure 4.1). A family of these dyes are commercially available as NHS esters (e.g. from Amersham). The original labelling reagents were bis-Nhydroxysuccinimide esters. These have the potential to cross-link and although the yields of cross-linked products would not be expected to be high at normal labelling concentrations, the mono NHS ester reagents eliminate this possibility and are also available. Certain phthalocyanine derivatives have useful long-wavelength fluorescence and some have been exploited, e.g. as labels for immunoassay (Kelly et al., 1991). Pyrene labels are too hydrophobic for general labelling purposes, but sulphonated derivatives, known as Cascade Blue TM, have been developed as hydrophilic labels emitting in the 422-430 nm range (Whitaker et al., 1991). Lucifer Yellow TM is another sulphonated fluorophore which has been used in immunochemical applications: it has a long Stokes' shift of ca 110 nm. Various coumarin derivatives are available as labelling reagents (Haugland, 1992). Since these require UV excitation they are more prone to interferences and hence have been used less often as labels. They do however have large

Figure 4.1 Western blot with Cy5. Tubulin detected with mouse anti-tubulin and Cy5-1abelled goat antimouse secondary antibody. Lanes were loaded with (right to left) 400, 200, 100, 50, 25, 12.5, 6.25, 3.1 and 1.6 ng purified tubulin, blotted onto PVDF. The 6.25 ng band was detectable. Image produced on a StormTM imager (Molecular Dynamics) and kindly provided by S. Pickett. Stokes' shifts and the blue light emission complements other colours in multiple label studies. A selection of amine-reactive fluorophore reagents is given in Figure 4.2, and key fluorescence data are provided in Table 4.1.

4.1.1.2 Protein fluorophores: the p h y c o b i l i p r o t e i n s

The phycobiliproteins are a broad class of fluorescent proteins from various species of algae, where they have a Iight harvesting function (for a review, see Glazer, 1994). Each protein molecule contains a number of open chain tetra-

54

Non-radioactive labelling

o~o.

o~o.

COOH

I 651s~ O

~~0

COOH

I NCS

0

fluorescein-5-isothiocyanate

5-carboxyfluoresceinNHS ester

I+

I

~

COOH O 65

~~-

,,

SO3H

SO2Cl

0

TexasRedTM

5-carboxytetramethylrhodamineNHS ester

F

F

HO3s~

O O

SO3H

l

Oo

o

"l.ro_~~ o

Cy5 monoNHS ester

BODIPYTM493/503NHS ester

~

O

o

~

NH2

N-O

O

O

Me

AMCA, NHS ester

Figure 4.2 Some useful amino-reactive fluorophore reagents.

pyrrole prosthetic groups, termed bilins, which are the fluorophores. The high quantum efficiency of these fluorophores and the packing of several (up to 34) of these in a single protein (without self-quenching) make these very bright

labels indeed. They have been used extensively in flow cytometry and fluorescence-activated cell sorting (FACS), but have also been exploited in immunohistochemistry and immunoassay (Kronick, 1988).

55

F l u o r e s c e n t l a b e l l i n g of p r o t e i n s a n d p e p t i d e s Table 4.2 Properties of phycobiliproteins Name

Molecular weight

Subunit structure

No. of fluors

B-phycoerythrin

250 000

(0~[~)6~

34

545

575

R-phycoerythrin

250 000

(~)6]t

34

567

578

Allophycocyanin

110 000

(0~) 3

6

650

660

Some properties of three phycobiliproteins of importance as labels are given in Table 4.2. The phycobiliproteins have short Stokes' shifts but broad excitation spectra, allowing efficient excitation at shorter wavelengths very distinct from the emission maxima. Thus, for example, phycoerythrin may be activated by the 488 nm laser line, emitting at 570 nm. Cy5 has been conjugated to R-phycoerythrin to give an intramolecular energy transfer conjugate whereby the energy of the excited phycobiliprotein is transferred efficiently to the Cy5 dye giving rise to Cy5 emission at 670 nm. R-phycoerythrin and its Cy5 conjugate are particularly useful for multi-label flow cytometry since, with fluorescein, they form a trio of labels, each with distinct emissions, but all excitable at 488 nm. Conjugation of phycobiliproteins is not as easy as labelling with conventional fluorophores, but does not present any particular problems providing that the proteins are not extensively modified. Methods such as those described in Chapter 3 may be used and pyridyldisulphideactivated phycobiliproteins are available (Molecular Probes). Allophycocyanine dissociates at high dilution but stable cross-linked preparations are available. Various conjugates with secondary antibodies and streptavidin are also available. Phycobiliproteins and their conjugates should not be frozen.

4.1.1.3 Time-resolved fluorophores: the lanthanide chelates

Time-resolved fluorescence was introduced in Section 1.5. Labelling via protein amino groups with europium and samarium DELFIA reagents is straightforward and discussed below (Section 4.3).

Excitation max. Emission max. (nm) (nm)

4.1.2 Choice of linkage chemistry Usually the choice of chemistry will be determined by the reagents that are available. The labelling chemistry is dominated by aminedirected reagents (succinimidyl esters, isothiocyanates, sulphonyl halides and dichlorotriazine derivatives) and thiol-directed reagents (iodoacetyl and maleimido derivatives). The relevant chemical background is given in Chapter 2. Thiol chemistry is relatively less important in this context, since the proteins used for locating and measuring biomolecules do not generally have free thiols and may be labelled adequately via amino groups. Note that iodoacetyl and maleimido reagents may be used for amine modification also, but a higher pH (>9.0) and longer reaction times are required. Isothiocyanates have been the most popular chemistry for many years, with fluorescein isothiocyanate (FITC) being the first port of call for many experimenters. Increasingly, the benefits of succinimidyl esters are being realized (faster reactions, reagents often more stable), and given that many are now available commercially, this chemistry is recommended, at least for the more water-soluble fluors. Sulphonyl halides are used primarily for work where a bond stable to acid hydrolysis is required (e.g. sequencing), and are not frequently used for general-purpose labelling. Triazine chemistry has also been used, for example the dichlorotriazine derivative of fluorescein (DTAF) may be used for labelling proteins. DTAF is often preferred for fluorescence polarization work.

4.1.3 Degree of substitution Determining the best degree of substitution involves the usual empirical compromise

Non-radioactive labelling

56

between maximizing the signal and minimizing the loss of biological activity, but with fluorescence there is the added complication of selfquenching. This phenomenon, also known as the 'inner filter effect', arises when two or more fluorophores are sited close together such that the second molecule absorbs energy from the first, reducing the intensity of the emission; it applies primarily to fluorophores with short Stokes' shift. Fluorescein is particularly prone to this phenomenon which means essentially that, as more fluorophores are added, there comes a point where the fluorescence actually declines. With large proteins such as antibodies, this may occur at a fairly high degree of modification (see Figure 4.3); with smaller molecules, it may occur at very low degrees of modification. For many applications, a low degree of modification of 1-3 mol mol-1 is perfectly adequate and it is advisable to aim for this window, unless higher sensitivity is shown to be necessary. Because of their size, antibodies can tolerate a higher degree of (molar) substitution (typically 6 - 1 0 m o l mol-1). Alternatively, a series of labelled molecules can be prepared and the best selected empirically. Measuring the degree of substitution accurately is not always easy (see below), but it is valuable to at least obtain an absorbance spectrum. To achieve the optimum degree of modification, it can be valuable to prepare a series by

varying the reagent concentration (see e.g. Figure 4.4). Alternatively, it may be convenient to prepare a single conjugate, and iterate towards the best degree of modification.

4 . 1 . 4 C h a r a c t e r i z a t i o n o f the p r o d u c t

Characterization can mean: (i) making a measurement which can act as a benchmark for future preparations; (ii) determining the degree of modification; or, ultimately, (iii) determining the sites of modification. Most workers will want to do (i), and (ii) is also advisable generally; (iii) is not appropriate.

4.1.4.1 Absorbance spectrum The simplest and most useful method to obtain the degree of modification is from the absorbance spectrum. This is only an approximation, since extinction coefficients for protein-linked fluorescent dyes are not generally available and in any case these will depend to a degree on the local chemical environment. But by using published extinction coefficients for the free dye, or determining it oneself, an estimate of the degree of substitution can be readily obtained. substitution (mole/mole)

5 1 O0

_

4 ~ 9(/1 c

_

3 -

r m u t,-.

-

80

_

60

2-

u In o

1 -

40

_

.> 20, ..,.,,

0

/

~

0.0

o

// ,

I

0.5

'

I

1.0

'

I

1.5

'

I

2.0

'

I

'

2.5

I

3.0

'

3.5

n,"

0

0.0

w

I,

!

l

i 5.0

,

,

,

,

i 10.0

,

,

,

v

i 15.0

Fluorophores/Protein (mol'mol)

,

,

r

,

i 20.0

Figure 4.3 Fluorescence yield of an antibody labelled with increasing amounts of fluorescein, showing selfquenching at high degrees of substitution. Reproduced with permission from Haugland (1993).

cone. fluoreseein-NHS

(mM)

Figure 4.4 Labelling of ras protein with fluorescein

NHS (5-carboxyfluorescein, N-hydroxysuccinimide ester). Dependence upon reagent concentration. Modification was carried out as described in Protocol 4.1.

F l u o r e s c e n t l a b e l l i n g of p r o t e i n s a n d p e p t i d e s

57

Protocol 4.1 P r o t e i n l a b e l l i n g w i t h f l u o r e s c e i n isothiocyanate

(FITC).

1. Prepare the protein at 1-5 mg ml-~ in 0.1 M NaHCO3/Na2CO3 buffer, pH 8.5. 2. Prepare a solution of FITC at 50 mM in DMSO. a 3. Add FITC to the protein to give a final concentration of 2 mM.b Mix and allow to react overnight at room temperature in the dark. 4. De-salt on a PD-10, or equivalent (see Appendix 6), into the buffer of choice, collecting the first coloured band to emerge. 5. Characterize as follows: record spectrum at 1:20 dilution, blanked against the column buffer. Calculate fluorescein concentration (F), using E495 of 68 000 M - 1 c m - 1 . Determine protein (P) by a standard method (e.g. Coomassie or BCA methods) allowing for carrier BSA if present, or from the spectrum (see Section 4.1.4.1; less accurate if carrier protein is present) or assume a column recovery factor (60-70% is reasonable). Calculate F/P. a Alternatively, the FITC stock may also be prepared in reaction buffer (but should be used immediately). b For an antibody, this concentration will give ca 6-10 mol mo1-1

Typically, dye absorbance peaks will be quite distinct from the protein absorption and hence the dye concentration may be obtained directly using the extinction coefficient. Most dyes will however absorb around 280 nm, and hence the protein concentration cannot be determined directly. However, from the spectrum of the free dye, one can determine the absorbance ratio, R, of OD (280 nm):OD (at dye absorbance maximum). This factor is then multiplied by the OD28o of the conjugate to obtain the absorbance contribution of the dye. The remainder may be assumed to be due to protein and thus the protein concentration may be derived. In a few cases, it is possible that the protein spectrum will overlap with that of the main dye peak. In this case, simultaneous equations may be set up and solved: PElp + DEld = OD~ PE2p + DE2p = OD2 (where P and D are the protein and dye concentrations, respectively, Elp and E2p are the extinctions of the protein at wavelengths 1 and 2, respectively, and I~ld and 1~2d are the extinctions of the dye at wavelengths 1 and 2, respectively; 1 cm pathlengths are assumed).

With these estimations, it is important, where possible, to use the absorbance spectrum of the free dye, not the dye reagent, since the spectrum in the UV may be substantially different. It is also useful to check that the protein concentration estimate is reasonable, i.e. consistent with the expected recovery from the de-salting step. Another possible cause for error is the presence of non-covalently linked dye, whether due to inadequate separation methodology, non-specific binding or the presence of dye aggregates. Such problems are more likely with dialysis than with de-salting columns, hence the latter is recommended. If in doubt, control reactions and control separations can be of value.

4.1.4.2 Fluorescence characterization

Because of the possibility of fluorescence quenching from various sources, perhaps the most obvious method, measuring the fluorescence, cannot directly give the degree of modification, but it can be of value to check that the labelling is successful. Fluorescence units are relative, and hence are only meaningful when compared with a calibration curve of the free dye.

58

Non-radioactive labelling / (A )

:::~l (B)

11.61

120|

11.5.~ f

36802

11. ....131

i "1~

.... 1 .... 1

10.11t 10.7"l

.... 1

lO.e-I lO.S~

~IU..

37936

*"%.. "Wt, _%..

t

........J.

lo.wI 1o.5t

10.44

lO.4t

10.3~ 10.2"I .

.... ]

....t

11.of~~ 10.9.1

11..1',,11,t

.

. . . . . . 14000 16000 18000

.

. . 22000

.

. . . . . . . . 26000 30000 34000 38000 42000 Mm4(1900 (m/z~

10"31,

Figure 4.5 MALDI mass spectra of a protein: (A) unlabelled and (B) labelled with tetramethyl rhodamine. The observed increase in molecular weight corresponds to 1.7 mol of attached dye. Spectra kindly provided by R. Davies.

4.1.4.3 Mass spectrometry Mass spectrometry is becoming a more widely used technique in protein chemistry and can be useful for determining the degree of substitution directly. The two techniques most used for protein work are matrix-assisted laser desorption/ ionization (MALDI) and electrospray (ES); these have been reviewed recently (Loo, 1995; Mann & Wilm, 1995). An example of the use of MALDI to characterize a rhodamine-labelled protein is given in Figure 4.5. This technique may not be totally quantitative in giving the ratios of various labelled species, but it is reassuring to see peaks with the correct mass in the spectrum.

4.1.4.4 Reactive group analysis Modification at protein thiol groups can be followed by measuring the loss of the thiol group using Ellman's reaction (Appendix 5). In principle, loss of amino groups may also be measured with, for example, TNBS (trinitrobenzene sulphonic acid; Fields, 1972); however this method would not be expected to be very accurate at low degrees of substitution.

4.2 LABELLING PROTEINS WITH ORGANIC FLUOROPHORES

4.2.1 Amine-directed reagents For an introduction to the relevant amine chemistry, see Section 2.3. For choice, NHS ester reagents should be used: these may be reacted at pH 7.0-8.0, using reaction times of 0.5-2 h. Protocol 4.2 describes the labelling of a protein with fluorescein NHS ester, which may be used as a model. The degree of modification may most conveniently be varied by varying the concentration of reagent: an example is shown in Figure 4.4. Isothiocyanates are a more traditional way of labelling with fluorophores (Protocol 4.1). These are less reactive than NHS esters, requiring either a higher pH, more prolonged reaction, or both. Typically reactions are carried out in the pH range 8.5-9.5 with overnight incubation. Some proteins are sensitive to pH > 9.0, and pH 8.5 is generally satisfactory.

4.2.2 Thiol-directed reagents Maleimido and iodoacetyl reagents may be used to modify thiol groups. This is a good route for

Fluorescent

labelling of proteins and peptides

59

Protocol 4.2 Protein labelling with fluorescein NHS ester. This procedure illustrates minimal labelling (1-2 mol mol-I) of a relatively dilute protein (1 mg ml-1). 1. Prepare protein in 50 mM sodium phosphate, pH 7.5 at ca 1 mg ml(1.0 ml). a 2. Prepare a fresh 20 mM solution of 5- (and 6-) carboxyfluorescein, succinimidyl ester b (Molecular Probes) in DMSO. 3. Add 20 B1 of the reagent to the protein, stirring with the pipette tip during addition. Final reagent concentration is 0.4 mM. Allow to react at room temperature for 1 h. 4. Equilibrate a Sephadex T M G25 column (Pharmacia PD-10; see Appendix 6) with PBS containing 1 mg ml-~ BSA C (heat-treated for 30 min at 56~ 5. Stop reaction by addition of 1/10th volume of 0.1 M ethanolamine, pH 8.5 (0.1 ml) and allow to react for a further 10 min. d 6. Apply the reaction to a PD-10 column, followed by 1.8 ml buffer. Then add further 1.6 ml and collect the coloured product band. 7. Characterize as follows: record spectrum at 1:20 dilution, blanked against the column buffer. Calculate fluorescein concentration (F), using E495 of 68 000 M-1 cm-!. Determine protein (P) by a standard method (e.g. Coomassie, or BCA) allowing for carrier BSA if present, or from the spectrum (see Section 4.1.4.1; less accurate if carrier protein is present) or assume a column recovery factor. Calculate D/P. 8. Store at 4~ with 0.05% NaN3 (away from light) or at -20~ Higher protein concentrations may be used. b Or pure isomers. c BSA is optional but helps improve protein recovery. d Optional if BSA not used (prevents reaction with BSA on column). a

labelling Fab' fragments at the bridge thiols, giving a small conjugate with retention of antigen binding assured (see Section 3.3.1.1). These reagents may be used at stoichiometric (or nearstoichiometric) amounts at high concentrations or at ca 10-fold excess over protein at lower concentrations (1 mg ml-1). Neutral or slightly basic (pH 7-8) buffers may be used, e.g. phosphate or Tris. The reaction may be monitored by measuring the loss of thiol groups using Ellm a n ' s reagent (Appendix 5) and a reaction time of 1 h should suffice in most cases.

4.2.3 Practical points Most fluorophores are light sensitive to varying extents. They should not be exposed to sunlight or other bright light sources and, while they can

be handled on the bench under normal laboratory lighting, it is good practice to protect the sample from light during long incubations or storage. Separation of labelled proteins from reaction mixtures by dialysis can be problematic with some fluorophores and in all cases gel filtration is recommended (it is also much quicker). Disposable columns are ideal (see Appendix 6). Many fluorophore reagents purchased from commercial sources are not pure. For watersensitive reagents, some hydrolysis products may be expected, but there are frequently other species present. In general, these do not affect labelling seriously but do confuse HPLC analysis of reaction mixtures. A simple method for determining the active ester content of NHS esters is described in Appendix 7. With amine-directed reagents, some workers

60

Non-radioactive

labelling

have reported loss of label from the protein upon storage, this being attributable to reversal of linkages to tyrosine. If this occurs (or as a precaution), treat the reaction mixture before desalting with 1.5 M hydroxylamine, pH 8.0 to give a final concentration of 0.1 M, and incubate for 1 h at room temperature (Brinkley, 1992). With fluorescence work especially, dust and particulates are likely to compromise results. Use of filtered water or buffers is advisable and particulates should be removed if they appear. Storage conditions will depend more on the protein than on the fluorophore, but storage in aliquots at - 2 0 ~ is suggested. In histochemical applications, freeing and thawing of fluorescently labelled antibodies has been reported to give increased backgrounds, and storage at - 2 0 ~ in 50% glycerol has been recommended (Wessendorf & Brelje, 1992).

Protocol 4.3 Europium

4.3 LABELLING WITH E U R O P I U M CHELATES The labelling reagent used for the DELFIA T M (dissociative) time-resolved fluorescence labelling is based on DTTA (isothiocyanatobenzyl)diethylenetriamine-N 1,N2,N3,N3-tetraacetic acid; Figure 4.6); a method for a typical protein labelling is given in Protocol 4.3. Europium chelates have mostly been used for antibody labelling (Madersbacher et al., 1993), but also for genetic

/~ /~ ~NCS r~Nh Nh N~

C00" C00- C00-

Eu3§

Figure 4.6 The europium chelate-labelling reagent, DTTA-Eu 3+ N'-(p-isothiocyanato)benzyl derivative)

chelate labelling of a protein.

1. Obtain the protein at 2-5 mg ml-~ in 50 mM NaHCO3, 0.9% NaC1, pH 8.5. 2. Add DTTA reagent to give a final concentration of 1 mM. The reagent may be added from a concentrated stock in water (but may not be stored in water) or added as a solid. 3. Incubate at room temperature for 16 h. 4. Equilibrate a Sephadex T M G25 desalting column (see Appendix 6) with 50 mM Tris-HC1, pH 7.5 (optionally containing 0.05% NAN3) as running buffer. Apply reaction mixture. 5. Collect the first eluting peak. a 6. Estimate the europium chelate concentration as follows. Take a portion of the product and dilute it by a factor of 1000. Count 10 ~1 of this with 100 ~tl of enhancement solution (Wallac) alongside a Eu standard curve. Calculate the Eu concentration from the standard curve. 8. Estimate protein concentration from the absorbance at 280 nm (or other method). If absorbance is used, correction needs to be made for the absorption of the label which has a molar extinction of 8 0 0 0 M - 1 cm -l. 9. Calculate the degree of substitution (europium chelate concentration/ protein concentration), b' c

Note: phosphate-containing buffers should be avoided. Optionally, portions of each fraction may be counted to obtain a europium elution profile. b With antibodies, this protocol should give in the order of 10 mol Eu mo1-1 protein. c The degree of modification can be adjusted by varying the reagent concentration.

a

COO-

F l u o r e s c e n t l a b e l l i n g of p r o t e i n s a n d p e p t i d e s conjugates such as Protein G (Markela et al., 1993) and streptavidin (Dahl6n, 1987).

4.4 LABELLING OF PEPTIDES

4.4.1 General comments As with peptide tagging, it may be more convenient to label the peptide during solid phase synthesis. However, where the peptide is difficult to synthesize, or where a number of labelling options are to be explored, it may be appropriate to modify the peptide in (aqueous) solution. Section 2.8 describes some chemical aspects that are peculiar to peptide modification. Compared with proteins, higher molar concentrations are often possible with peptides, resulting in faster, more efficient reactions. In addition, HPLC characterization is possible and useful. Separation of reaction products from excess reagents requires some thought, however. Use of HPLC (see Appendix 12) may be appropriate but frequently the lack of homogeneity of labelling reagents, and the presence of hydrolysis products, give rise to a forest of peaks which may be hard to interpret. Gel filtration on Sephadex T M G10 is therefore recommended: this should remove all low molecular weight material and, provided the coupling reaction goes to completion, give pure product. Subsequently, HPLC may be used to characterize and, if necessary, further purify the product.

4.4.2 Energy transfer substrates FRET (fluorescence resonance energy transfer), or internally quenched, substrates have been used for the assay of proteases for which simple peptide chromogenic or fluorogenic substrates cannot be derived (Carmel et al., 1973). Here a donor fluorophore and an acceptor (quencher) molecule are positioned either side of the cleavage site of a peptide substrate. Uncleaved, the donor fluorescence is low due to intramolecular

61

quenching; but, when cleaved, the full fluorescence of the donor is revealed. Most synthesis schemes involve labelling during solid phase peptide synthesis (see e.g. Maggiora, 1992), but it is possible to adopt a chemical modification approach (Garman, 1994; Geohegan, 1996) and this serves well to exemplify peptide labelling approaches. Figure 4.7 shows a scheme starting from a peptide containing a single amino group on one side of the bond cleaved by the protease, and a single thiol group on the other side. Any peptide that does not contain a lysine may be used directly, requiring a free N-terminus and the 'mutation' of a suitable amino acid to cysteine (or addition of cysteine to the Cterminus). If lysine is present, this may be used for attachment or changed to arginine (if acceptable). The approach allows various combinations of donor and acceptor molecules to be attached using amino- and thiol-directed reagents. This H2N O

LSH

O H2N

Ls 0

0 N-FI 0

0 F~"N

H

'~'

LS

O N-F1 0

Figure 4.7 Scheme for preparation of an energy transfer protease substrate by double labelling of a peptide. The small arrow indicates the protease cleavage point.

62 Protocol

Non-radioactive 4.4

labelling

Preparation

of a fluorescein/

tetramethylrhodamine

FRET

protease

substrate. The starting point is a peptide with a free N-terminus, no other amines and a cysteine (see Figure 4.7). 1. Dissolve the peptide in 0.1 M NaH2POa/Na2HPO4 buffer, pH 7.5, to a concentration of 2 mMa. (Allow for non-peptidic material in calculating the concentration.) If the peptide does not dissolve, reduce the ionic strength of the buffer and/or the peptide concentration. Water may be used provided the resulting pH is in the range 6-8. Alternatively, co-solvents such as DMSO may be added (see also Section 2.8.1). 2. Prepare a 10 mM solution of fluorescein-5-maleimide (Molecular Probes) in DMSO. Add one molar equivalent b to the peptide and allow to react for 10 minc. 3. Measure the thiol content using Ellman's method (Appendix 5): take 10 ~1 of the reaction, dilute to 1.0 ml in PBS (or above phosphate buffer) in a cuvette and zero the spectrophotometer. Add 20 ~tl of 2 mM DTNB and record the OD at 412 nm. This should be zero (check also unreacted peptide). 4. Prepare a 50 mM solution of tetramethylrhodamine NHS ester d (Molecular Probes) in DMSO. Add to the fluoresceinylated peptide to a final concentration of 10 mM and react for 30 min at room temperature. 5. Prepare a Sephadex T M G 10 column of volume greater than 10 times the reaction volume in a disposable column (Biorad, or barrel of PD-10 or NAP column with upper frit repositioned). Equilibrate in 50 mM Tris-HC1, pH 7.5, or running buffer of choice e. 6. Apply the reaction mixture to the column. Add buffer to elute and collect the first eluting coloured band. 7. Run spectrum to confirm that peaks corresponding to both fluorescein (495 nm) and tetramethylrhodamine (520 nm) are present. Analyse by reverse-phase HPLC (Appendix 12) and characterize by mass spectrometry. " Peptides with free thiols are prone to dimerization. Handle in degassed buffers and react the thiol as soon as possible after dissolution. It is advisable to check the free thiol content (see Appendix 5) before reaction. b Because of uncertainties in the peptide and fluorescein-5-maleimide concentration, it is advisable to react, say, 0.75 equivalents of reagent, measure the thiol content and then add smaller reagent aliquots until the thiol is all reacted. c The maleimido-thiol reaction is fast at these concentrations, but will require longer incubations at lower concentrations. a Use of pure 5- or 6-isomer of the tetramethylrhodamine will give a purer product and aid HPLC characterization. e If insolubility problems have been encountered, there is a possibility of the product precipitating on the column. Experiment to find a suitable buffer: water may be a good choice.

has the a d v a n t a g e that several candidate substrates m a y be p r e p a r e d with relative ease and the m o s t active selected. F l u o r e s c e i n is a suitable d o n o r and this m a y be used with

t e t r a m e t h y l r h o d a m i n e , d i m e t h o x y f l u o r e s c e i n or eosin as acceptor. The size of these fluorophores is not a p r o b l e m p r o v i d e d that the a t t a c h m e n t sites are selected with care (too close and activity

F l u o r e s c e n t l a b e l l i n g of p r o t e i n s a n d p e p t i d e s may be affected, too far away and the degree of quenching will be low). A 10-20 amino acid spacing is a suitable window to aim for, but use should be made of whatever structurefunction data may be available. Protocol 4.4 describes the synthesis of a substrate using fluorescein as donor, attached via the cysteine thiol, and tetramethylrhodamine as acceptor, attached at the N-terminus. Donor and acceptor may easily be reversed in position using the appropriate reagents. It is desirable to react the thiol first: this may be achieved with equimolar quantities, allowing progression to the amino coupling step without prior purification.

REFERENCES Brinkley, M. (1992). A brief survey of methods for preparing protein conjugates with dyes, haptens and cross-linking agents. Bioconjugate Chem. 3, 2-13. Carmel, A., Zur, M., Yaron, A. & Katchalski, E. (1973). Use of substrates with fluorescent donor and acceptor chromophores for the kinetic assay of hydrolases. FEBS Lett. 30, 11-14. Dahl6n P. (1987) Detection of biotinylated DNA probes by using Eu-labeled streptavidin and time resolved fluorometry. Anal. Biochem. 164, 78-83. Fields, R. (1972). The rapid determination of amino groups with TNBS. Meth. Enzymol. 25, 464-468. Garman, A.J. (1994). Fluorescence energy transfer substrates. Patent application WO 94/28166. Geoghegan, K.F. (1996). Improved method for converting an unmodified peptide to an energy-transfer substrate for a proteinase. Bioconjugate Chem. 7, 385-391. Glazer, A.N. (1994). Phycobiliproteins- a family of valuable, widely used fluorophores. J. Appl. Phycol. 6, 105-112. Haugland, R.P. (1992). Handbook of fluorescent probes and research chemicals. Molecular Probes, Eugene, OR. Haugland, R.P (1993). Environmental effects of bioconjugation. In Methods in nonradioactive detection (ed. G.C. Howard), pp. 62-68. Appleton and Lange. Kelly, T.A., Hunter C.A., Schindele, D.C. & Pepich, B.V. (1991). Aluminium phthalocyanine-streptavidin: new, sensitive fluorescent tracer for immunoassay. Clin. Chem. 37, 1283-1286. Khanna, P.L. & Ullman, E.F. (1980) 4',5'-dimethoxy6-carboxyfluorescein: a novel dipole-dipole coupled fluorescence energy transfer acceptor

63

useful for fluorescence immunoassays, Anal. Biochem. 108, 156-161. Kronick, M.N. (1988). Phycobiliproteins as labels in immunoassay. In Non-isotopic immunoassay (ed. T.T. Ngo), Plenum Press, New York. Loo, J.A. (1995). Bioanalytical mass spectrometry: many flavors to choose. Bioconjugate Chem. 6, 644-665. Madersbacher, S., Shu-Chen, T., Schwarz, S., Dirnhofer, S., Wick, G. & Berger, P. (1993). Timeresolved immunofluorometry and other frequently used immunoassay types for follicle-stimulating hormone compared by using identical monoclonal antibodies. Clin. Chem. 39, 1435-1439. Maggiora, L.L. (1992). A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis. J. Med. Chem. 35, 3727-3730. Mann, M. & Wilm, M. (1995). Electrospray mass spectrometry for protein characterization. Trends Biochem. Sci. 20, 219-224. Markela, E., St~thlberg, T.H. & Hemmil~i, I. (1993). Europium-labelled recombinant protein G. J. lmmunol. Meth. 161, 1-6. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J. & Waggoner, A.S. (1993). Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chem. 4, 105-111. Southwick, P.L., Ernst, L.A., Taurillo, E.W., Parker, S.R., Mujumdar, R.B., Mujumdar, S.R., Clever, H.A. & Waggoner, A.S. (1990). Cyanine dye labeling reagents - carboxymethylindocyanine succinimidyl esters. Cytometry 11, 418-430. Wessendorf, M.W. & Brelje, T.C. (1992). Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas Red and cyanine 3.18. Histochemistry 98, 81-85. Whitaker J.E., Haugland, R.P., Moore, P.L., Hewitt, P.C., Reese, M. & Haugland, R.P. (1991). Cascade Blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. Biochemistry 198, 119-130.

FURTHER READING Chen R.F. & Scott, C.H. (1985). Atlas of fluorescence spectra and lifetimes of dyes attached to proteins. Anal. Lett. 18, 393-421. Haughland, R.P. (1992). Handbook of fluorescent probes and research chemicals. Molecular Probes, Eugene, OR. Hemmil~i, I.A. (1991). Applications of fluorescence in immunoassays. John Wiley, Chichester. Lacowicz J.R. (1983). Principles of fluorescence spectroscopy. Plenum, New York.

CHAPTER

FIVE

Use of tags in the labelling and detection of biomolecules

5.1 INTRODUCTION The identification of the very strong interaction between the vitamin biotin and egg white avidin not only explained the vitamin deficiency induced by the consumption of an excess of raw eggs, it also opened up a new approach to the labelling and detection of biomolecules (Green, 1975; Wilchek & Bayer, 1988). Now no longer did one need to covalently conjugate a large molecule such as an enzyme, one could simply modify with biotin, bind the biomolecule to its target (be it in a gel, filter, tissue section, microtitre plate well etc.), then incubate with a conjugate of avidin with the label of choice and develop the signal. This approach has been used for a wide variety of biomolecules (Wilchek & Bayer, 1990). For signalling purposes (as opposed to capture to solid phases), avidin has now been largely replaced by streptavidin, which is a less sticky protein in many assay systems, giving improved signal-to-noise ratios.

In a variant scheme, streptavidin, being tetravalent, may be used as a bridge, linking biotin on the target biomolecule to biotinylated label molecules. The popularity of biotin has given rise to a range of biotinylation reagents and conjugates of streptavidin and every major label type, and is often the bioscientist's first thought when a labelling need is identified. Other tags such as digoxigenin and other haptens are also now becoming popular. But it is important to be clear about the advantages and disadvantages of the tagging approach, since it is sometimes used in situations where covalent conjugates would give equivalent or improved results with less overall effort.

5.1.1 Advantages of tagging (i)

Stability. Biotin and the various hapten tags that have been developed are stable molecules; many labels, particularly enzymes,

U s e of tags in t h e l a b e l l i n g a n d d e t e c t i o n of b i o m o l e c u l e s

(ii)

(iii)

(iv)

(v)

are not. Therefore, if the incubation of the biomolecule with its target or binding partner involves a hostile environment (for example, many nucleic acid hybridization protocols), tagging makes sense. Ease of use. When using protein labels, tagging the biomolecule with for example biotin is a simpler procedure than protein conjugation. Small size. It is often stated that the small size of biotin and other tags is an advantage: it is less likely to destroy the activity of the biomolecule and, for histochemical studies, allows better penetration of tissue samples. However, there are now many examples of active covalent enzyme conjugates of different biomolecules (several examples in this book) to show that the size of the label is not an issue, and, in any case, size and penetration issues could be said to apply equally when the labelled streptavidin (or other anti-tag molecule) is brought into play. The size of tags may be of importance in the hybridization of long DNA probes, especially to membranebound targets, where large molecules attached to the probe may be expected to hinder duplex formation. Solution phase assays. Though not strictly a labelling issue, a further advantage of tags is that assays may be designed that permit biomolecule interactions to take place free in solution. The complex between the tagged biomolecule and its target is then captured onto a solid phase coated with the anti-tag molecule. The two main advantages are the liquid phase assay kinetics obtained (though subsequent capture may be slow) and the fact that for different biomolecules, a common coated solid phase may be employed. Sensitivity. Multiple tagging allows a degree of amplification of signal.

5.1.2 Disadvantages of tagging (i)

Extra steps. The protocols involve the extra steps of incubation with the signal conjugate and washing to remove unbound

65

conjugate. Also, though called noncovalent labelling, to bring this about two covalent conjugates are in fact required (biomolecule-tag and anti-taglabel). Though the latter conjugates may be available commercially, they are sometimes expensive. (ii) Backgrounds. Non-specific binding of conjugate can give rise to higher backgrounds. Some tissues contain naturally occurring biotin and erroneous signals can result. For some haptens, cross-reacting substances may be present. (iii) Characterization. For some tags, the tagged biomolecule may be difficult to characterize. Biotin, for example, has no strong and distinct absorbance to measure. This is not true for some other tags, e.g. fluorescein and dinitrophenyl (DNP), where the colour is a useful feature.

5.1.3 When do tags make sense? For each of the advantages and disadvantages listed above, some will be key issues in some situations but irrelevant in others. For DNA labelling and sequencing applications, the use of tags often makes considerable sense, especially for enzyme labels, where direct incorporation by polymerases and enzyme-dNTP derivatives would not be viable, and where harsh hybridization protocols are employed. Biotin is not necessarily the best choice, however, and other tags are increasingly being used. Because of the importance of tagging in molecular biology applications, tagging nucleic acids is discussed separately in Chapters 6 and 7. For proteins, tags are also useful where there are a number of proteins to be screened (e.g. antibodies) and preparation of a number of individual covalent conjugates would be time consuming. Once the best protein has been selected, switching to a covalent conjugate may give advantages, in particular a shorter protocol and, probably, lower background signals. Tags also make sense for recombinant proteins, since several different types of tag may now readily be attached as fusion proteins (see below).

66

Non-radioactive labelling Table 5.1 Some hapten tags

5.2 WHICH TAG? BIOTIN AND THE ALTERNATIVES Despite the importance attached to the high affinity of the biotin-avidin interaction, estimated to be 10 -15 M (Green, 1975), it is interesting to see how unimportant this feature has proved to be in practice, since other pairs of binding partners of much lower affinity have proved to be equally useful. Part of the reason is that the very high affinity may not always be achieved when the biotin is derivatized (Garlick & Giese, 1988); but also the binding to streptavidin (now preferred to avidin, see below) is about two orders of magnitude lower than avidin, as judged by faster dissociation rates (Green, 1990): despite this, streptavidin is generally not inferior to avidin in performance. Furthermore, the affinity of biotinylated proteins to streptavidin is a further order of magnitude lower (Vincent & Samuel, 1993), ca 10 -12 M. Many hapten-anti-hapten tagging systems have now been shown to give at least as good sensitivity, although the affinities involved are only in the range for a good antibody, i.e. 10 -9 to 10- ~~ M. Interestingly, a monoclonal antibody to biotin is at least equally effective as streptavidin (McQuaid & Allan, 1992). Nor is the on-rate of the biotin-avidin abnormally fast compared with hapten antibody interactions, the latter having been shown to be close to the diffusion-controlled limit (Absolom & van Oss, 1986). Therefore, although biotin is very well precedented, and hundreds of different reagents and conjugates are commercially available, its exceptional affinity is not of paramount importance. When selecting a tag, consideration should therefore be given as to whether other tags may have advantages in particular applications. Alternative hapten tags are listed in Table 5.1. Of these, digoxigenin (DIG) has been extensively used. For all applications, a coloured or fluorescent tag is useful for quantitation and following the probes in experimental protocols. Fluorescein-labelled DNA probes have been used widely and these provide the dual potential of direct fluorescence detection or anti-fluorescein-mediated enzyme (or other label) detection.

Abbreviation

Supplier

Digoxigenin Nitroiodophenyl Fluorescein

DIG NIP FL

Dinitrophenol

DNP

Boehringer Promega Amersham, Genosys Pierce, Dako

Hapten tag

Much depends of course on the quality of the antibody. Monoclonals generally give better backgrounds than polyclonals. When selecting an antibody or conjugate from commercial sources, a track record in the application of interest should be sought.

5.3 AVIDINS AND OTHER TAG BINDING AGENTS

5.3.1 Avidin and streptavidin Avidin and streptavidin conjugates of many common labels are available commercially from a variety of sources, and, when a new label is developed, it is frequently first made available in the form of a streptavidin conjugate. Such conjugates are generally easy to make, and the protocols in previous chapters can be used or adapted for streptavidin labelling. Streptavidin is often preferred to avidin for label conjugates, which tends to exhibit high non-specific binding, believed to be related to its high pl (10) and the presence of oligosaccharide. The recently described 'neutravidin' (Pierce), which has been deglycosylated (Hiller et al., 1987) and has a pI of 6.3, shows promise as a lower cost alternative to streptavidin.

5.3.2 Anti-tag antibodies Antibodies to the main hapten tags mentioned above are commercially available. If there is a choice, monoclonal antibodies are recommended since these tend to give lower noise levels. Enzyme conjugates of other tags are also

Use of tags in the labelling and detection of biomolecules available, for example DIG (Boehringer), DNP (Dako) and fluorescein (Genosys, Amersham). Other labels may be linked to antibodies by methods described in previous chapters. It should be noted that fluorescein is generally derivatized at either the 5 or the 6 position (the 5-isomer is the more common). Depending on the antibody selected, it may be important to choose a derivative of the correct fluorescein isomer.

67

have quite deep biotin binding sites, of the order of 15 A (Wilchek & Bayer, 1990). Protocol 5.1 describes the use of this reagent to modify proteins. It is very rapid and, using a disposable column to purify the product, takes less than an hour to carry out. DIG and fluorescein may also be introduced in an analogous fashion by means of the NHS

0

jl.

HN

5.4 METHODS FOR TAGGING BIOMOLECULES

NH

0

Given the importance of tags in molecular biology, labelling of DNA and RNA is treated separately in Chapters 6 and 7. The following section applies principally to proteins, glycoproteins and peptides, though the chemistry is applicable to other biomolecules that exhibit the appropriate functional groups (amino, carboxyl, thiol, vicdiol), such as derivatized nucleic acids and haptens. It concentrates on three main tags: biotin, DIG and fluorescein.

0

COOH O

6

5

0

5.4.1 Amino-directed reagents The biotin carboxyl group, not required for (strept)avidin binding, presents a suitable position for preparing labelling reagents and the NHS ester has been the standard biotinylation reagent (Bayer et al., 1976). However, the long chain analogue (Figure 5.1A) is now generally preferred since it makes the biotin more exposed for interaction with avidin or streptavidin, which

O OHcH ~ O

O

N-O O

O

oH

O

Figure 5.1 Amino-direct tagging reagents:

(A)

biotin; (B) fluorescein; (C) digoxigenin (DIG).

Protocol 5.1 B iotinylation of protein amino groups with biotin-LC-NHS. 1. Obtain antibody or other protein in 0.1 M sodium phosphate buffer, pH 7.4 (or PBS) at 1-5 mg m1-1. 2. Prepare a 10 mM solution of biotin-LC-NHS (Figure 5.1A) in DMF. 3. Add 1/20th volume of reagent to the protein solution and react for 30 min at room temperature. 4. Purify by gel filtration on a disposable column (Appendix 6) into PBS, or buffer of choice.

68

Non-radioactive labelling

0

H-NAN-H O

0

N O

O

H_NAN.H O

0

Figure 5.2 Thiol-directed biotinylation reagents.

Protocol 5.2 Biotinylation of thiol groups using maleimido or iodoacetyl reagents. 1. Obtain protein at 1-5 mgml-~ in 50 mM Tris-HC1 buffer, pH 8.0. 2. Take aliquot and determine thiol concentration (Appendix 5). 3. To the protein solution, add iodoacetyl-LC-biotin (Pierce, 1 mgml-~)a to provide a 2- to 5-fold excess of reagent over SH groups. 3. React at room temperature for 1-2 h. 4. Monitor thiol concentration at intervals (Appendix 5): the reaction may be terminated when the thiols have been reacted. 5. Remove reagents by passage through a de-salting column (Appendix 6). " A similar procedure may be used for maleimido reagents.

esters (Figure 5.1). Fluorescein may also be introduced using the isothiocyanate, FITC (see Protocol 4.1). DNP may be attached to proteins using dinitrophenyl fluoride.

Maleimido derivatives of both fluorescein and digoxigenin are available. Fluorescein is also available as the iodoacetyl derivative. General comments on the use of thiol reactive labels are given in Section 2.4 (see also Protocol 5.2).

5.4.2 Thiol-directed reagents 5.4.3 Carboxyl-directed reagents Several reagents for introducing biotin to protein thiols are available (Figure 5.2), including a maleimido and an iodoacetyl derivative (Yamamoto et al., 1984). Where thiols are present, this presents a more easily quantifiable route to biotinylation, since thiols before and after reaction with these reagents may readily be measured with Ellman's reagent. In some circumstances, where chemical precision is desired, this advantage may even justify deliberate introduction of thiols for those proteins that do not have them (see Section 3.3.1).

For biotin, hydrazide reagents have been used for carbodi-imide-mediated coupling to carboxyl groups, either based on biotinylaminocaproic acid or on biocytin (Figure 5.3). Likewise, the DIG hydrazide may also be used. For fluorescein, several amine derivatives are available (Molecular Probes). The fluorescein carboxyl group is not affected by water-soluble carbodiimides under normal conditions.

Use of tags in the labelling and detection of biomolecules 5.4.6 Characterization of biotinylated proteins

5.4.4 Carbohydrate-directed reagents Hydrazide reagents are also well suited for reaction with periodate-oxidized carbohydrate groups of glycoproteins (Figure 5.3). Protocol 5.3 (Bayer & Wilchek, 1992) describes a procedure for biotin, and hydrazides of fluorescein (Molecular Probes) and DIG (Boehringer) may also be coupled by the same route.

5.4.5 Tyrosine-directed reagent p-Diazobenzoyl biocytin has been used for the biotinylation of tyrosines or histidines (Bayer & Wilchek, 1992). Histidine modification is also possible with this reagent.

O H-NLN-H H

N"~~O 0

NH2 NH NH2 I

O H-NLN-H O H

0

Figure 5.3 Biotin hydrazide reagents.

69

In general, apart from the cases described above, measuring the degree of biotinylation is not straightforward. For proteins biotinylated at amino groups, treatment of the protein with proteinase K yields free biotin which may be determined by fluorometric or other assays (Wilchek & Bayer, 1990), but these are not straightforward procedures. Fluorescence polarization has been proposed (Shah et al., 1994): here the product is allowed to compete with fluoresceinylated biotin for an anti-biotin monoclonal; the polarization indicates the unbound fluoresceinylated biotin content and may be related to the functional biotin content of the derivatized protein. Another fluorescence method exploits the quenching of the intrinsic fluorescence of avidin upon binding biotin (Der-Balian et al., 1990). Most workers rely on functional characterization. An easy procedure is to take samples of the product, mix with varying amounts of streptavidin (e.g. 1-, 2-, 4- and 8-fold excess) and analyse, for example on a rapid gel filtration HPLC column which can distinguish between complex and free streptavidin. The highest excess sample that does not give rise to a free streptavidin peak will indicate the approximate number of functional biotins present. Alternatively, a series of derivatives may be prepared with differing concentrations of reagents (3- to 5-fold differences give a suitable spread of conditions) and the best member of the series

Protocol 5.3 B iotinylation of glycoprotein carbohydrate groups. 1. Provide protein solution at 0.5-3 mg m1-1 in PBS. 2. Add 1/10th volume of a freshly prepared solution of 0.1 M sodium periodate and allow reaction to proceed at room temperature for 30 min. 3. De-salt (Appendix 6) or dialyse the product into PBS. 4. Add 1/10th volume of 20 mg ml biocytin hydrazide (Pierce) in PBS (heating required to prepare the biotin hydrazide stock solution) and react for 2 h at room temperature. 5. De-salt (Appendix 6) or dialyse the product into PBS.

70

Non-radioactive labelling

selected on the basis of performance in the test system or a system that closely models it. Mass spectrometry is an attractive option for characterization (see Section 4.1.4.3).

been reviewed (Uhl6n et al., 1992; Flaschel & Friehs, 1993). As a routine method of tagging proteins, such methods are slow compared with chemical labelling. However, if the protein is anyway to be produced by recombinant technology, or the tagged protein is required on a large scale, then this approach is very attractive.

5.5 TAGGING OF PEPTIDES The tagging of peptides obviously calls for an element of chemical design. Where a single amino group or a single thiol group is present (or can be arranged to be present), and is known to be present at a biologically tolerant position, then the amino- and thiol-directed reagents described above can be used (Schvartz et al., 1991). The amino terminus frequently proves to be a convenient and acceptable attachment site. Introduction of the tags during solid phase synthesis gives more choice of attachment position and can lead to more biologically active peptides. This is exemplified by the biotinylation of endothelin- 1 (Natarajan et al., 1992). In general however, in the absence of good structureactivity information, it is advisable to tag at the termini. The N-terminus may conveniently be tagged on-column using amine-directed biotin NHS esters or carboxyl coupling methods. An alternative approach is to introduce a cysteine residue at a convenient point and use a thioldirected approach. Solid phase synthesis also opens up the possibility of adding a fusion protein type of tag as described below.

5.6 FUSION PROTEIN TAGS

5.6.1 General comments

Many expression systems have been described for making recombinant tagged proteins in the form of fusion proteins. The main rationale for using the fusion protein route is to facilitate purification; however, there is increasing interest in tags that may be used to identify or measure the protein in subsequent biochemical assays and investigations, and several tags can fulfil both functions well. Fusion protein tags have

5.6.2 Choice of fusion tag

Of the many different types that have been described, the following are selected as being of particular interest for our purpose. 5.6.2.1 Antigenic tags

Essentially any protein or peptide sequence for which one can obtain an antibody may be fused, N- or C-terminally, to the protein of choice. Peptide tags are particularly useful in this respect and an almost infinite variety is possible. Examples of peptide tags are the 'FLAG TM' peptide, DYKDDDDK (Kodak; Hopp et al., 1988) and the 'c-myc' tag (Newton et al., 1994). 5.6.2.2 Biotin mimetics

The peptide AWRHPQFGG has been shown to have useful affinity for streptavidin and an expression system for C-terminal fusion proteins with this tag, known as 'Strep-Tag', has been described (Schmidt & Skerra, 1993). Kits are available from Biometra. 5.6.2.3 Biotinylation sequences

In E. coli, proteins may be post-translationally modified with biotin if they are fused with a 13 amino acid biotinylation consensus peptide (Schatz, 1993). This system, called 'PinPoint TM', has been commercialized by Promega. 5.6.2.4 6-His tags

Tags comprising consecutive histidines have an affinity for certain metal chelates, and tagged proteins may be purified on nickel chelate columns (Hochuli et al., 1988; Hochuli & Piesecki, 1992). 6-His tags may conveniently be detected

U s e of tags in t h e l a b e l l i n g a n d d e t e c t i o n of b i o m o l e c u l e s using a conjugate of a nickel chelate and the signal of choice. Various products related to this systems are available from Qiagen. A simple preparation of a nickel chelate-HRP conjugate is given in Chapter 8 (Protocol 8.1). Alternatively, detection via an anti-histidine tag antibody is possible (Zentgraf et al., 1995).

REFERENCES Absolom, D.R. & van Oss, C.J. (1986). The nature of the antigen-antibody interaction and the factors affecting its association and dissociation. CRC Crit. R ev. Immunol. 6, 1-46. Bayer, E.A. & Wilchek, M. (1992). Labeling and detection of proteins and glycoproteins. In Nonradioactive labeling and detection of biomolecules (ed. C. Kessler), pp. 91-100. Springer,

Berlin. Bayer, E.A., Wilchek, M. & Stutelsky, E. (1976). Affinity cytochemistry: the localization of lectin and antibody receptors on erythrocytes via the avidin-biotin complex. FEBS Lett. 68, 240-244. Der-Balian, G.P., Gomez, B., Masino, R.S. & Parce, J.W. (1990). A fluorometric method for determining the degree of biotinylation of proteins. J. Immunol. Meth. 126, 281-285. Flaschel, E. and Friehs, K. (1993). Improvement of downstream processing of recombinant proteins by means of genetic engineering methods. Biotech. Adv. 11, 31-78. Garlick, R.K. and Giese, R.W. (1988). Avidin binding of radiolabeled biotin derivatives. J. Biol. Chem. 263, 210-215. Green, N.M. (1975). Avidin. Adv. Prot. Chem. 29, 85-133. Green, N.M. (1990). Avidin and streptavidin. In Avidin-biotin technology, eds, M. Wilchek & E.A. Bayer. Meth. Enzymol. 184. 51-67. Hiller, Y., Gershoni, J.M., Bayer, E.A. and Wilchek, M. (1987). Biotin binding to avidin. Oligosaccharide chains not required for ligand association. Biochem. J. 248, 167-171. Hochuli, E. and Piesecki, S. (1992). Interaction of hexhistidine fusion proteins with nitrilotriacetic acid-chelated Ni 2+ ions. Methods 4, 68-72. Hochuli, E., Bannwarth, W., D6beli, H., Gentz, R. and St0ber, D. (1988). Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Biotechnology 6, 1321-1325. Hopp, T.P., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, D.P., Urdal, D.L. and Conlon, P.J. (1988). A short polypeptide marker sequence useful for recombinant protein identifi-

71

cation and purification. Biotechnology 6, 1204-1210. McQuaid, S. and Allan, G.M (1992). Detection protocols for biotinylated probes: optimization using multistep techniques. J. Histochem. Cytochem. 40, 569-574. Natarajan, S. Festin, S.M., Hedberg, A., Liu, E.C.-K., Floyd, D.M. and Hunt, J.T. (1992). Site-specific biotinylation. Int. J. Peptide Protein Res. 40, 567-574. Newton, C.R., MacCallum, L.J., McPheat, W., Carr, H., Foster, H.R., Abbott, W.M., Needham, M., Dinsmore, A. and Copley, C.G. (1994). Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunoaffinity tag. Prot. Express. Purif 5, 449-457. Schatz, P.J. (1993). Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology 11, 1138-1143. Schmidt, T.G.M. and Skerra, A. (1993). The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purifiation of a functional IgG Fv fragment. Protein. Eng. 6, 109-122. Schvartz, I., Gitlin, G., Amarant, T., Ittoop, O. and Hazum, E. (1991). Biotinylated endothelin as a probe for the endothelin receptor. Peptides 12, 1229-1233. Shah, D., Salbilla, V., Richerson, R. and Brown, W. (1994). Determination of biotin in biotin-conjugated protein by measuring fluorescence polarization. Clin. Chem. 40, 2112-2113. Uhl6n, M., Forsberg, G., Moks, T., Hartmanis, M. and Nilsson, B. (1992). Fusion proteins in biotechnology. Curr. Opin. Biotechnol. 3, 363-369. Vincent, P. and Samuel, D. (1993). A comparison of the binding of biotin and biotinylated macromolecular ligands to an anti-biotin monoclonal antibody and to streptavidin. J. Immunol. Meth. 165, 177-182. Wilchek, M. and Bayer, E.A. (1988). The avidinbiotin complex in bioanalytical applications. Anal. Biochem. 171, 1-32. Wilchek, M. & Bayer, E.A. (eds) (1990). Avidinbiotin technology. Meth. Enzymol. 184. Yamamoto, K., Sekine, T. and Sutoh, K. (1984). Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface. FEBS Lett. 176, 75-78. Zentgraf, H., Frey, M., Schwinn, S., Tessmer, C., Willeman, B., Samstag, Y. and Velhagen, I. (1995). Detection of histidine-tagged fusion proteins by using a high-specific mouse monoclonal anti-histidine tag antibody. Nucl. Acids Res. 23, 3347-3348.

CHAPTER

SIX

Labelling of oligonucleotides

6.1 INTRODUCTION Non-radioactively labelled oligodeoxyribonucleotides now have a wide range of established applications ranging from sequencing primers, PCR primers, probes for filter and in situ techniques and homogeneous assays. Of the nonisotopic labels, fluorescent and enzyme labels dominate, with fluorescence used for sequencing and other low to moderate sensitivity methods and enzymes being the label of choice for high sensitivity applications. Tags such as biotin and DIG are also popular, although the rationale for using tags with oligonucleotide probes is less compelling compared with full length probes (see Section 5.1). As probes, oligonucleotides have a number of advantages compared with full-length probes and are to be recommended whenever sequence information is available. They can be made easily and inexpensively in relatively large

quantities. They can be made reproducibly and chemically characterized. In contrast, long probes vary from batch to batch and are heterogeneous. There need be no concerns about sensitivity; for Southern blots and other applications, it is now accepted that a 1:1 enzyme oligonucleotide can deliver the same sensitivity of a cloned probe of several kilobases with many labels incorporated (Giles et al., 1990; Ruth, 1994; see also Figure 6.1). In general, detection protocols are simpler compared with tagged full-length probes. Oligonucleotide probes are favoured for in situ applications (Wisden & Morris, 1994). Fluorescence techniques are beginning to dominate the detection of PCR products for situations where gel electrophoresis is inconvenient. A good example is the 'Taqman xM' approach (Perkin Elmer) where the 5' ~ 3' exonuclease activity of Taq polymerase is used to degrade a probe labelled with a fluoroph0re and quencher, resulting in an increase in fluorescence (Livak et al., 1995).

L a b e l l i n g of o l i g o n u c l e o t i d e s

Figure 6.1 Southern blot of DNA fingerprint of three human genomic HinfI digests, loaded with 2 lag DNA per lane and hybridized with (A) MSI minisatellite probe labelled with 32p (6 h exposure) or (B) MSI alkaline phosphatase oligonucleotide probe developed with Lumi-Phos T M chemiluminescent substrate (2 h exposure). Data kindly provided by E. Booth. General issues regarding the choice of label are addressed in Chapter 1. For blotting applications enzyme labels have been widely used and have excellent sensitivity (Urdea et al., 1988). Alkaline phosphatase is to be preferred since it is more stable than HRP, and can be developed using a wide range of substrates. In particular, the use of dioxetane substrates (Section 1.7.2.4) with alkaline phosphatase offers better sensitivity than 32p-labelled probes, in a shorter time period (Bronstein et al., 1989).

6.1.1 Chemical modification of synthetic DNA Native DNA has fewer sites that are suitable for the chemical attachment of labels compared with proteins. For this reason, the labelling chemistry is dominated by methods that introduce a label, a tag or a reactive group either during chemical synthesis or by an enzyme-mediated reaction after synthesis. The relative ease of automated oligonucleotide synthesis (Brown & Brown, 1991) and the wide availability of DNA synthesizers means that the chemical approach is not only convenient, but also within the scope of the non-chemist. A great variety of phosphoramidite reagents are available commercially to enable amino group handles, biotin tags, fluorophores

73

etc. to be incorporated, requiring little more than attachment of the reagent to a spare phosphoramidite port and appropriate programming of the synthesizer. By far the most convenient and important linkage point is the 5' terminus. However, other sites of attachment include the 3' terminus, the bases, in particular N-4 of cytosine and N-6 of adenine, and the phosphodiester link. Though base and backbone linkages (and other strategies) offer the potential of multiple tagging, good sensitivities for routine blotting applications can be achieved with 1:1 enzyme to oligonucleotide conjugates. Space does not permit a comprehensive survey of all possible labelling methods. These have been reviewed (Goodchild, 1990). Here we focus on options for introducing labels at the 5' end since (i) many useful reagents are available commercially, (ii) oligonucleotides labelled at this position have a good track record in diagnostic applications, and (iii) various labelling kits are based on 5' labelling. There is also evidence that oligonucleotides tagged with biotin (Cook et al., 1988) or labelled directly with alkaline phosphatase (J.R. Parker, unpublished results, 1989) show greater sensitivity when the linkage is made at the terminus, rather than via a base in the middle of the sequence. There will however occasionally be a need to consider other labelling strategies for a variety of reasons; hence these are reviewed here, in brief, with references. The other method of importance is post-synthesis labelling based on enzymic extension at the 3' or 5' end. This approach is appropriate when an unmodified oligonucleotide is available and it is not considered desirable to resynthesize the same oligonucleotide with a label or handle attached.

6.2 ON-COLUMN LABELLING USING PHOSPHORAMIDITE REAGENTS The development of a biotin phosphoramidite reagent (Alves et al., 1989) marked the start of the development of many such reagents designed to introduce the desired tag or label directly

74

Non-radioactive labelling

during automated solid phase synthesis. The main advantage of this approach is that no offcolumn manipulations are necessary (though HPLC purification is advisable), hence the approach is simple and convenient. Many specialist phosphoramidites are now available, especially for introducing fluorophores and tags. Of the important tags, only DIG has refused to comply due to the chemical complexity of the molecule. Many labelling phosphoramidites are now available in 'multi-addition' form. The generic design for such reagents is given in Figure 6.2. The key feature is the pendant dimethoxytrityl (DMT)-protected hydroxyl group which can be programmed (as for a nucleoside phosphoramidite) to be the site of attachment of further molecule of reagent, a process that can be repeated ad libitum. Multiple addition is probably not necessary for most situations, but these reagents do provide more options and also give the opportunity for labelling elsewhere in the sequence. For those familiar with oligonucleotide synthesis using phosphoramidites, these speciality phosphoramidite reagents are straightforward to use. They are typically introduced in automated synthesis at one of the spare phosphoramidite ports on the synthesizer and may be handled and programmed as for normal nucleoside phosphoramidites. Manufacturer's recommendations on, for example, concentrations should be followed. Some of these reagents are not as stable as phosphoramidites of normal nucleosides. Typically, once dissolved in acetonitrile, they only last 1 week on the synthesizer (or less). If used infrequently, remove the bottle and store at - 2 0 ~ under argon. It should be noted that oligonucleotides derivatized with

DMT

Figure 6.2 Generic

. L NIL I jP \

CN

design for multi-addition phosphoramidite reagent for introducing tags and labels. X is the function to be introduced.

multi-addition reagents (such as shown in Figure 6.2) should be synthesized 'trityl-on', because during ammonia de-protection there is the possibility of hydroxyl attack on the neighbouring phosphate, leading to cleavage of the phosphodiester bond. The trityl-on approach is anyway to be recommended in most cases for the usual reason that the trityl group allows easy purification of full-length product on reverse-phase HPLC.

6.2.1 Biotin phosphoramidites Biotinylated oligonucleotides have a wide range of applications, including as probes (Saiki et al., 1989), sequencing primers (Hultman et al., 1989) and solid-phase capture tags. Biotin phosphoramidites provide a more direct and convenient route to 5'-biotinylated oligonucleotides than the earlier off-column biotinylation of 5' amino oligonucleotides (Chollet & Kawashima, 1985; Cook et al., 1988). A number of such reagents have been described; some of these are shown in Figure 6.3. The first such reagent, reagent I (Alves et al., 1989), contained a protecting dimethoxytrityl group on the biotin ureido nitrogen in order to prevent phosphitylation by a second reagent molecule at this position. This feature was retained by Pon (1991) whose reagent includes a C-6 spacer group (reagent IV). Reagents II (Misiura et al., 1990) and III contain a pendant DMT protected hydroxyl group, a feature which permits multiple biotinylations by successive reaction cycles. It should be noted that these reagents lack a protecting group on the biotin. It has been shown that branching can occur at unprotected biotin when introduced in the middle of an oligonucleotide (Pieles et al., 1990), and it has been found also that these reagents can give heterogeneous products due to reaction at biotin (I.J. Hodgson, M.J. McLean & A.J. Garman, unpublished results, 1991). For most applications, more than one biotin is unnecessary, and it is not necessarily the case that the sensitivity is proportional to the number of biotin groups (Misiura et al., 1990), probably due to steric effects when the streptavidin binds. Overall, reagent IV gives high yields of homogeneous product and is recom-

Labelling of o l i g o n u c l e o t i d e s

75

O DMT-

H -N

N H

N

O--~_CN

O

.j~o,_~ II

O

o

o._,_cN

DMT

O

H-NAN'H

O_~p~N.__~

O

N

DMT"N A N ' H

O_~_CN

O'-'~_CN

O

DMT

II!

IV

Figure 6.3 Biotin phosphoramidite reagents. For description, see text.

~

o --o o} H N ~

,N

\ O""~.. CN

FAM

oCl~

el0

N

~

CI

O C I ~ c I O

c~-.~..~- o HN~ 7 ' ~ . ~ , ~

O-

O~_._ CN HEX

NO2 H NO.,,,,. ~~ N~

c,.~~ ~ ol

H

CI

-( m-( o-~ O% , 0--~_ CN 0

bMT

,

O--~C N

TET

DNP

Figure 6.4 Three phosphoramidite reagents for introducing the fluorophores and the DNP multi-addition reagent. mended for most applications. A reagent combining biotin protection and the multi-addition feature has recently been described (Neuner, 1996).

6.2.2 DNP phosphoramidite Dinitrophenyl (DNP) is a 'classic' hapten that has been proposed as an oligonucleotide tag (Will et al., 1991). A multi-addition phosphoramidite reagent (Grzybowski et al., 1993) is shown in Figure 6.4; this is available from

76

Non-radioactive labelling

Cruachem. A useful advantage of DNP is that its yellow colour aids purification and tracking. DNP is hydrophobic and retards the product on reverse-phase HPLC. Detection of DNP is via antibody-enzyme conjugates which are available from, for example, Dako.

from Clontech. It should be noted that fluorescence quenching will occur when several fluoresceins are added close together. Multi-addition may be of value when the fluorescein is used as a tag and detected via an anti-fluorescein antibody. A pyrene phosphoramidite reagent has also been described (Mann et al., 1992).

6.2.3 Fluorophore phosphoramidites Fluorescein and two chlorinated fluorescein derivatives have been widely used for labelling oligonucleotides, principally for sequencing purposes. The reagents, which are additionally protected with pivaloyl groups, are known by the names 6-FAM, TET and HEX; these are shown in Figure 6.4. The fluorophores known as ROX, TAMRA and JOE are not available as phosphoramidites but may be linked to 5' amino groups by means of active esters (see Section 6.3.5). For wavelength data, see Appendix 4. Phosphoramidites for introducing the cyanine dyes are also available (Glen Research). A multi-addition fluorescein phosphoramidite and an acridine phosphoramidite are available thiol-reactive

protein . . . . .

oligonucleotide

HS.....

Cp~

As with normal oligonucleotides, modified oligonucleotides may often be successfully used without purification, provided that stepwise coupling yields are satisfactory (>99%). However, it is advisable to purify labelled oligonucleotides, especially those which have been fluorescently labelled: conventional HPLC purification schemes may be used (see Appendix 12). For 5'-amino oligonucleotides that are intended for protein labelling, purification is unnecessary since failure sequences (if capped) will not bear an amino group and hence will be

oligonucleotide

P ~ thiol-~

6.2.4 Purification of modified oligonucleotides

~ 2-iminothiolane,

\other thiolatingagents

0 ~ N..... oligonucleotid~lCKC,~~StMr ~ ~ 0

SMCC

H2N~

0. . l ~ 0 . , , ~ OH

COOH o

HNANH

fluorescein rea

H HN N.....

oligonucleotide

N.....

oligonucleotide

YS

O

~1 O-- P- O-oligonucleotide OH

biotin reagents

Figure 6.5 Scheme to illustrate some of the labelling and tagging options provided by a 5' amino-derivatized oligonucleotide.

Labelling of oligonucleotides unable to conjugate. They can be removed when the conjugate is purified on a size-exclusion column (this is a useful advantage compared with other labelling positions).

L.A. F

H

6.3.1 Introduction of a primary amine at the 5' terminus

A range of different phosphoramidite reagents have been described for introducing 5' amino groups (Connolly, 1987). These differ in the amino group protection used (trityl, monomethoxytrityl, trifluoroacetyl), the number of methylenes introduced by the linker, and the phosphoramidite protection. The most widely used is Aminolink-2 T M (Perkin Elmer), structure I in Figure 6.6. Protocol 6.1 describes its use. Other phosphoramidites that have been used to introduce amino groups via different length

I

F,~,,., , . N ~ o / P F ]~ 0

5..

6.3 LABELLING AT INTRODUCED PRIMARY AMINO GROUPS Extension of the 5' terminus to provide a primary amino group, linked via a simple alkyl spacer group, opens up an enormous range of labelling options, based for the most part on labelling strategies devised for proteins (Agrawal et al., 1986). Figure 6.5 surveys some of the options. To an extent, such derivatives may be conceptually treated in the same way as protein amino groups and most of the labelling strategies described in earlier chapters of this book can be applied. Figure 6.6 shows some of the reagents that may be used to introduce the amino group handle. Aminoalkyl groups may also be incorporated into oligonucleotides using H-phosphonate reagents (Sinha & Cook, 1988). Compared with proteins, primary amino derivatized oligonucleotides have the advantage that they may be designed such that only one is present, and hence only one labelled species should result. Furthermore, its reactivity is not sequence dependent, so that it is possible to derive generic methods that will apply to essentially all oligonucleotides.

77

DMT\

0~ N H O/ II Imoc F

. Fmoc ~ N ~ H

I

O

).,

N"

o~

.......

N/~/~/~ H IV

0 ,~

CN

o

O

o J-..) DMT - 0 - ~

~CN

-MMT

III

O

0

I

P\

0/

I

\

H N ~Fmoc

0 P

.A.

"0 ~

CN

Fmoc =

~

o ~

Figure 6.6 Reagents for introducing the amino function during solid phase oligonucleotide synthesis. I, Aminolink-2; II, multi-addition reagent; III, Fmoc-3'-amino-modifier CPG; IV, DMT-5amino-dU-CEP.

alkyl spacers have been described (Cardullo et al., 1988).

6.3.2 Introduction of primary amines within the sequence

Incorporation of bases containing primary amine handles provided an early route to making

78

Non-radioactive labelling

P r o t o c o l 6.1 P r e p a r a t i o n o f a 5' a m i no- d e r i v ati z e d oligonucleotide. 1. Dissolve 250 mg Aminolink-2T M reagent (Perkin Elmer) in anhydrous acetonitrile (3.3 ml) and attach bottle to a spare phosphoramidite port on the DNA synthesizer. 2. Program the synthesizer with the desired sequence, specifying 'trityl on'. 3. After the synthesis, perform the ammonia de-protection step as usual. This removes also the TFA protecting group. 4. The product may then be purified by anion-exchange HPLC. However, it is generally convenient to proceed directly to the coupling step and purify after coupling. In this case, it is important that all ammonia is efficiently removed and it is therefore recommended that the oligonucleotide be de-salted (Appendix 6) before reacting at the amino group.

conjugates with enzymes (Jablonski et al., 1986; Ruth 1991) and other labels (Telser et al., 1989). Phosphoramidite reagents such as IV (Figure 6.6; available from Peninsula), containing a protected amino group, have been used. The protecting group is removed during the routine ammonia oligonucleotide deprotection. Incorporation of an amino group in this fashion opens up in principle the same wide range of labelling opportunities described for 5' amine labelling above. A similar approach employs a 3' phosphoramidite of an N-4 cytidine derivative bearing an amino handle (Li et al., 1987). Another reagent for introducing amino groups is reagent II in Figure 6.6 (Clontech). This is of the multi-addition design and may therefore be introduced as many times as desired and at any point in the sequence (Nelson et al., 1989b). Interrupting the DNA sequence in this way is likely to destabilize the duplex, and hence this reagent is perhaps best regarded as a route to multiple labelling at or near the termini.

6.3.3 Introduction of a primary amine at the 3' terminus Apart from 3' tailing reactions (Section 6.6.1), there are a number of strategies should one wish to derivatize at the 3' end. An amino group can be attached at the 3' end during solid phase synthesis by use of a special CPG support containing a pendant-protected amine (Figure 6.6, III; Nelson et al., 1989a).

The oligonucleotide is built up on the monomethoxytrityl-protected hydroxyl; cleavage and de-protection gives an aminohexyl function. Other chemical strategies for incorporation of amino groups at the 3' end include: (i) oligonucleotide synthesis starting with a ribonucleoside, followed by periodate oxidative cleavage of the vic-diol, reaction with 1,6-diaminohexane and borohydride reduction (Agrawal, 1994) and (ii) an H-phosphonate approach permitting multiple attachments of diaminohexane at the phosphodiester groups (Agrawal, 1994).

6.3.4 Enzyme labelling at introduced amines This scheme (see Figure 6.7) was originally developed for fingerprinting applications (Giles et al., 1989; Garman, 1991). The starting point for this approach (Figure 6.7) is a 5' amino oligonucleotide (though the method may be modified for any primary amine derivative). This is extended by reaction with 2-iminothiolane to give a free thiol which is subsequently reacted with maleimido-activated enzyme. An excess of oligonucleotide should be used (on a molar basis, it is less expensive than alkaline phosphatase); this ensures that the free enzyme content of the crude product is minimal and thus a simple gel filtration purification to remove unconjugated oligonucleotide yields a preparation of high purity. Applications which require very low free enzyme content should use a higher ratio of oligonucleotide to enzyme.

Labelling of o l i g o n u c l e o t i d e s Though 2-iminothiolane is a convenient (onestep) thiolating reagent to employ, in principle any of the thiolating reagents discussed in Chapter 3 are suitable. In similar circumstances, Nacetyl-DL-homocysteine thiolactone has been successfully used (Kumar et al., 1991). Likewise other thiol reactive groups may be employed; SlAB gives good results with alkaline phosphatase for example. Protocols 6.2 and 6.3 describe the conjugation procedure for alkaline phosphatase and HRP. Conjugates of this type may also be prepared using kits (e.g E-LINK TM, Genosys). Protocol 6.4 describes the use of alkaline phosphataselabelled probes for filter hybridization with chemiluminescent detection. Alternatively, enzymes may be attached by amines introduced at other positions in the oligonucleotide. Attachment to in-chain amino groups introduced by reagents such as IV (Figure 6.6), has been employed quite widely (Jablonski et al., 1986; Ruth, 1991), in particular for the SNAP TM series of alkaline phosphataselabelled probes (Syngene). DNA fingerprinting

O enzyme]--- NH

has also been demonstrated (Edman et al., 1988). The original method involves the use of a homobifunctional cross-linking agent, disuccinimidyl suberate, to link together probe and enzyme amino groups. Use of a homobifunctional reagent may be expected to give low yields of product, and reduced yields compared with a heterobifunctional approach has been reported for 5' labelling (Reyes & Cockerell, 1993).

6.3.5 Fluorescent labelling at primary amino groups Primary amino groups may be reacted with a variety of amine-reactive fluorophore reagents, as described in Chapter 4, and dyes commonly used for DNA analysis (see Figure 6.8). Examples of the use of isothiocyanates and NHS esters are given in Protocols 6.5 and 6.6, respectively.

o

O

N--O O

NH2

N

9+

H2N

o II O - P -O -oligonucleotide OH

~

O

O

NH2

O

[enzyme

HS

N~

O O

-

H

NH: N

H

O

Figure 6.7 Scheme for labelling oligonucleotides with an enzyme.

P -O -oligonucleotide OH

O

O

79

o OH

80

Non-radioactive labelling

Protocol 6.2 Preparation of an oligonucleotidealkaline phosphatase conjugate. 1. Synthesize the amino-oligonucleotide using Aminolink-2 T M phosphoramidite (Perkin Elmer) as described in Protocol 6.1. 2. Prepare maleimido-activated alkaline phosphatase as follows. Take 0.2 ml stock alkaline phosphatase (10 mgml -j, Boehringer, DNA grade) a and add 0.6 ml 0.1 M triethanolamine-HC1, pH 7.4, containing 1 mM MgC12 and 1 mM ZnSO4. Prepare a fresh solution of SMCC (Pierce) in dry DMF (6.7 mgm1-1) and add 12 pl to the enzyme. Leave to react at room temperature for 30 min. Equilibrate a NAP-25 de-salting column (Pharmacia) with PBS. Apply sample and allow to soak on. Apply 1.95 ml PBS. Apply 1.6 ml and collect the product, b Keep on ice and use within the same day. 3. Thiolate the oligonucleotide as follows. Prepare a fresh solution of 2iminothiolane (Pierce) in 0.2 M sodium bicarbonate buffer, pH 9.0 and immediately add 0.3 ml of this to 0.2 ml of the amino-oligonucleotide (ca 0.04 pmol). Discard unused reagent. Incubate the reaction at 37~ for 30 min. Equilibrate a NAP-25 T M de-salting column (Pharmacia) with PBS. Apply sample and allow to soak on. Apply 2.3 ml PBS. Apply 1.6 ml and collect the product. Proceed immediately with next step. 4. Add the oligonucleotide to the activated enzyme and incubate overnight at 4~ c 5. Prepare a column (20-60 ml) of Biogel P-100F (Biorad) and equilibrate in 50 mM Tris-HC1, pH 7.5 containing 0.1% BSA and 0.2% NAN3. 6. Concentrate the reaction mixture using a microconcentrator (Amicon) and apply to the column. Collect the first eluting peak which comprises the conjugate. Store aliquotted at 4~ Other sources may be used provided they are free of nucleases. b When carrying out the enzyme modification for the first time, it is advisable to check the maleimido content as described in Protocol 3.4. c Conjugation make take place at room temperature for 2 h.

a

Protocol 6.3 Preparation of an oligonucleotide-HRP conjugate. 1. Synthesize the amino-oligonucleotide using Aminolink-2 T M phosphoramidite (Perkin Elmer) as described in Protocol 6.1. 2. Prepare maleimido-activated HRP as follows. Make a solution of 4.0 mg HRP (Biozyme) in 0.1 M triethanolamine-HC1, pH 7.4 (0.8 ml). Prepare a fresh solution of SMCC (Pierce) in dry DMF (15 m g m l - ' ) and add 100 pl to the enzyme. Leave to react at room temperature for 40 min. Equilibrate a NAP-25 T M de-salting column (Pharmacia) with PBS. Apply sample and allow to soak on. Apply 1.9 ml PBS. Apply 1.6 ml and collect the product, a Keep on ice and use within the day. 3. Thiolate the oligonucleotide as follows. Prepare a fresh solution of 2iminothiolane (Pierce) in 0.2 M sodium bicarbonate buffer, pH 9.0, and immediately add 0.3 ml of this to 0.2 ml of the amino-oligonucleotide. Discard unused reagent. Incubate the reaction at 37~ for 30 min. Equilibrate a NAP-25 T M de-salting column (Pharmacia) with PBS. Apply sample and allow to soak on. Apply 2.3 ml PBS. Apply 1.6 ml and collect the product, b Proceed immediately with next step.

Labelling of oligonucleotides

81

4. Add the oligonucleotide to the activated enzyme and incubate overnight at 4~ c 5. Prepare a column (20-60 ml) of Biogel P-100F (Biorad) and equilibrate in 50 mM Tris-HC1, pH 7.5, containing 0.1% BSA. 6. Concentrate the reaction mixture using a microconcentrator (Amicon) and apply to the column. Collect the first eluting peak which comprises the conjugate. Store aliquotted at -20~ a When carrying out the enzyme modification for the first time, it is advisable to check the maleimido content as described in Protocol 3.4. b The oligonucleotide need not be purified before conjugation. c Conjugation may take place at room temperature for 2 h.

Protocol 6.4 Hybridization and detection of alkaline

phosphatase-labelled oligonucleotides (nylon membranes) a. Solutions 1. Phosphate buffer concentrate: 0.5 M NaH2POa/Na2HPO4 buffer, pH 7.2. 2. 10% sodium lauryl sulphate (SDS) 3. 20• SSC: 3 M NaC1 (175.3 g 1-1), "0.3 M sodium citrate (88.2 g l-l). 4. Prehybridization buffer: add 10 ml 10% SDS to 990 ml of the above phosphate buffer concentrate. 5. Wash solution 1:0.01 M NaH2POa/Na2HPO4 buffer, pH 7.2, 0.1% SDS. To prepare, dilute 20 ml of phosphate concentrate to 990 ml, then add 10 ml 10% SDS (dilute before adding SDS). 6. Wash solution 2:13.8 g 1-1 maleic acid, 8.7 g 1-1 NaC1 adjusted to pH 7.5 with conc. NaOH. 7. Blocking agent: 100 g 1-1 casein (Hammarsten, BDH cat. no. 44020) in wash solution 2. Dissolve by heating at 50-70~ for 1 h. Autoclave and store at -20~ 8. Hybridization buffer: 900 ml prehybridization buffer and 100 ml blocking agent. 9. 0.1 M MgC12 (20.3 g l-l). 10. CDP-Staff M assay buffer: 0.1 M diethanolamine, pH 9.5, containing 1 mM MgC12. To prepare, dilute 10 ml diethanolamine (free base) to ca 900 ml, add 10 ml 0.1 M MgC12, adjust pH to 9.5 with 6 M HC1 and make up to 1 1. Method 1. Prehybridize by wetting in 1X SSC and placing, DNA side down, in prehybridization buffer (500 ml) at 50~ b. Gently agitate for 20 min at 50~ 2. To a suitable container (e.g. sandwich box) add hybridization buffer (160 ml) and the alkaline phosphatase-oligonucleotide conjugate to a final concentration of 1 nM. Mix well. 3. Transfer the membrane, DNA side down, into the hybridization chamber, ensuring that there are no trapped bubbles. Handle the membrane with plastic forceps. Gently agitate for 20 min at 50~ c. 4. Wash by transferring the membrane, DNA side down, into prewarmed wash solution 1 (500 ml). Gently agitate for 10 min at 50~ 5. Repeat step 4 with fresh wash solution 1. 6. Rinse by transferring the membrane, DNA side down, into wash solution 2 (500 ml) at room temperature. Gently agitate for 10 min at room temperature.

82

Non-radioactive

labelling

7. Repeat step 6 with fresh wash solution 2. 8. Rinse the membrane in CDP-Star T M assay buffer at room temperature. 9. Dilute an appropriate amount of CDP-Star T M concentrate with CDPStar assay buffer to give a 1:100 dilution, d Add the membrane to this and incubate at room temperature for at least 5 min. 10. Remove the membrane and drain carefully. Sandwich the membrane between two polyester sheets, using a ruler to squeeze out excess substrate. Avoid contaminating the outside of the polyester sheets with substrate. 11. Secure the sandwich with a piece of tape along each edge and place in a light-proof casette against X-ray film. Intensifying screens are not required. 12. Develop the film after 0.5-1 h. The chemiluminescent signal will continue for several days allowing repeat exposures to optimize the image, e See Appendix 9 for colour detection. b Up to 10 membranes may be processed simultaneously, provided care is taken that they do not stick together. They should be handled individually during transfer steps and during the detection steps they should not contact each other but be processed individually. c More prolonged hybridization and wash times will adversely affect enzyme activity. a Containers exposed to CDP-Star T M should be cleaned thoroughly after use. e The probes may be stripped from the membrane by agitating them for 15 min in 0.1% SDS at 80~ Rinse in 1• SSC prior to rehybridization. a

Ol

Cl

~ o

t

N

~

{~

but nevertheless applications have focused on the (less demanding) detection of PCR-amplified products using coated microtitre plate capture and labelled oligonucleotide probes (Dahl6n et al., 1991 a) or use of labelled PCR primers (Dah16n et al., 1991b). Multiple labelling is recommended for best sensitivity in this format (which has a relatively low surface area). Figure 6.9 shows the labelling reagent; this may be coupled to oligonucleotides bearing amino groups by procedures analogous to that described in Protocol 6.5 (Wallac, 1994; see also Protocol 4.3).

COOH

JOE

N~

COOH

d

o~oCOOH

~

~

6.3.7 Introduction of tags at oligonucleotide amino groups

o

TAMRA

ROX

Figure 6.8 Some fluorophore active esters for labelling amino-derivatized DNA.

6.3.6 Europium chelate labelling at primary amino groups Compared with conventional fluorophores, the DELFIA europium chelates are more sensitive,

Though phosphoramidites are a good route for tag attachment, this has not proved possible for DIG. 3' Tailing is also an option (Section 6.6.1), but chemical linkage is attractive, giving defined conjugates with a simple active ester coupling reaction. NHS reagents for adding DIG, fluorescein and biotin are described in Section 5.4.1 (Figure 5.1), but in addition many other NHS esters are available. For biotin, the reagent with

Labelling of oligonucleotides

83

Protocol 6.5 Labelling-aminolinked oligonucleotides with fluorescein ~. 1. Obtain the amino-oligonucleotide (e.g. amino-linked oligonucleotide, 20-30 nmol) in water (120 lad and add 1 M NaHCO3 buffer, pH 9.0 (60 lal). 2. Prepare a 1% solution of fluorescein isothiocyanate (FITC) in DMF. Add 20 lal of this to the oligonucleotide and react for 3 h at room temperature in the dark. 3. Apply to a Sephadex T M G25 NAP-5 T M column (Pharmacia) equilibrated in PBS, or buffer of choice. Add a further 0.3 ml buffer and allow to soak on. Add 0.4 ml buffer collecting the eluting product. 4. Purify the product on reverse-phase HPLC (see e.g. Appendix 12). If possible, monitor the fluorescein absorbance at 495 nm. Select the principle peak (which should be visibly yellow). 5. Record the absorbance spectrum, noting the A260 and A495. 6. Calculate the fluorescein content by using e495 of 68000. Calculate the fluorescein contribution to the A260 using an extinction coefficient of 62 000. Deduct this contribution from the observed OD to obtain the oligonucleotide contribution and calculate the concentration ( 1 0 D unit = 33 lag). The fluorescein:oligonucleotide ratio should be unity but may be somewhat lower due to uncertainties in the extinction coefficients and approximations made in the calculations. a This procedure can be used with other fluorophore isothiocyanates.

Protocol 6.6 Reaction of amino-linked oligonucleotides with NHS reagents. This procedure may be used with all types of NHS esters, including various fluorescent dyes, biotin and DIG reagents a. 1. Obtain the amino-oligonucleotide (e.g. amino-linked oligonucleotide, ca 50 nmol) in water (100 lal) and add 1 M NaHCO3 buffer, pH 8.5 (50 lal). 2. Prepare a 20 mM solution of NHS ester in DMF or DMSO. Add 20 lal of this to the oligonucleotide and react for 3 h at room temperature. 3. Apply to a Sephadex T M G25 NAP-5 T M column (Pharmacia) equilibrated in PBS, or buffer of choice. Add a further 0.3 ml buffer and allow to soak on. Add 0.4 ml buffer collecting the eluting product. 4. Purify the product on reverse-phase HPLC (see e.g. Appendix 12). a For DIG NHS ester, freshly prepare reagent at 1 mg ml-1 in ethanol and add one volume of this (150 ktl) to the oligonucleotide (Boehringer, 1994).

a C6 spacer (biotin-X-NHS) is r e c o m m e n d e d . A general m e t h o d for using NHS reagents with a m i n o - d e r i v a t i z e d oligonucleotides is given in Protocol 6.6.

/ ~ COOH SCN

k _ COOH / - - COOH N

k--- COOH

Figure 6.9 A europium chelate-labelling reagent (C401-101).

84

Non-radioactive labelling

6.4 LABELLING AT INTRODUCED THIOLS

6.4.1 Incorporation of thiol groups at the 5' end

Thiols may be introduced by means of a phosphoramidite reagent bearing a trityl-protected thiol (Connolly & Rider, 1985; Figure 6.10). The trityl group permits conventional trityl-on HPLC purification. Since the thiol group is not stable for long-term storage, the trityl group is left on and removed when required by treatment with AgNO3. The excess silver is precipitated out with dithiothreitol and the product recovered by centrifugation.

6.4.2 Incorporation of thiol groups at the 3' end

This has been achieved by means of a disulphide-containing spacer linking the 3' nucleotide to the CPG support (Zuckermann et al., 1987). The disulphide is cleaved post-synthesis by dithiothreitol.

6.5 OTHER CHEMICAL APPROACHES Several approaches have been devised for labelling on the phosphodiester oligonucleotide backbone. Though these involve departures from standard phosphoramidite chemistry, they do allow labelling at specific internucleotide sites. Using H-phosphonate chemistry, oxidation in the presence of a primary amine gives a phosphoramidate, allowing the introduction of a label or chemical handle via the amine ( Y a m a n a & Letsinger, 1985; Agrawal & Tang, 1990). Phosphorothioates are generally of interest for antisense work rather than detection, but the sulphur of the phosphorothioate linkage does offer the opportunity to introduce labels by means of thiol-reactive derivatives (Fidanza et al., 1994; see also Section 6.6.2). Unmodified oligonucleotides bearing a 5' phosphate group may be extended using carbodi-imide activation (Chu et al., 1983). For 3' labelling, a wide variety of other functions may be positioned at the 3' end via CPG derivatives analogous to Figure 6.6, structure III. Many such supports are available (e.g. Penninsula).

6.6 ENZYMIC LABELLING 6.4.3 Attachment of labels at introduced thiols

A number of maleimido and iodoacetyl derivatives of fluorophores and tags are available from several sources (see Chapters 4 and 5). Proteins which have been derivatized with thiol-reactive groups (see Section 3.3.2) may be conjugated as described in Protocol 6.2.

6.6.1 Enzymic labelling at the 3' end: tailing

The enzyme terminal deoxynucleotidyl transferase (terminal transferase) has been widely used to add labelled nucleotides to the 3' end (Riley et al., 1986) and, for native oligonucleotides, represents the labelling method of choice. In this way fluorophores, tags and chemical handles may be incorporated. The enzyme offers three main possibilities. (i)

I

DMT- S ~

0/P\ 0 ~CN

Figure 6.10 C6 thiol modifier.

Addition of relatively short tails of the modified nucleotide. (ii) Addition of longer tails containing an unmodified nucleotide with the modified nucleotide randomly incorporated. (iii) Addition of a single nucleotide by means of a dideoxynucleoside triphosphate (ddNTP).

L a b e l l i n g of o l i g o n u c l e o t i d e s Only method (iii) will give a defined product, but has the obvious drawback of only incorporating one label: many applications such as in situ hybridization benefit from a higher signal. Approaches (i) and (ii) give multiple additions but the products will be heterogeneous. Long tails are also sometimes associated with higher non-specific signals. Protocol 6.7 describes a general method for tailing with dATP and D I G - 1 1 - d U T P (H61tke et al., 1992) that may be adapted for the incorporation of tags, labels or chemical handles. It is

Protocol

6.7

85

important not to reduce the concentration of enzyme since this will lead to a greater heterogeneity in the product, with some unreacted oligonucleotide (this is because the enzyme is processive). A kit for 3' tailing with fluorescein is available (Vistra TM, Amersham). For the addition of a single modified nucleotide, Protocol 6.7 may be used substituting 1 mM DIG- 11-ddUTP instead of DIG- 11-dUTP and omitting the dATP. A primary amine may be introduced by means of aminoalkyl dUTP derivatives, and this

3' tailing with DIG.

Solutions

1. 5• labelling buffer: 1 M potassium cacodylate a (NB: toxic), 0.125 rvi Tris-HC1, 1.25 mgm1-1 BSA, pH 6.6. b 2. 25 mM COC12. 3. DIG-11-dUTP: 1 mM in water. 4. dATP: 10 mM in TE (10 mM Tris-HC1, 1 mM EDTA, pH 8.0). 5. Terminal transferase: 50 Uktl- 1 (Boehringer). 6. Glycogen: 20 mg ml- 1 in redistilled water. 7. 0.2 M EDTA, pH 8.0. 8. 4 M LiC1. Method

1. Into a plastic microfuge tube add the following, in order: 4 ktl labelling buffer 4 ktl COC12 solution 1 ktl DIG- 11-dUTP Oligonucleotide to be labelled: up to 100 pmole in up to 9 ktlc 1 ktl dATP 1 ~tl terminal transferase sterile distilled water to make volume up to 20 ktl 2. Incubate for 15 min at 37~ then place on ice. 3. To stop the reaction, add 1 ktl glycogen solution, followed by 1 ktl EDTA. 4. Purify by ethanol precipitation: d add 2.5 ktl 4 M LiC1 and 75 lal prechilled (-20~ ethanol. Mix and place at -70~ for 30 min or -20~ for 2 h. 5. Centrifuge at 12000 g for 15 min at 4~ Remove the supernatant and wash with chilled 70% ethanol. Centrifuge at 12000 g for 5 min and remove supernatanc 6. Dry in vacuo and redissolve in 20 gl sterile redistilled water. Store at -20~ a Due to the toxic nature of cacodylate and the time-consuming preparation method of the buffer (see Eschenfeldt et al., 1987), use of Boehringer buffer (supplied with the enzyme) is suggested. b Phosphate buffers must be avoided (cobalt phosphate will precipitate). c Do not increase oligonucleotide concentration: increase volume to cater for higher quantities. d Or purify on a spin column (e.g. Biorad).

86

Non-radioactive labelling

Connolly, B.A. (1987). The synthesis of oligonucleotides containing a primary amino group at the 5'terminus. Nucl. Acids Res. 15, 3131-3139. Connolly, B.A. and Rider, P. (1985). Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of 6.6.2 Enzymic labelling at the 5' end thiol-specific probes. Nucl. Acids Res. 13, 4485-4502. A useful way of enzymically attaching a formal Cook, A.F., Vuocolo, E. and Brakel, C.L. (1988). thiol group is to introduce a 5' terminal thioSynthesis and hybridization of a series of biotinylated oligonucleotides. Nucl. Acid Res. 16, phosphate group using T4 polynucleotide kinase 4077-4095. and )rS-ATP. Such groups may be reacted with Dahl6n, P.O., Iiti/i, A.J., Skagius, G., Frostell, A., thiol reactive reagents, e.g. iodoacetyl fluoresNunn, M.F. and Kwiatkowski, M. (1991 a). cein (Vistra T M kit, Amersham). Detection of human immunodeficiency virus type I by using the polymerase chain reaction and a time-resolved fluorescence based hybridization assay. J. Clin Microbiol. 29, 798-804. Dahl6n, P.O., Iiti~i, A.J., Mukkala, V-M., Hurskainen, REFERENCES P. and Kwiatkowski, M. (1991b). The use of europium (Eu3§ labelled primers in PCR amplification of specific target DNA. Mol. Cell. Agrawal, S (1994). Functionalization of oligonucleoProbes 5, 143-149. tides with amino groups and attachment of amino specific reporter groups. Meth. Mol. Edman, J.C., Evans-Holm, M.E., Marich, J.E. and Ruth, J.L. (1988). Rapid DNA fingerprinting Biol. 26, 93-120. using alkaline phosphatase-conj ugated Agrawal, S. & Tang, J.-Y. (1990). Site-specific funcoligonucleotides. Nucl. Acids Res. 16, 6235. tionalization of oligonucleotides for non-radioacEschenfeldt, W.H., Puskas, R.S. and Berger, S.L. tive labelling. Tetrahedron Lett. 31, 1543-1546. (1987). Homopolymeric tailing. Meth. Enzymol. Agrawal, S., Christodoulou, C. & Gait, M.J. (1986). 152, 337-442. Efficient methods for attaching non-radioactive labels to the 5'-end of synthetic oligodeoxy- Fidanza, J.A., Ozaki, H. & McLaughlin, L.W. (1994). Functionalization of oligonucleotides by the ribonucleotides. Nucl. Acids Res. 14, 6227-6245. incorporation of thio-specific reporter groups. Alves, A.M., Holland, D. & Edge, M.D. (1989). A Meth. Mol. Biol. 26, 93-120. chemical method of labelling oligodeoxyribonucleotides with biotin: a single step procedure Garman, A.J. (1991). Probes. European patent application 422861. using solid phase methodology. Tetrahedron Giles, A.F., Booth, K.J., Parker, J.R., Garman, A.J., Lett. 30, 3089-3092. Carrick, D.T., Akhavan, H. and Schaap, A.P. Boehringer (1994). The DIG system user's guide for (1990). Rapid, simple, non-isotopic probing of filter hybridization. Boehringer Mannheim Southern blots for DNA fingerprinting. Adv. GmbH. Forens. Haemogen. 3, 40-42. Bronstein, I., Voyta, J.C. and Edwards, B. (1989). A comparison of chemiluminescent and colori- Goodchild, J. (1990). Conjugates of oligonucleotides and modified oligonucleotides: a review of their metric substrates in a hepatitis B virus DNA synthesis and properties. Bioconjugate Chem. 1, hybridization assay. Anal. Biochem. 180, 95-98. 165-187. Brown, T. & Brown, D.J.S. (1991). Modem machineaided methods of oligodeoxyribonucleotide Grzybowski, J., Will, D.W., Randall, R.E., Smith, C.A. and Brown, T. (1993). Synthesis and antisynthesis. In Oligonucleotides: a practical body-mediated detection of oligonucleotides approach (ed. F. Eckstein). IRL Press, Oxford. containing multiple 2,4-dinitrophenyl reporter Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, groups. Nucl. Acids Res. 21, 1705-1712. P.C. and Wolf, D.E. (1988). Detection of nucleic acid hybridization by non-radiative fluorescence H61tke, H-J., Seibl, R., Scmitz, G.G., Walter, T., Rtiger, R., Sagner, G., Burg, J., Mtihlegger, K. resonance energy transfer. Proc. Natl. Acad. Sci. & Kessler, C. (1992). Labeling and detection of USA 85, 8790-8794. nucleic acids. In Nonradioactive labeling and Chollet, A. and Kawashima, E.H. (1985). Biotindetection of biomolecules (ed. C. Kessler). labelled synthetic oligodeoxyribonucleotides: Springer, Berlin. chemical synthesis and uses as hybridization Hultman, T., St~hl, S., Homes, E. and Uhl6n, M. probes. Nucl. Acids Res. 13, 1529-1541. (1989). Direct solid phase sequencing of Chu, B.C.F., Wahl, G.M. and Orgel, L.E. (1983). genomic and plasmid DNA using magnetic beads Derivatization of unprotected oligonucleotides. as solid support. Nucl. Acids Res. 17, 4937-4946. Nucl. Acids Res. 11, 6513-6529. approach is the basis of the TDT-LINK T M kit for alkaline phosphatase attachment (Genosys).

Labelling of oligonucleotides Jablonski, E., Moomaw, E.W., Tullis, R.W. and Ruth, J.L. (1986). Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucl. Acids Res. 14, 6115-6128. Kumar, A., Advani, S., Dawar, H. and Talwar, G.P. (1991). A simple method for introducing a thiol group at the 5'-end of synthetic oligonucleotides. Nucl. Acids Res. 19, 4561. Li, P., Medon, P.P., Skingle, D.C., Lanser, J.A. and Symons, R.H. (1987). Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens. Nucl. Acids Res. 15, 5275-5287. Livak, K.J., Marmaro, J. and Todd, J.A. (1995). Towards fully automated genome-wide polymorphism screening. Nature Genet. 9, 341-342. Mann, J.S., Shibata, Y. and Meehan, T. (1992). Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'phosphate. Bioconjugate Chem. 3, 554-558. Misiura, K., Durrant, I., Evans, M.R. and Gait, M.J. (1990). Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides. Nucl. Acids Res. 18, 4345-4354. Nelson, P.S., Frye, R.A. and Liu, E. (1989a). Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations. Nucl. Acids Res. 18, 7187-7194. Nelson, P.S., Sherman-Gold, R. and Leon, R. (1989b). A new and versatile reagent for incorporating multiple primary aliphatic amines into synthetic oligonucleotides. Nucl. Acids Res. 17, 7179-7186. Neuner, P. (1996). New non-nucleosidic phosphoramidite reagent for solid phase synthesis of biotinylated oligonucleotides. Bioorg. Med. Chem. Lett. 6, 147-152. Pieles, U., Sproat, B.S. and Lamm, G.M. (1990). A protected biotin containing deoxycytidine building block for solid phase synthesis of biotinylated oligonucleotides. Nucl. Acids Res. 18, 4355-4360. Pon, R.T. (1991). A long chain biotin phosphoramidite reagent for the automated synthesis of 5'biotinylated oligonucleotides. Tetrahedron Lett. 32, 1715-1718. Reyes, R.A. and Cockerell, G.L. (1993). Preparation of pure oligonucleotide-alkaline phosphatase conjugates. Nucl. Acids Res. 21, 5532-5533. Riley, L.K., Marshall, M.E. and Coleman, M.S. (1986). A method for biotinylating oligonucleotide probes for use in molecular hybridizations. DNA 5, 333-337. Ruth, J.L. (1991). Oligodeoxynucleotides with reporter groups attached to the base. In Oligonucleotides and analogues: a practical approach (Ed.

87

F. Eckstein), pp. 255-282. Oxford University Press, Oxford. Ruth, J.L. (1994). Oligonucleotide-enzyme conjugates. Meth. Mol. Biol. 26, 93-120. Saiki, R.K., Walsh, P.S., Levenson, C.H. and Erlich, H.A. (1989). Genetic analysis of amplified DNA with immobilised sequence-specific oligonucleotide probes. Proc. Natl. Acad. Sci. USA 86, 6230-6234. Sinha, N.D. and Cook, R.M. (1988). The preparation and application of functionalized synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected aminohexanol and mercaptopropanol or -hexanol. Nucl. Acids. Res. 16, 2659-2669. Telser, J., Cruickshank, K.A., Morrison, L.E. and Netzel, T.L. (1989). Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: comparison of biotin, fluorescein and pyrene labels by thermodynamic and optical spectroscopic methods. J. Am. Chem. Soc. 111, 6966-6976. Urdea, M.S., Warner, B.D., Running, J.A. Stempien, M., Clyne, J. and Horn, T. (1988). A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent, and enzyme-labelled synthetic oligodeoxyribonucleotide probes. Nucl. Acids Res. 16, 4937--4956. Wallac (1994). Eu-labelled oligonucleotides are stable and sensitive as probes and primers. DELFIA Reasearch Systems Application Note, Wallac Oy, Finland. Will, D.W., Pritchard, C.E. and Brown, T. (1991). The synthesis of oligonucleotides that contain 2,4-dinitrophenyl reporter groups. Carbohydrate Res. 216, 315-322. Wisden, W. & Morris, B.J. (eds) (1994). In situ hybridization protocols for the brain. Academic Press, London. Yamana, K. and Letsinger, R.L. (1985). Synthesis and properties of oligonucleotides beating a pendant pyrene group. Nucl. Acids Symp. Ser. 16, 169-173. Zuckermann, R., Corey, D. and Schultz, P. (1987). Efficient methods for the attachment of thiolspecific probes to the 3'-ends of synthetic oligodeoxyribonucleotides. Nucl. Acid Res. 15, 5305-5321.

FURTHER READING Agrawal S. (ed.) (1994). Protocols for oligonucleotide conjugates. Humana Press. Eckstein, F. (ed.) (1991). Oligonucleotides and analogues: a practical approach. IRL Press, Oxford.

CHAPTER

SEVEN

Labelling of long nucleic acid probes

7.1 INTRODUCTION The labelling of nucleic acids has long been dominated by radioisotopes, particularly 32p, and for full-length probes this is still largely the case. This is despite the development of a plethora of non-radioactive labelling methods and detection strategies that have been developed, particularly in the last 10 years or so. Possibly the choice of non-isotopic approaches has been too great, and methods described before all the difficulties have been overcome. Continuity with the past is also a factor, and many researchers have little time to spend on introducing new methodologies. Nevertheless, good, proven alternatives do exist that offer considerable advantages, providing faster results, more stable probes and more convenient protocols. As a result, more researchers are investing in these methods, particularly in the field of in situ hybridization, where exposure times of

weeks and months can be avoided. Given also the increasing pressures to limit use of radioactivity, non-isotopic alternatives are now Leing more extensively used in other hybridization applications. The wide range of labelling methods have been reviewed elsewhere (see e.g. Kessler, 1995). Here the objective is to introduce some of the more commonly used and proven techniques, and to describe what is involved practically in the various labelling schemes.

7.1.1 Labelling strategies There are two major strategies that have been employed for the non-isotopic labelling of long (non-synthetic) DNA probes: (i) enzymic (polymerase-mediated) incorporation of labelled or tagged nucleotides; (ii)chemical modification of the nucleic acid. Enzymic labelling is somewhat unusual in that, strictly speaking, it consists of synthesizing

Labelling of long n u c l e i c a c i d p r o b e s

89

denaturation

denaturation

extension / tag incorporation

labelling

t?

y

denaturation

hybridisation to target

hybridisation to target ta~l

~r

t~g

tag detection step

g Figure 7.1 Direct A and indirect B labelling of DNA. a labelled molecule de novo, using DNA as a template. Both the enzymic and chemical strategies can be further divided into direct approaches, where the label is directly attached, and indirect, where a tag is attached to a probe which is then subsequently revealed after hybridization by an anti-tag signal conjugate (Figure 7.1).

7.2 LABELLING BY ENZYME-MEDIATED INCORPORATION OF MODIFIED NUCLEOTIDES Enzymic labelling has been most widely used and is capable of being the most sensitive. Many nucleoside triphosphates with a wide

range of labels and tags attached to the base unit are commercially available (Table 7.1 and Figure 7.2), and these may be incorporated by all the principal enzymic labelling methods used with radiolabels. These include the random priming method (Feinberg & Vogelstein, 1983), the nick translation method (Rigby et al., 1977) and labelling using PCR (Lo et al., 1988). RNA probes may also be prepared by transcription. What may be incorporated into the nucleic acid by this approach can be divided into direct labels (e.g. fluorophores), tags (biotin, haptens) and chemical 'handles' (for subsequent chemical modification). Provided that the nucleotide analogue is designed with care, polymerase systems are relatively insensitive to the nature of the signal

90

Non-radioactive labelling

Table 7.1 Fluorescent deoxynucleoside triphosphates Reagent

Excitation max. (nm)

Emission max. (nm)

Fluorescein-12-dUTP, -14-dATP

492

520

Tetramethylrhodamine-6-dUTP (TAMRA) Tetramethylrhodamine-12-dUTP (TAMRA) R 110-dUTP R6G-dUTP AMCA-6-dUTP Cy3-dCTP Cy5-dCTP Eu-dCTP

550 550 502 528 318 550 649 340

580 580 530 555 382 570 670 613

Possible sources

Boehringer, Amersham, Molecular Probes, Perkin Elmer Boehringer Molecular Probes, Perkin Elmer Perkin Elmer Perkin Elmer Boehringer Amersham Amersham Wallac

For RNA labelling, ribonucleoside triphosphate analogues are also available for most of the above. Wavelengths given are approximate only. group introduced (though Taq polymerase is but also because of the availability of very senmore discriminating), and many different fluoro- sitive chemiluminescent and fluorogenic subphores, chemilumophores and other groups may strates for this enzyme. be introduced. The key parameter is the incorporation rate, which is a function of the concentration ratio of 7.2.1 Enzymic incorporation of tags the analogue to the parent dNTP and the degree of recognition of the analogues by the polymerase The synthesis of DNA probes containing biotin being used. The higher the incorporation rate, (Langer et al., 1981) was a key event in the the more sensitive the probe will be; however, evolution of non-isotopic labelling of nucleic too high a rate and hybridization will be acids. This work employed a dUTP derivative, adversely affected. There is therefore an opti- containing biotin linked to the uracil base via a mum incorporation rate which, though it varies 4-atom spacer. The length of the spacer group slightly from analogue to analogue, is in the and the base to which it was attached then order of 1 per 30 bases. Because of reduced became a focus of interest. A shorthand develrecognition of the analogue by the polymerase, oped where these analogues became known as it requires concentrations of the modified dNTP biotin-n-dNTP, where N designates the nucleosimilar to that of the native dNTP to achieve side and n indicates the number of atoms in the spacer (ignoring its composition, which does this. vary). Of the biotin analogues investigated, bioIt is not practical to incorporate enzyme labels directly by means of modified nucleoside tin-11-dUTP (Figure 7.2) is probably the most triphosphates (since they are used at typically frequently used, but biotin-14-dATP and biotin0.2 mM) but they may be introduced via a tag 14-dCTP are also used. There are slight preferanti-tag system (see Figure 7.1). Three tags ences depending on the polymerase employed have been widely employed; biotin, DIG and (see subsequent protocols), but dUTP derivafluorescein. Since enzymes provide the most tives are commonly employed for introducing sensitive labels, polymerase-mediated labelling many tags and labels. Different length chains with tags is of central importance. A general have been investigated for different tags and introduction to tags is given in Chapter 6. A labels, and different enzymes. It is hard to discussion of signal enzymes and detection sys- generalize on the relationship between length tems that may be used is given in Chapter 1. As and incorporation rate, but very long spacers for immunoassay, alkaline phosphatase and HRP (>11 atoms) are not necessary for routine dominate, but here alkaline phosphatase has a applications. Of the hapten tags, DIG and fluorescein have clear edge, partly because it is more heat stable,

Labelling of long n u c l e i c acid p r o b e s

o

O

O

O

II

II

o

/'~N~ ~ ` ` ~

HO-P-O- P - 0 OH OH OH II

91

I~1/~~/I~l O~ OJ ~ ~ !

OH

/

OH O O

o

o

o

II

II

II

I

I

HO-P-O-P-O"P'O "--'~O OH OH OH OH

O N

/

OH

o

H.N,~'-N.H 0

0 H

o II

o II

o II

HOP 'O-O-POH -'O-PO O 'O-H -~ H

or

0

/

OH Figure 7.2 Labelled nucleoside triphosphates" (A) DIG- 11-dUTP; (B) fluorescein- 12-dUTP; (C) biotin-11dUTP. been most widely used. These have the potential to detect less than 0.1 pg, i.e. single copy human genes in 1 mg genomic DNA. DIG may be introduced using DIG-11-dUTP and fluorescein with fluorescein-11-dUTP. All three give probes that are stable for many months or years at -20~ Fluorescein-labelled probes have the advantage that they may be detected directly or via enzyme anti-fluorescein conjugates. It is also very useful to assess the degree of labelling of the newly synthesized probe, either by visual inspection with a long-UV light box, or quantitatively with a fluorimeter. In contrast, DIG- and biotin-labelled probes need to be characterized

functionally by spotting dilutions of the probe on a membrane and developing with the chosen signal development system. Fluorescence detection is of course also possible with hapten tags, using a fluorescently labelled anti-tag protein. In fluorescence in situ hybridization (FISH), combinatorial labelling has been exploited for the simultaneous detection of seven different probes (Ried et al., 1992). Overall, of the tags that can be used, DIG and fluorescein are recommended. Potentially, many other hapten tags could be devised (the perfect hapten tag has yet to be developed), but the DIG and fluorescein systems are well supported with many reagents and kits available.

92

N o n - r a d i o a c t i v e labelling

7.2.2 Tag detection

and dioxetane substrates is straightforward and gives good results. For a fuller discussion of enzyme labels, see Section 1.7.

Biotin and hapten tags may be detected after hybridization with the appropriate anti-tag conjugates. For biotin, streptavidin conjugates should be employed, rather than avidin, since these give much better backgrounds. Even so, 7.2.3 Enzymic incorporation of labels backgrounds can often be more of a problem with the biotin system compared with DIG and Labelling with fluorescent groups is now a stanfluorescein, especially with dioxetane chemiludard technique for FISH techniques. Apart from minescent substrates. Furthermore, with tissues fluorescein (above), many other labels, in parand colony lifts, endogenous biotin can give rise ticular fluorescent labels, can be incorporated to erroneous results (Wang et al., 1993). DIG by means of nucleoside triphosphate analogues can give more sensitive detection than biotin in (Ballard & Ward, 1993). Some of the more in situ hybridizations (McQuaid et al., 1995). important derivatives are listed in Table 7.1. The success of hapten tags depends crucially As well as the more conventional fluoroon the quality and nature of the antibody used, phores, the cyanine dyes Cy-3 and Cy-5 may but good antibodies proven for this application be introduced by means of dUTP analogues are available. In general, conjugates of monoand optimized conditions for nick translation clonal antibodies give better backgrounds than and PCR labelling have been reported (Yu et those of polyclonal antibodies, especially for in al., 1994). Europium chelate dCTP derivatives situ hybridizations. Biotin can also be regarded are also available (Wallac). as a hapten: anti-biotin antibodies are available, When the labelled nucleoside triphosphate of which may be conjugated to the signal of choice. interest cannot be obtained commercially, In general, anti-biotin antibodies do not offer recourse may be made to analogues bearing significant advantages, although good results in chemical 'handles'. Amino-derivatized analoin situ hybridizations have been reported gues are particularly useful (Figure 7.3). These (McQuaid & Allan, 1992). may be reacted with, for example, an NHS ester For the most sensitive filter hybridization of the label, and then incorporated into the applications, the use of alkaline phosphatase nucleic acid (Yu et al., 1994), or, alternatively, with dioxetane substrates (see Section 1.7.2.4) first incorporated into the nucleic acid and then is ideal, offering excellent sensitivities with labelled (Folsom et al., 1989). Aminohexyl derishort development times. The only major convatives of ATP and dATP are available (Life cern with this system is that care is needed to Technologies). avoid unsightly backgrounds ('spots and clouds') on longer exposures. Use of the newer AMPPD derivatives (CSPD and CDP-Star) with the appropriate hybridization and detection con- 7.2.4 Enzymic labelling" practical points ditions should however minimize or eliminate Commercial kits are available for many of the these problems. The precipitating chromogenic substrate labelling methods described below. They are an reagent BCIP/NBT may also be used for both filter and in situ hybridizations. See Chapter 1 HN ~ NH2 for a discussion on signal enzyme substrates. N "J'~"N BCIP/NBT is less sensitive than the dioxetane 0 0 0 substrates, but more seriously, stripping and N N II ii ii reprobing is very difficult (heating the filter in H O -OP - O 'O OH P - O 'HOH P'O-~ DMF will remove the coloured enzyme product, OH but this is not recommended). In contrast, stripping and reprobing with alkaline phosphatase Figure 7.3 6-Aminohexyl-dATP.

Labelling of long n u c l e i c acid p r o b e s excellent way to start and give the best chance of producing usable probes consistently. Also, new improvements in methodology may be made available to the user by this route. However, kits can be expensive and give the user less flexibility in scale and choice of label used. Premixed nucleoside triphosphate cocktails are also commercially available (e.g. Boehringer), covering DIG, fluorescein and biotin labelling. A method for the hybridization and chemiluminescent detection of DIG-labelled probes is given in Protocol 7.1 and may be used as a model for other tagged DNA probes. A number of variant hybridization solutions have been used; for example, a high-SDS-containing buffer gives very low backgrounds (Engler-Blum et al., 1993). For best quality results, protocols need to be followed closely. Buffers should be fresh or properly stored, and times and incubation temperatures followed exactly. Maintain normal precautions against nucleases (sterile buffers, use of EDTA, general cleanliness). Most labelling reactions should be carried out in polypropylene tubes (e.g. Eppendorf) and screwtop tubes are preferred. After operations that agitate the sample (mixing, boiling etc.), the tube contents should be spun briefly to recover the contents at the bottom of the tube. The membrane blocking agent is a crucial ingredient for low backgrounds and this should be prepared carefully: it needs to be totally in solution. Casein (BDH Hammerstan grade) is a suitable agent, though some manufacturers claim superior results can be obtained with their proprietary formulations. Hybridizations may conveniently be carried out in sandwich boxes or sealed plastic bags. Adequate buffer should be used to ensure that the membranes are always covered and gentle agitation should be used to prevent the membranes sticking. Avoid trapping air under the membranes and do not allow them to dry out at any stage. Handle the membranes carefully with plastic forceps and gloved hands.

dNTP (typically a dUTP derivative; see Figure 7.2); thus the key parameter is concentration of this derivative and its parent dNTP. For dUTP derivatives, a ratio of 35% modified dUTP to 65% dTTP is successful. Others may be slightly different, for example Eu-dCTP has been used at 25-33%. Incorporation of modified nucleosides can slow down the labelling and more prolonged incubations are necessary: overnight is usually suitable. Protocol 7.2 gives a scheme for DIG labelling. Other labels and tags may be incorporated by analogous procedures.

7.2.6 Nick translation labelling of DNA Nick translation (Rigby et al., 1977) can be readily adapted to introduce labelled nucleotide analogues (Langer et al., 1981). For most purposes, random primer labelling is to be preferred, since it gives probes of higher activity and because nick translation requires careful optimization of the ratio of nicking and polymerase activities. Standard protocols may be adapted by the inclusion of the modified dNTP. For DIG, the labelling mix should contain 40 laM DIG-11dUTP and 10 laM of the other dNTPs (Boehringer, 1994). For biotin, 10 ~tM biotin- 14-dATP, 10 ~M dATP and 20 laM of the remaining dNTPs has been used (Rashtchian & Mackey, 1992). Cyanine dye-labelled dUTP may be used at equimolar quantities at 20 ~tM (Yu et al., 1994). Characterization of probes may be achieved as described in the random primer labelling (Protocol 7.2) or by fluorescence where possible.

7.2.7 Labelling of DNA using PCR The PCR (Saiki et al., 1988) provides a useful way of making probes (Schowalter & Sommer, 1989), giving the following advantages. (i)

7.2.5 Random primer labelling of DNA Incorporation of modified nucleotides requires simple substitution of a suitably modified

93

Very little template is required; genomic DNA may be used as a template. (ii) Probes produced are of defined length. (iii) Consistent labelling. (iv) The ability to cope with a wide range of input DNA concentrations.

94

Non-radioactive labelling

Protocol 7.1 Hybridization and chemiluminescent detection of DIG-labelled probes. Hybridization Solutions 1. 20• SSC: 0.3 M sodium citrate, 3 M NaC1, pH 7.0. 2. Hybridization solution: 5• SSC containing 2% blocking reagent (Boehringer), 0.1% N-lauroyl sarcosine, Na salt, 0.02% SDS. 3. Hybridization/formamide soluation: above solution diluted 1:1 with formamide. Method All hybridization operations are conveniently carried out in sealed plastic bags in waterbaths. 1. Prehybridize the membrane (nylon, Boehringer) by incubating it at 68~ for 1 h, using 20 ml hybridization/formamide solution per 100 cm 2 of membrane. 2. Denature the probe by heating at 96~ for 10 min (a PCR cycler block is convenient), followed by 30 min in an ice/salt bath. 3. Hybridize overnight with ca 25 ng probe (freshly denatured) in 2.5 ml hybridization/formamide solution per 100 c m 2 membrane at 42~ 4. Wash the membrane twice, 5 min per wash, with 2 x SSC containing 0.1% SDS (at least 50 ml per 100 c m 2 membrane) at room temperature. 5. Wash twice, 15 min per wash, with 0.1 • SSC containing 0.1% SDS at 68~ using prewarmed solution. 6. Membranes may be used for detection directly or air-dried for subsequent detection.

Detection with CPSD TMa Solutions 1. CSPD chemiluminescent substrate (Boehringer). 2. Buffer 1:0.1 M maleic acid, 0.15 M NaC1 adjusted to pH 7.5 with NaOH. 3. Washing buffer: to the above maleic acid buffer, add Tween 20 to 0.3%. 4. Blocking stock solution (10X): 10% blocking reagent (Boehringer) in maleic acid buffer. Dissolve reagent by autoclaving and store at 4~ 5. Buffer 2: dilute blocking stock solution 1:10 in maleic acid buffer. 6. Buffer 3:0.1 M Tris-HC1, 0.1 M NaC1, 50 mM MgC12, pH 9.5. Method All detection steps are performed in sealed plastic bags, resealed after each addition. Ensure that all air bubbles are squeezed out at each stage. During incubations, the bags are gently rocked (e.g. Denley A600 rocker). 1. Rinse the membranes for 1-5 min in washing buffer. 2. Incubate for 30 min in buffer 2 (100 ml). 3. Dilute the anti-DIG alkaline phosphatase conjugate to 75 mUm1-1 (1:10000) in buffer 2. 4. Incubate membrane for 30 min in above diluted conjugate. 5. Wash twice, 15 min per wash, in washing solution (100 ml). 6. Equilibrate for 2-5 min in buffer 3. 7. Dilute CPSD T M (25 mM) 1:100 in buffer 3. Add the diluted CPSD T M to the membrane and immediately expose to X-ray film. The optimum exposure must be determined emperically: 2 h is a suggested starting point. The membrane may be re-exposed. a

See Appendix 9 for colour detection.

Labelling of long nucleic acid probes

95

Protocol 7.2 Random primer labelling with DIG (Boehringer, 1994). Solutions

1. TE buffer: 10 mM Tris-HC1, 1 mM EDTA, pH 8.0. 2. Hexanucleotide mix: 6 2 . 5 0 D units ml -~ (2 mg m1-1) random hexanucleotides in 0.5 M Tris-HC1, 100 mM MgC12, pH 7.2 containing 1 mM dithioerythritol, 2 mgm1-1 BSA. 3. dNTP mix: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM DIG-11-dUTP, pH 7.5 (Boehringer). 4. DNA polymerase: 2 units ~tl-~ Klenow enzyme, large fragment, labelling grade, E. coli (Boehringer). 5. 0.2 M EDTA, pH 8.0. 6. 4 M LiC1. 7. DNA dilution buffer: 50 lagml-~ herring sperm DNA in 10 mM TrisHC1, 1 mM EDTA, pH 8.0. 8. Blocking buffer concentrate: dissolve blocking reagent (Boehringer) at 10% in buffer 1. Shake and heat to dissolve, but do not boil. Autoclave and store at -20~ or 4~ 9. Buffer 1:0.1 M maleic acid, 0.15 M NaC1, pH 7.5. 10. Buffer 2: blocking reagent concentrate, diluted 1:10 in buffer 1 (final concentration is 1:100). 11. Buffer 3:0.1 M Tris-HC1, 0.1 M NaC1, 50 mM MgC12, pH 9.5. Method

1. Into a sterile microfuge tube on ice, add the following in the order specified: (i) DNA templatea' b (10 ng to 3 ~tg) in up to 15 ~tl; (ii) 2 ~tl hexanucleotide mixC; (iii) 2 ~tl dNTP mix; (iv) sterile distilled water to take volume to 19 ~tl; (v) 1 ~tl DNA polymerase. 2. Incubate the reaction at 37~ for 1 h. Prolonged incubation times, up to 20 h, will increase the yield of labelled probe by 2- to 4-fold. Terminate the reaction by addition of 2 ~tl of EDTA. 3. Add 1/10th the volume of 4 M LiC1 and 2.5-3.0 volumes of cold ethanol (chilled at -20~ Mix well and incubate at - 7 0 ~ for 30 min. 4. Spin in a microcentrifuge at 13000 g for 15 min at 4~ Decant off the supernatant. 5. Add 100 ~tl chilled ethanol and repeat step 3. 6. Dry the pellet and dissolve in 50 ~tl TE buffer. The probe should be stored at -20~ preferably aliquotted. The probe should be functionally characterized, as follows. 7. Prepare 10-fold dilutions of the control DIG-DNA (Boehringer) in DNA dilution buffer, from 10 ngml-1 to 0.1 pg ml-~. Likewise prepare 10-fold dilutions of the experimental probe to be characterized. 8. Spot 1 ~tl of each onto a positively charged nylon filter, the dilutions of the probe running parallel to the control. Fix by cross-linking with UV light. 9. Wash the membrane in buffer 1. 10. Incubate the membrane in buffer 2 for 30 min. 11. Dilute the anti-DIG alkaline phosphatase 1:5000 in buffer 2 and incubate the membrane in this for 30 min, ensuring the membrane is completely covered. 12. Wash the membrane twice in buffer 1. 13. Incubate the membrane in buffer 3 for 2 min. This activates the alkaline phosphatase.

96

Non-radioactive labelling

14. The membrane can now be developed in the substrate of choice, for example BCIP/NBT (see Appendix 9) or CPSD (see Protocol 7.1). 15. The functional yield of probe may be estimated by eye by comparing the intensities of the probe spots with the control spots. " If low melting point agarose is present, the DNA should be isolated by gel filtration rather than ethanol precipitation. b Labelling efficiency depends greatly on template purity. Purification by phenol chloroform extraction is suggested. Template DNA should be heat-denatured before labelling (100~ for 10 min, place immediately on ice). Circular plasmid DNA should be digested to make linear, followed by denaturation. c High and consistent yields are reported using a proprietary labelling mixture, 'High Prime', comprising premixed dNTPs and a 6-mer oligonucleotide random primer mix (Boehringer).

The method generally works best for shorter PCR products, ideally around 0.5-1 kb. A good alternative approach for making larger amounts of probe is to carry out a normal PCR reaction and then label the PCR product using the random primer method. This gives probes of about 0.5 kb which is an ideal length for hybridization.

way of visualizing the products when run out on gels. All that is required is to add some fluorescent dUTP derivatives into the PCR reaction. In this way, many more fluorophores can be incorporated into the product than the alternative approach of using fluorescent primers.

7.2.7.2 PCR labelling with modified primers 7.2.7.1 PCR labelling with nucleoside triphosphate analogues As with other polymerase-mediated reactions, nucleotides substituted with tags such as biotin (Lo et al., 1988) or DIG (Lion & Haas, 1990; Lanzillo, 1991) may be incorporated. Protocol 7.3 describes a PCR labelling procedure for introducing DIG (based on Lion & Haas (1990) and Tsai et al. (1995)). In principle, other tags may be introduced in the same way. However, Taq polymerase is more particular than other polymerases and reduced yields may result in some cases. For biotin labelling, biotin14-dATP has been proposed (Rashtchian & Mackey, 1992): this should be used at 40% biotin-14-dATP:60% ATP, and with a prolonged extension time in the cycle (3 min at 72~ Biotin-21-dUTP has also been employed (Finckh et al., 1991); these workers also prepared single-stranded product by employing a second round of PCR with only one primer present. PCR probes may be prepared also from an RNA template (Vil~ek & Herring, 1994). Fluorescent labels are easy to incorporate by PCR. With this labelling approach, fluorescently labelled PCR products may be used as probes, but such labelling is useful purely as a simple

The ease with which oligonucleotides may be derivatized or labelled (Chapter 7) allows a wide range of options for producing labelled PCR products via modified primers. The use of fluorescent primers is now well established, but many other options are possible. An interesting example is the use of 5' amino-linked primers to give an amino-derivatized PCR product that may be conjugated to a protein (Joerger et al., 1995). PCR with one tag (for solid-phase capture) on the primer and incorporation of a different tag (for detection) during amplification has also been explored (HolmstrCm et al., 1993).

7.2.8 Labelling of RNA RNA probes have the advantages of being single stranded and that the DNA-RNA hybrid formed is of high stability, leading to high-sensitivity detection, down to 0.1 pg homologous DNA. Conventionally they are prepared from DNA cloned into a suitable transcription vector containing a phage promoter such as SP6 or T7, using the appropriate RNA polymerase. Once cloned, the run-off transcription of RNA probes is a rapid and efficient process, giving probes of defined length. RNA probes may be reduced in

L a b e l l i n g of long n u c l e i c acid p r o b e s

Protocol 7.3 Preparation

of DIG-labelled

07

probes by

PCR. Solutions 1. 10• dNTP mix: 2 mM dATP, 2 mM dCTP, 2 mM dGTP, 1.3 mM dTTP, 0.7 mM DIG- 11-dUTP (Boehringer). 2. 10 X PCR buffer: 100 mM Tris-HC1, 500 mM KC1, 15 mM MgCI2, 0.1% gelatin, pH 8.3. 3. Taq polymerase 2.5 U ~tl-1. 4. Sterile distilled water. Method 1. Into a polypropylene Eppendorf vial, place the following: (i) 5 ~tl 10• PCR buffer; (ii) 5 ~tl 10x dNTP mix; (iii) 50 pmol of each primer; (iv) DNA template (up to 1 [tg); (v) water to make volume to 49 ~tl; (vi) 1 ~tl Taq polymerase. 2. Overlay with mineral oil and place in thermal cycler. 3. Programme the temperature cycler as follows a, b: (i) Initial denaturation: 95~ for 90 s. (ii) Cycle: denaturation, 95~ for 30 s; annealing, 55~ for 30 s; extension, 72~ for 60 s. (iii) Number of cycles: 35. 4. Analyse by agarose gel electrophoresis. The product will run somewhat slower than an equivalent unlabelled product.

a Annealing conditions should be 5-10~ below the Tm of the primer. This will depend on primer sequences and should be adjusted accordingly. The formula 4 X (G+C) + 2 • (A+T) - 3 may be used to derive the optimum annealing temperature or the reader is referred to texts on PCR (e.g. Newton, 1995). b When using genomic DNA or other complex DNA as template, a 'hot start', i.e. adding the enzyme to the mix after the initial denaturation step, is recommended to improve specificity. size for in situ work by controlled alkaline hydrolysis (Boehringer, 1994). Biotin- or hapten-tagged NTP derivatives may be incorporated in this reaction (H61tke & Kessler, 1990). For biotin labelling, biotin-14-CTP has been used (Rashtchian & Mackey, 1992). Protocol 7.4 gives a procedure for making DIG-labelled RNA probes (Boehringer, 1994). It may be adapted to incorporate NTPs bearing other tags or labels.

7.3 L A B E L L I N G BY C H E M I C A L MODIFICATION

7.3.1 The Renz/ECL method This is a direct labelling strategy (PollardKnight et al., 1990) where the DNA probe is

labelled with HRP. Kits and reagents are available from Amersham International. HRP is not a very heat stable enzyme and hence a key feature of this approach is the hybridization step which has been modified to maintain HRP activity. In sensitivity, the method does not match some of the enzymic approaches and it is not well suited, for example, to single-copy human gene detection. It is however an easy labelling method and adequate for lower sensitivity applications such as plaque lifts and colony hybridizations. It is not suitable for in situ hybridization. The labelling itself (Figure 7.4 and Protocol 7.5) is based upon the electrostatic interaction between the (negatively charged) DNA probe and a conjugate of HRP and (positively charged) polyethyleneimine conjugate (Renz & Kurz, 1984). This complex is then made covalent by treatment with glutaraldehyde. The hybridization takes place in a urea-containing buffer at

08

Non-radioactive

labelling

Protocol 7 . 4 P r e p a r a t i o n o f D I G - l a b e l l e d

RNA

(Boehringer, 1994). Solutions a

All buffers should be treated with DEPC to inactivate ribonucleases. 1. 10• transcription buffer: 0.4 M Tris-HC1, 0.1 M NaC1, pH 8.0, containing 60 mM MgC12, 100 mM dithiothreitol, 20 mM spermidine and 1 U ~tl-~ RNAase inhibitor. 2. 10X NTP mix: 10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM DIG-11-UTP in Tris-HC1, pH 7.5. 3. DNAase I: 10 U ~tl- l DNAaseI (RNAase-free). 4. RNAase inhibitor: 20 U ~tl-~. 5. RNA polymerase: SP6, T7 or T3 RNA polymerase, 20 Ulal -~. 6. Diethylpyrocarbonate (DEPC)-treated water: sterile water treated with 0.1% diethylpyrocarbonate. 7. 200 mM EDTA, pH 8.0. 8. 4 M LiC1, or 3 M sodium acetate (not ammonium acetate). Method

The template DNA must first be linearized with a restriction enzyme cutting downstream of the cloned insert. 1. To a sterile microfuge tube on ice add the following in the order specified: (i) 1 ~tg purified template DNA; (ii) 2 ~1 10• NTP mix; (iii) 2 lal 10X transcription buffer; (iv) DEPC-treated water to 18 gl; (v) 2 ~1 RNA polymerase. 2. Mix gently and centrifuge briefly. Incubate at 37~ for 2 h. 3. Stop the reaction with 2 ~tl EDTA. 4. Precipitate the RNA with 1/10th volume of LiC1 and 2.5-3.0 volumes of chilled ethanol (-20~ Mix and incubate at -70~ for 30 min. or - 20~ overnight. 5. Centrifuge at 13000 g for 15 min in a microcentrifuge at 4~ 6. Decant the supernatant and wash the pellet with 100 ~tl chilled ethanol. Centrifuge as above and decant off the supernatant. 7. Dry the pellet and resuspend in 100 ~tl DEPC-treated water. Use immediately or store a t - 2 0 ~ 8. The probe may be analysed by agarose gel electrophoresis with ethidium bromide detection. Labelling efficiency may be checked by spotting dilutions b onto nylon filters, as described in Protocol 7.2. At the end of the transcription reaction, template DNA may be digested. Add 2 ~tl DNAase I and incubate at 37~ for 15 min. However, since RNA produced greatly exceeds the DNA template, this step is optional. b Use control DIG-RNA (Boehringer) and dilution buffer: DEPC-treated water, 20X SSC and formaldehyde, 5 : 3 : 2.

a

42~ the stringency being adjusted in the posthybridization wash steps by adjusting the salt concentration but keeping the temperature at 42~ The final development of the signal is by means of the enhanced chemiluminesce (ECL) reaction involving luminol as substrate (see Chapter 1).

7.3.2 Sulphonation (the ChemiprobeT M system) The labelling reaction in this (indirect) labelling system (Nur et al., 1989) is the creation of an antigenic group by a sulphonation reaction at cytosine residues, with post-hybridization detection of the sulphonated DNA with a specific

L a b e l l i n g of long n u c l e i c a c i d p r o b e s Double-strandedDNA probe

99

, I Boil, snap cool

Negatively-chargeddenatured probe

+++++ ++ +8+ +8:>+ +

\ /T

++

DNA labelling reagentpositively-chargedcomplexes of peroxidase

V

_

Peroxidaseloosely bound to probe

-+8:>

-

I Add glutaraldehyde

c9

&5

cP

Labelled probe - peroxidase

complexes covalently linked to

DNA

I Hybridization Probe DNA boundto target

Detection reagent 1

Detection reagent2

O~+ H20

Lum~zed product Enhancer Light

Results on film

Figure 7.4 ECL labelling scheme. Figure supplied courtesy of Amersham International. antibody conjugated to the signal of choice, generally alkaline phosphatase. Several workers have achieved good sensitivity with this method (e.g. Lebacq et al., 1988) but this method is not widely used. Reagents are available from Orgenics. With a diamine present, the reaction of cytosine with sodium bisulphite can be used to introduce a primary amine reactive group (Viscidi et al., 1986). This amine can in principle then be the attachment point for a wide variety of tags and labels, though this labelling route approach has not been pursued extensively. It has been

used to introduce europium chelate labels (Hurskainen et al., 1991; L6vgren et al., 1995). Extensive modifications of this type can impair hybridization efficiency; for europium chelates a substitution rate of 4-8 per 100 bases was found to be optimal.

7.3.3 Photolabelling methods Aryl azido compounds have been widely used for cross-linking studies with proteins and nucleic acids. Activation with UV light (ca

100

Non-radioactive labelling

Protocol 7.5 Renz/ECL labelling of DNA a (Amersham, 1993). 1. Dilute the DNA to 10 ngml-~ in sterile distilled water (10 lal).b The salt concentration should not be less than 10 mM. Denature in a boiling water bath for 5 min, then cool rapidly on ice. 2. Add 10 pl DNA labelling reagent (HRP-polyethyleneimine, Amersham) and mix thoroughly. 3. Add 10 pl glutaraldehyde solution (Amersham), mix and incubate at 37~ for 20 min. 4. Use probe within 15 min, or store in 50% glycerol at -20~ (up to 6 months).

Renz/ECL probe hybridization Reagents 1. Hybridization buffer and blocking agent (Amersham). 2. Wash buffer: 0.5• SSC containing 6 M urea and 0.4% SDS. Store at 4~ for up to 3 months. Method 1. Add NaC1 to the hybridization buffer to the desired concentration: 0.5 M is generally suitable but this may require adjustment for some probes. 2. Add blocking reagent to 5% and suspend evenly. Mix at room temperature for 1 h, then heat at 42~ for 0.5-1 h, until dissolved. 3. Prehybridize the filter in a suitable volume of the above solution for at least 15 min at 42~ with gentle agitation. 4. Add probe to the prehybridization buffer, withdrawing a portion of the buffer to dilute the probe. Hybridize overnight at 42~ with gentle agitation. For high concentration targets, shorter hybridization times (1-4 h) are recommended. 5. Wash in wash buffer for 20 min at 42~ The stringency may be adjusted by varying the SSC concentration (not the temperature). 6. Repeat this wash step. 7. Wash the membrane twice in 2• SSC at room temperature for 5 min per wash. The membrane may be kept in this buffer for up to 30 min. 8. Proceed to the ECL detection step. This is described in Appendix 9. c If more probe is required, increase volumes in proportion (but not concentrations). b RNA probes may also be labelled by this method. c Do not allow filters to dry out. a

320 nm) causes generation of highly reactive aryl nitrenes which are able to insert into any nearby bond. The reaction is characterized by its almost complete lack of selectivity and low labelling efficiency. Nevertheless, this approach did provide a convenient early method for labelling, particularly with biotin (Forster et al., 1985), and photoreactive labelling reagents for biotin (photobiotin) and DIG are available. Nitro analogues have also been described which employ longer wavelength excitation and thereby minimize DNA damage. Photolabelling is not widely used for preparing labelled probes but can be used for preparing

labelled molecular weight size ladders, where the lower incorporation rate can be tuned to give bands of similar intensities as those of sample bands.

REFERENCES Amersham (1993). ECL direct nucleic acid labelling and detection systems. Amersham International. Ballard, S.G. & Ward, D.C. (1993). Fluorescence in situ hybridization using digital imaging microscopy. J. Histochem. Cytochem. 41, 1755-1759.

Labelling of long nucleic acid probes Boehringer (1994). The DIG system user's guide for filter hybridization. Boehringer Mannheim GmbH. Engler-Blum, G., Meier, M., Frank, J. & Mtiller, G.A. (1993). Reduction of background problems in nonradioactive northern and southern blot analyses enables higher sensitivity than 32p-based hybridizations. Anal. Biochem. 210, 235-244. Feinberg, A.P. and Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. Finckh, U., Lingenfelter, P.A. and Myerson, D. (1991). Producing single-stranded DNA probes with Taq DNA polymerase: a high yield protocol. Biotechniques 10, 35-39. Folsom, V., Hunkeler, M.J., Haces, A. and Harding, J.D. (1989). Detection of DNA targets with biotinylated and fluoresceinylated RNA probes. Anal. Biochem. 182, 309-314. Forster, A.C., McInnes, J.L., Skingle, D.C. and Symons, R.H. (1985). Nonradioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucl. Acids Res. 13, 745-761. HolmstrCm, K., Rossen, L. and Rasmussen, O.F. (1993). A highly sensitive and fast nonradioactive method for detection of polymerase chain reaction products. Anal. Biochem. 209, 278-283. H61tke, H-J. and Kessler, C. (1990). Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucl. Acids Res. 18, 5843-5851. Hurskainen, P., Dahl6n, P., Ylikoski, J., Kwiatkowski, M., Siitari, H. and L6vgren, T. (1991). Preparation of europium labeled DNA probes and their properties. Nucl. Acid. Res. 19, 1057-1061. Joerger, R.D., Truby, T.M., Hendrickson, E.R., Young, R.M. and Ebersole, R.C. (1995). Analyte detection with DNA-labelled antibodies and polymerase chain reaction. Clin. Chem. 41, 1371-1377. Kessler, C. (1995). Methods for nonradioactive labeling of nucleic acids. In Nonradioactive probing, blotting, sequencing (ed. L.J. Kricka), pp. 41-109. Academic Press, London. Kricka, L.J. (ed.) (1995). Non-isotopic probing, blotting, sequencing. Academic Press, London. Langer, P., Waldrop, A. and Ward, D.S. (1981). Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637. Lanzillo, J.L. (1991). Chemiluminescent nucleic acid detection with digoxigenin-labeled probes: a model system with probes for angiotensin converting enzyme which detect less than one attomole of target DNA. Anal. Biochem. 194, 45-53. Lebacq, P., Squalli, D., Duchenne, M., Pouletty, P. and Joannes, M. (1988). A new sensitive non-isotopic

101

method using sulfonated probes to detect picogram quantities of specific DNA sequences on blot hybridization. J. Biochem. Biophys. Meth. 15, 255-266. Lion, T. and Haas, O.A. (1990). Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction. Anal. Biochem. 193, 335-337. Lo, Y.M.D., Mehal, W.Z. and Fleming, K.A. (1988). Rapid production of vector-free biotinylated probes using the polymerase chain reaction. Nucl. Acid Res. 16, 8719. L6vgren, T., Hurskainen, P. & Dahl6n, P. (1995). Detection of lanthanide chelates by timeresolved fluorescence. In Nonisotopic probing, blotting, sequencing (ed. L.J. Kricka), pp. 165183. Academic Press, London. McQuaid, S. and Allan, G.M (1992). Detection protocols for biotinylated probes: optimization using multistep techniques. J. Histochem. Cytochem. 40, 569-574. McQuaid, S., McMahon, J. and Allan, G.M. (1995). A comparison of digoxigenin and biotin labelled DNA and RNA probes for in situ hybridization. Biotechn. Histochem. 70, 147-154. Newton, C.R. (ed.) (1995). PCR: essential data. John Wiley, Chichester. Nur, I., Reinhartz, A., Hyman, H.C., Razin, S. and Herzberg, M. (1989). ChemiProbe, a non-radioactive system for labeling nucleic a c i d - principles and applications. Ann. Biol. Clin. 47, 601-606. Pollard-Knight, D., Read, C.A., Downes, M.J., Howard, L.A., Leadbetter, M.R., Pheby, S.A., McNaughton, E., Syms, A. and Brady, M.A.W. (1990). Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase. Anal. Biochem. 185, 84-89. Rashtchian, A & Mackey, J. (1992). The biotin system: labeling and detection of nucleic acids. In Non-radioactive labelling and detection of biomolecules (ed. C. Kessler), pp. 70-90. Springer,

Berlin. Renz, M. and Kurz, C. (1984). A colorimetric method for DNA hybridization. Nucl. Acid Res. 12, 3435-3444. Ried, T., Baldini, A., Rand, T.C. and Ward, D.C. (1992). Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 1388-1392. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA-polymerase I. J. Mol. Biol. 113, 237-251. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988). Primer-directed enzymatic

102

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labelling

amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Schowalter, D.B. and Sommer, S.S. (1989). The generation of radiolabelled DNA and RNA probes with polymerase chain reaction. Anal. Biochem. 177, 90-94. Tsai, S-T., Jin, Y-T. and Wu, T-C. (1995). Synthesis of PCR-derived, digoxigenin-labeled DNA probes for in situ detection of Epstein-Barr early RNAs in Epstein-Barr virus-infected cells. J. Virol. Meth. 54, 67-74. Vil(:ek, S. and Herring, A. (1994). A single-tube method for the preparation of digoxigeninlabeled probe from RNA by PCR. Biotechniques 16, 1020. Viscidi, R.P., Connelly, C.A. and Yolken, R.H. (1986). Novel chemical method for the preparation of nucleic acids for nonisotopic hybridization. J. Clin. Microbiol. 23, 311-317. Wang, D., Waye, M.M.Y., Taricani, M., Bucking-

ham, K. and Sandham, H.J. (1993). Biotin containing protein as a cause of false positive clones in gene probing with streptavidin/biotin. Biotechniques 14, 209-211. Yu, H., Chao, J., Patek, D., Mujumdar, R., Mujumdar, S. and Waggoner, A.S. (1994). Cyanine dye dUTP analogs for enzymatic labeling of DNA probes. Nucl. Acids Res. 22, 3226-3232.

FURTHER READING Boehringer (1994). The DIG system user's guide for filter hybridization. Boehringer Mannheim GmbH. Kessler, C. (ed.) (1992). Non-radioactive labelling and detection of biomolecules. Springer, Berlin. Kricka, L.J. (ed.) (1995). Nonisotopic probing, blotting, sequencing. Academic Press, London.

CHAPTER

EIGHT

The labelling of small biomolecules

8.1 INTRODUCTION This chapter is intended to cover low molecular weight biomolecules (< ca 1000 Da), other than small peptides and nucleic acids. So here we include steroids, drugs, metabolites, hormones and other small molecules. Clearly, to describe labelling schemes comprehensively, the diversity of this 'catch-all' category poses a major problem, making it difficult to generalize. Also, particularly for attaching some fluorophores and tags, there may be a requirement for substantial organic chemistry, which takes us outside the scope of this book. What follows is some general considerations which apply to many molecules, together with some illustrative examples. Most of the chemistry is based on that described in earlier chapters, particularly Chapters 2-5.

8.2 LABELLING WITH ENZYMES AND OTHER PROTEIN LABELS

8.2.1 Introduction

The concept of labelling small molecules with a large protein such as an enzyme may seem ambitious at first sight. Yet it is possible, as is illustrated by the success of competition enzyme immunoassays for small molecules (haptens). The size of the label is much less important than where it is positioned, and provided that the site of attachment on the small molecule is chosen with care, and an adequate spacer group included, there is no reason why a successful conjugate should not result. The field of coupling small molecules to proteins is dominated by the widespread need to prepare conjugates for immunization purposes. This field has been reviewed (Erlanger, 1980;

104

Non-radioactive labelling

Tijssen, 1985; Brinkley, 1992), and the hapten carrier conjugation literature is a possible source of ideas for methods of derivatizing the molecule of interest. Indeed, it is possible that for the molecule of interest, an antibody to it will have been raised, and information on the derivatization method employed will be of value in designing a protein label conjugate. This information may be in the primary literature or, if an antibody is available commercially, enquiries to the supplier may reveal the carrier conjugation method used. Labelling ligands for receptor binding studies will require structure-activity information so that a tolerant attachment site can be selected. It needs to be borne in mind that the demands of carrier conjugation and of protein labelling are different. While a carrier with 50 haptens attached may make an adequate immunogen, a signal enzyme with 50 haptens attached will make a very poor conjugate. Even if the enzyme retains activity (which is unlikely), the sensitivity in any competitive immunoassay will be far from optimal. For best sensitivity, the hapten to enzyme ratio should be unity (though <1, i.e. oligomerized enzyme, may be advantageous). Carrier conjugation methodology is dominated by methods where the degree of modification is considered less important and usually not measured. So while it may be worthwhile to consult this literature for deciding how to derivatize the molecule, it may be advisable to select a different conjugation chemistry, in particular chemistry that offers a greater degree of control and the ability to measure the degree of substitution. A further consideration for competitive immunoassays is the selection of the linker group. This should be different from the linker group used to prepare the immunogen, since otherwise the interaction between antibody and the enzyme-hapten conjugate may be enhanced by antibody-linker interactions ('bridge recognition'), thereby decreasing the sensitivity of the resulting immunoassay. The heterobifunctional reagent approaches discussed in Chapter 3 offer plenty of scope for varying the linker. As a starting point let us assume that we have a molecule that has some functional groups, say amino, carboxyl or hydroxyl. (If there is more

than one group, then we need to be concerned about specificity and side reactions. For example, if there are both carboxyl and amino groups present, linking via the carboxyls would be inappropriate since, when these are activated, they may be attacked by the amino groups. Given the range of groups and chemical environments possible, it is not possible to cover this aspect adequately here.) For functional groups present in proteins, the principles discussed in Chapter 2 may be used as a guide. Let us also assume that we have some knowledge of what parts of the molecule are important for activity and have selected a site for attachment that is remote from the 'business end' of the molecule. In the case of haptens where we wish to retain antigenicity, we will want to link at the same point as the molecule was linked to the carrier for immunization. We also need to consider what length of linker group is required; if the literature is silent on this point it is best to opt for the longest that may be conveniently incorporated. Heterobifunctional strategies that involve derivatization of both components (for example Section 3.3) give longer overall links. Figure 8.1 shows some of the more straightforward schemes that will be discussed below.

8.2.2 Primary (aliphatic) amines Water-soluble carbodi-imides provide an easy one-step method of coupling amines to protein carboxyls. The disadvantage of this approach is that it may be more difficult to measure and control the degree of modification, and also this approach does not introduce any spacer group (which may be crucial if the amino group is close to the functional part of the molecule). A more controlled approach uses an amine-reactive heterobifunctional cross-linking agent, as described in Section 8.2.5. Amines may also be coupled to periodateoxidized proteins. This method (see Section 3.4) is particularly useful for coupling amines to HRP. As an example, Protocol 8.1 (M. Abbott, personal communication, 1995) describes the preparation of a conjugate of HRP with a nickel-chelating ligand, NTA,

The labelling of small biomolecules

succi nic .-" (~o /,~ OH anhydride 0

~OH primary

0

alcohol

105

0

OH ] carboxylic

\

~ carbodi/ . acid ~mide/

%(

OH OH vic-diol

ent

.ooc_

Figure 8.1 Some options for coupling small biomolecules, R, containing various reactive groups, to proteins.

Protocol 8.1 Preparation of an HRP-Ni chelate

conjugate. 1. Prepare a solution of NTA reagent (Qiagen) in 0.3 M sodium bicarbonate/carbonate buffer, pH 9.4. 2. Dissolve 1.0 mg of periodate-activated HRP (Pierce) in 0.1 ml water. 3. Add 0.25 ml of the NTA reagent to the activated enzyme and incubate for 1 h at room temperature. 4. Add 10 ~1 5 M sodium cyanoborohydride in 1 M NaOH and allow reduction to take place for 15 min at room temperature. 5. Quench the reaction by adding 20 ~tl 3 M ethanolamine, pH 9.0 and leaving for a further 15 min. a 6. Dilute the mixture to 1.0 ml with distilled water and dialyse against PBS for 1 h. 7. Add NiSO4 to the dialysis buffer to a final concentration of 1 mg m l and dialyse for a further hour to coordinate the nickel. 8. Dialyse further against several changes of PBS. Aliquot the conjugate and store at -20~ a

Steps 6-8 are peculiar to the NTA ligand. For other amine components, the product may be isolated directly after step 5, preferably on a de-salting column (Appendix 6).

106

N o n - r a d i o a c t i v e labelling

containing a primary amine (N-(5-amino-1carboxypentyl)iminodiacetic acid).

8.2.3 Hydroxyl groups Relatively unreactive to most cross-linking agents, hydroxyls are generally derivatized to

provide a more reactive group for conjugation. Primary hydroxyls may be oxidized to a carboxylic acid, but a more convenient way of providing a carboxyl is reaction with a cyclic anhydride such as succinic anhydride (Figure 8.1). Several haptens of interest are commercially available as hemisuccinates. This route has the added attraction of providing a (short)

N

0

O

II

c

R.-U'--OH

L v

II

N

O

R

JLo ,

0

JL O-N~ R

O

HO-N NH

O

N

NH O=( NH

Figure 8.2 Preparation of an NHS active ester reagent.

P r o t o c o l 8.2 A c t i v a t i o n o f c a r b o x y l g r o u p s . 1. Dissolve the carboxyl component in DMSO a (fresh bottle, or dried over molecular sieves) at as high a concentration as possible. 2. Add 1.1 molar equivalents of N-hydroxysuccinimide (Aldrich; molecular weight 115) and dissolve, adding further solvent if necessary. 3. Add 1.05 molar equivalents of dicyclohexylcarbodi-imide (caution: toxic/sensitizer; Aldrich; molecular weight 206.3) dissolved in a minimum volume of DMSO. 4. Stopper the reaction tube and leave to react at room temperature. Look for precipitation of the dicyclohexyl urea formed by the reaction. This may appear within several minutes or several days, depending on the concentration and reactivity of the carboxyl. Leave to react for about five times as long as it took for the precipitate to first appear. 5. Centrifuge the reaction (polypropylene Eppendorf tubes are good for small volumes, or filter for larger volumes). The clear supernatant contains the crude active ester. Calculate the molar concentration and use directly for protein labelling. 6. Store at -20~ a

Or DMF. Other solvents may be considered provided they are aprotic and do not contain reactive functional groups.

T h e l a b e l l i n g of s m a l l b i o m o l e c u l e s linker group. The carboxyl may then be linked to the signal protein as described below. For thiol-based coupling chemistry, hydroxyls may be derivatized with p-maleimidophenyl isocyanate (PMPI; Annunziato et al., 1993). Hydroxyls may also be activated with tosyl chloride and converted to thiols (Ebert et al., 1977).

8.2.4 Carboxyls: in situ active ester coupling For molecules that contain carboxyl groups (and are free from amino groups and other potentially reactive nucleophiles), it is possible and convenient to prepare a stable NHS ester with dicyclohexyl carbodi-imide (Figure 8.2). Though ideally it is desirable to isolate the product as a solid, it is generally acceptable to use the reagent in solution without purification. A generic method is given in Protocol 8.2.

8.2.5 Thiol-based couplings This scheme, described for protein conjugates in Section 3.3, involves the derivatization of one component with a thiol (if not already present) and the other component with a thiol-reactive group, followed by conjugation. As an example, the coupling of the amine of thyroxine T4 to alkaline phosphatase is shown in Figure 8.3 and this conjugation is described in Protocol 8.3. Though more involved, this approach has a number of advantages. Firstly, measuring the thiol content allows the degree of modification to be estimated: for biomolecules that are difficult to estimate when conjugated (e.g. no distinctive spectrum), this is a significant advantage. Secondly, it gives rise to a long spacer arm.

8.2.6 Characterization of the conjugate 8.2.6.1 Absorbance spectrum

Where the biomolecule shows a strong and distinct absorbance spectrum, this will be the method of choice (see Section 4.1.4.1). How-

107

ever, this will generally not be possible, especially for low degrees of modification. 8.2.6.2 Functional group analysis

Loss of protein functional groups is a possible approach, especially for coupling to thiols which will generally be few in number. Loss of protein amines may be measured, for example using trinitrobenzene sulphonic acid (TNBS; Fields, 1972). However, this method will not be accurate when only a small proportion of amino groups are reacted, and it is often the case that the biomolecule introduced interferes in the determination (and is not easily controlled for). 8.2.6.3 Radioactive tracers

Radioactive tracers can be used if available. Though this approach may appear inelegant (and against the principles of this book!), one needs only a few thousand counts added to the conjugation reaction to determine the fraction that becomes conjugated, and the product, especially when diluted to working concentrations, is of such low activity that it may (depending on local regulations) be regarded as non-radioactive. Alternatively a repeat conjugation without tracer could be carried out subsequently. 8.2.6.4 Mass spectrometry

Mass spectrometry is a powerful general approach (as described in Section 4.1.4.3 for fluorophores) which has been applied to hapten carrier conjugates (Adamczyk et al., 1994). 8.2.6.5 Amino acid analysis

This may be used for peptides where the amino acid composition is distinctive. More generally however, conjugates prepared using the crosslinking agent MBS (Section 3.3.2) may be analysed by amino acid analysis, since each cross-link gives rise upon acid hydrolysis to a residue of S-succinyl cysteine (R. Camble, personal communication, 1986). This elutes before Asp and Glu on conventional ion-exchange amino acid analysis. N-Bromoacetyl derivatives of peptides (or

108

N o n - r a d i o a c t i v e labelling HO

0

0

0 N--O

0

ON

HO . 0 0

H S ~ N I

0

0

OH

HO..,~.O 0

I

i

§

I 0

OH

Figure 8.3 Coupling of an amine component (thyroxine T4) to an enzyme label. other amine-containing biomolecules) that are coupled to protein cysteine residues may be quantified by the S-carboxymethyl cysteine produced upon acid hydrolysis (Kolodny & Robey, 1990).

8.2.6.6 Electrophoretic methods The use of non-denaturing gels has been explored for characterizing hapten-carrier conjugates and should in principle be applicable to

other protein conjugates (Kamps-Holtzapple et al., 1993).

8.2.6.7 Functional analysis Obtaining evidence of conjugation by the functional property of the conjugate you are looking for is not satisfactory, but there may be no easy alternative. In this case, it is useful to prepare a series of conjugates with varying degrees of modification and select the one with the best

T h e l a b e l l i n g of s m a l l b i o m o l e c u l e s

109

Protocol 8.3 Preparation of a thyroxine T4 alkaline phosphatase conjugate. 1. Suspend T4 (17.3 mg, 19.5 ~tmol) in dry DMF (4.0 ml). 2. Add to a solution of SMCC (Pierce, 6.5 mg, 19.5 ~tmol) in 1.0 ml DMF and stir the reaction at room temperature for 1 h. The T4 is pulled into solution as it reacts and the solution clears within 10 min. Solutions containing T4 should be protected from light to minimize loss of iodine. 3. The solution may be used in the next step without purification. It may be stored at -20~ 4. Transfer by gel filtration the alkaline phosphatase (Boehringer, 5 mg, 0.5 ml) into de-gassed PBS buffer pH 7.4 using a PD-10 column or equivalent to give 1.2 ml product. 5. To 0.6 ml of this, add 0.2 ml 2-iminothiolane (Sigma, 1.84 mg ml-~, fresh in PBS) and incubate at 37~ for 30 min. 6. Desalt on a PD-10 to give 1.2 ml product. Analyse a portion immediately at 1:20 dilution for (i) protein concentration by OD at 280 nm and (ii) thiol content using Ellman's reagent. The protein concentration should be ca 8 ~tM and the degree of substitution 2-3 mol mol-1. See Appendices 5 and 6. Proceed to the next step without delay. 7. To this solution, add 12 ~tl of the T4 reagent to give a final concentration of 50 laM and allow to react at ambient temperature for 1.5 h. 8. Remove excess reagent on a PD-10 gel filtration column, or equivalent, to give 2.0 ml of product. 9. Analyse a portion of the product for thiols as above. This should be essentially zero indicating complete reaction. Run a UV spectrum: T4 is indicated by a shoulder at 330 nm on the main protein 280 nm peak. This may be used to estimate the T4 content but since the spectra mutually overlap, this requires solving simultaneous equations and is anyway inaccurate since the T4 absorption is weak. Loss of thiol function gives an adequate estimate of the degree of substitution. 10. Add heat-treated BSA to 1 mg m1-1 and sodium azide to 0.1% and store at 4~ Under these conditions, the conjugate is stable for at least a year.

performance. To obtain the series, the best parameter to vary is the reagent concentration (depending on the chemistry used). Three or four reactions with the concentration varied by a factor of 3- to 5-fold between reactions is suggested.

8.3 L A B E L L I N G W I T H L O W M O L E C U L A R W E I G H T LABELS AND TAGS The attachment of fluorophores and tags to small biomolecules is, in many cases, a natural extension of coupling methods described above. Where functional groups allow it, fluorophore

and tag reagents designed primarily for proteins may successfully be employed. By way of illustration, DIG has been coupled to ampicillin using the active ester reagent (Weigel et al., 1994), and fluorescein attached to folate using FITC and a diaminopentane spacer (Van Houten et al., 1985). Such couplings may be regarded as an organic chemistry undertaking, but in many cases may be undertaken successfully without the infrastructure of the organic chemistry laboratory. Indeed, where economics dictate a small synthesis scale (perhaps in the order of 1 mg), some deviation from more traditional organic chemistry approaches is in any case appropriate. Generalizing such syntheses is difficult, but usually,

110

Non-radioactive labelling

stoichiometric amounts of reagents (or a small excess of the reagent) are dissolved in a suitable solvent (DMSO and DMF are often used) and the reaction followed by thin layer chromatography (TLC) or HPLC. The first step of Protocol 8.3 uses such an approach. Protocol 8.2 gives the activation of a carboxyl component with carbodi-imide: N-hydroxysuccinimide may be replaced by a primary or secondary amine of choice to give an amide. Amines must be in the free base form: addition of an equivalent of triethylamine will achieve this where the hydrochloride is used. HPLC may then be used to isolate the product. Aqueous buffers may be used as solvent (as for protein reactions), provided allowance is made for any reagent hydrolysis. Thus, for example, a benzodiazepine drug containing a primary amine has been successfully labelled on a submilligram scale with the isothiocyanate europium chelate labelling reagent, using an aqueous coupling at pH 9, followed by HPLC purification (Takeuchi et al., 1993). Various other strategies for labelling small biomolecules with lanthanide chelates have been devised (Mikola et al., 1995).

REFERENCES Adamczyk, M., Buko, A., Chen, Y-Y., Fishpaugh, J.R., Gebler, J.C. & Johnson, D.D. (1994). Characterization of protein-hapten conjugates. 1. Matrix-assisted laser desorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods. Bioconjugate Chem. 5, 631-635. Annunziato, M.E., Patel, U.S., Ranade M. & Palumbo P.S. (1993). p-Maleimidophenyl isocyanate: a

novel heterobifunctional linker for hydroxyl to thiol coupling. Bioconjugate Chem., 4, 212-218. Brinkley, M. (1992). A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2-13. Ebert, C., Ebert, G. & Knipp, H. (1977). On the introduction of disulfide crosslinks into fibrous proteins and bovine serum albumin. In Protein cross-linking: nutritional and medical consequences (ed. M. Friedman). Plenum Press, New

York. Erlanger, B. (1980). The preparation of antigenic hapten-carrier conjugates: a survey. Meth. Enzymol. 70, 85-104. Fields, R. (1972). The rapid determination of amino groups with TNBS. Meth Enzymol. 25, 464-468. Kamps-Holtzapple, C., Carlin, R.J., Sheffield, C., Kubena, L., Stanker, L. & Deloach, J.R. (1993). Analysis of hapten-carrier protein conjugates by non-denaturing gel electrophoresis. J. Immunol. Meth. 164, 245-253. Kolodny, N. and Robey, F.A. (1990). Conjugation of synthetic peptides to proteins: quantification from S-carboxymethylcysteine released upon acid hydrolysis. Anal. Biochem. 187, 136-140. Mikola, H., Takalo, H. & Hemmil~i, I. (1995). Syntheses and properties of luminescent lanthanide chelate labels and labeled haptenic antigens for homogeneous immunoassays. Bioconjugate Chem. 6, 235-241. Takeuchi, T., Yoshida, M., Kabasawa, Y., Matsukawa, R., Tamiya, E. & Karube, I. (1993). Time-resolved fluorescence receptor assay for benzodiazepines. Anal. Lett. 26, 1535-1545. Tijssen, P. (1985). Preparation of enzyme-antibody or other enzyme-macromolecule conjugates. Lab. Techn. Biochem. Mol. Biol. 15, 279-296. Van Houten, J.L., Smith, R., Wymer, J., Palmer, B. & Denaro, M. (1985). Fluorescein-conjugated folate as an indicator of specific folate binding to Paramecium. J. Protozool. 32, 613-616. Weigel, L.M., Belisle, J.T., Radolf, J.D. & Norgard, M.V. (1994). Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence. Antimicrob. Agents Ther. 38, 330-336.

APPENDIX 1

List of suppliers

Company

Relevant products

UK/Europe

North America

Amersham

Kits, labelling reagents

Amersham International plc, Little Chalfont, Bucks HP7 9NA, UK. Tel: 01494 544000. Fax: 01494 542929

Amersham Life Science Inc., 2636 S. Clearbrook Drive, Arlington Heights, IL 60005, USA. Tel: 800 341 7543. Fax: 708 437 1640

Bio-rad

Chromatography

B io-rad Laboratories Ltd, Bio-rad House, Maylands Avenue, Hemel Hempstead, Herts HP2 7TD, UK. Tel: 01442 232552. Fax: 01442 259118

B io-rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547, USA. Tel: 510 741 1000. Fax: 510 741 1060

Biometra

Molecular biology, biotin mimetic

Biometra GmbH, Rudolf Wissell Str., D 37079 G6ttingen, Germany. Tel: 551 506860. Fax: 551 5068666

B iometra Inc., 550 N Reo St., Tampa, FL 336091013, USA. Tel: 813 287 5132. Fax: 817 287 5163

Biozyme

Enzymes

Biozyme Laboratories Ltd., Unit 6, Gilchrist-Thomas Estate, Blaenavon, Gwent NP4 9RL, UK. Tel: 01495 790678. Fax: 01495 791780

Biozyme Laboratories International Ltd, 9939 Hibert St., Suite 101, San Diego, CA 92131-1029, USA. Tel: 619 549 4484. Fax: 619 549 0138

112

Non-radioactive

labelling

Appendix 1 (Continued)

Company

Relevant products

UK/Europe

North America

Boehringer Mannheim

DIG labelling, enzymes, substrates

Boehringer Mannheim UK, Bell Lane, Lewes, East Sussex BN7 1LG, UK. Tel: 01273 480444. Fax: 01273 480266

Boehringer Mannheim Corporation, GHS Hague Road, PO Box 50528, Indianapolis, IN 462500528. Tel: 317 845 2000. Fax: 317 845 2090

Clontech

Molecular biology labelling reagents

Cambridge Bioscience, 25 Signet Court, Newmarket Road, Cambridge CB5 8LA, UK. Tel: 01223 424222. Fax: 01223 360732

Clontech Laboratories Inc., 4030 Fabian Way, Palo Alto, CA 94303, USA. Tel: 415 424 8222. Fax: 415 424 1064

Cruachem

Phosphoramidites

Cruachem Ltd, Todd Campus, West of Scotland Science Park, Acre Road, Glasgow G20 0UA, UK. Tel: 0141 945 0055. Fax: 0141 946 6173

45150 Business Court, Suite 550, Sterling, VA 22170, USA. Tel: 703 689 3390. Fax: 703 689 3392

Dako

Conjugates, immunodetection

DAKO Ltd, 16 Manor Courtyard, Hughenden Ave., High Wycombe, Bucks HP13 5RE, UK. Tel: 01494 452016. Fax: 01494 441846

DAKO Corporation, 6392 Via Real, Carpinteria, CA 93013, USA. Tel: 805 566 6655. Fax: 805 566 6688

Genosys

Labelling kits, reagents

Genosys Biotechnologies (Europe), London Road, Pampisford, Cambridge CB2 4EF, UK. Tel: 01223 839000. Fax: 01223 839200

Genosys Biotechnologies, Inc., 1442 Lake Front Circle, Suite 185, The Woodlands, TX 773803600 USA. Tel: 800 234 5362. Fax: 713-363-2212

Genzyme

Enzymes, streptavidin

Genzyme Diagnostics, 50 Gibson Drive, Kings Hill, West Mailing, Kent, ME 19 6HG, UK. Tel: 01732 220022. Fax: 01732 220024/5

Genzyme Corporation, One Kendall Square, Cambridge, MA 02139, USA. Tel: 617 252 7500. Fax: 617 252 7700

Glen Research

DNA synthesis

Cambio Ltd, 34 Millington Road, Cambridge CB3 9HP, UK. Tel: 01223 66500. Fax: 01223 350069

Glen Research, 44901 Falcon Place, Sterling, VA 22170, USA. Tel: 703 437 6191. Fax: 703 435 9774

Igen

Electrochemiluminescence systems

Igen, Institute of Research and Igen, Inc., 16020 Industrial Drive, Gaithersburg, MD Development, Birmingham Research Park, Vincent Drive, 20877, USA. Tel: 301 984 8000. Fax: 301 230 0158 Birmingham, UK. Tel: 01214 147033. Fax: 01214 725738

JBL

Substrates, Attophos T M

Europa Bioproducts Ltd, Pond Green, Wicken, Ely, Cambridge CB7 5XX, UK. Tel: 01353 721118. Fax: 01353 624589

JBL Scientific, 277 Granada Drive, San Luis Obispo, CA 93401, USA. Tel: 805 544 8524. Fax: 805 543 1531

Appendix

1 - List of s u p p l i e r s

113

Appendix 1 (Continued)

Company

Relevant products

UK/Europe

North America

Life Technologies

DNA labelling products

Life Technologies Ltd, PO Box 35, Trident House, Renfrew Road, Paisley PA3 4EF, UK. Tel: 041 814 6100. Fax: 041 887 1167

Life Technologies Inc., 8400 Helgerman Court, PO Box 6009, Gaithersburg, MD 20884-9980, USA. Tel: 301 840 8000

Molecular Dynamics Imaging equipment

Molecular Dynamics, 5 Beech House, Chiltern Court, Asheridge Road, Chesham, Bucks, HP5 2PX, UK. Tel: 01494 793377. Fax: 01494 793222

Molecular Dynamics, 928 East Arques Avenue, Sunnyvale, CA 94086, USA. Tel: 800 333 5703. Fax: 408 773 1493

Molecular Probes

Fluorescent reagents

Cambridge Bioscience, 25 Signet Court, Newmarket Road, Cambridge CB5 8LA, UK. Tel: 01223 424222. Fax: 01223 360732

Molecular Probes Inc., P.O. Box 220~0, Eugene, OR 97402-0414, USA. Tel: 503 465 8300. Fax: 503 344 6504

New England Nuclear

Catalysed reporter deposition

NEN Life Science Products, Wedgwood Way, Stevenage, Herts., SG1 4QN, UK. Tel: 01438 734027. Fax: 01438 734379

NEN Life Science Products, 549 Albany Street, Boston, MA 02118, USA. Tel: 617 482 9595. Fax: 617 542 8463

Packard

Detection instrumentation, HTRF

Canberra Packard Ltd, Brook House, 14 Station Road, Pangbourne, Berks RG8 7DT, UK. Tel: 44 1734 844981. Fax: 44 1734 844059

Packard Instrument Company, Inc., 800 Research Parkway, Meriden, CT 06450, USA. Tel: 800 323 1891. Fax: 203 639 2172

Perkin Elmer

DNA synthesis, Tropix Perkin Elmer, Kelvin Close, Perkin Elmer, 850 Lincoln reagents Birchwood Science Park North, Center Drive, Foster City, Warrington, Cheshire WA3 CA 94404, USA. 7PB, UK. Tel: 01925 825650. Fax: 01925 282502

Pharmacia

Chromatography, SPDP Pharmacia B iotech Ltd, 23 Grosvenor Road, St Albans, Herts ALl 3AW, UK. Tel: 01272 814000. Fax: 01272 814001

Pharmacia B iotech, Inc., 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855-1327. Tel: 800 526 3593. Fax: 908 457 0557

Pierce

Cross-linking agents, Pierce and Warriner, 44 Upper other protein chemistry Northgate St, Chester CH1 4EF, products UK. Tel: 01244 382525. Fax: 01244 373212

Pierce Chemical Company, 3747 North Meridian Road, PO Box 117, Rockford, IL 61105, USA. Tel: 815 968 0747. Fax: 815 968 7316

Promega

PinPoinff M, molecular biology reagents

Promega Corporation, 2800 Woods Hollow Road, Madison WI537115399, USA. Tel: 608 274 4330. Fax: 608 277 2516

Promega UK Ltd., Delta House, Chilworth Research Centre, Southampton SO 16 7NS, UK. Tel: 01703 760225. Fax: 01703 767014

114

Non-radioactive

labelling

Appendix 1 (Continued) i

i

Company

Relevant products

UK/Europe

North America

Qiagen

Molecular biology products, Ni chelate reagents

Qiagen Ltd, Boundary Court, Gatwick Road, Crawley, West Sussex RH10 2AX, UK. Tel: 01306 740444. Fax: 01306 875885

Qiagen Inc., 9600 De Soto Avenue, Chatsworth, CA 91311, USA. Tel: 800 426 8157. Fax: 800 718 2056

Sigma

Biochemicals, reagents, Sigma Chemical Company, enzymes Fancy Road, Poole, Dorset BH17 7NH, UK. Tel: 01202 733114. Fax: 01202 715460

Vector

Enzyme substrates

Vector Laboratories Ltd, 16 Wulfric Square, Bretton, Peterborough PE3 8RF, UK. Tel: 01733 265530

Vector Laboratories Inc., 30 Ingold Road, Burlingame, CA 94010, USA. Tel: 415 697 3600. Fax: 415 697 0339

Wallac

Time-resolved fluorescence, instrumentation

EG and G Ltd Milton Keynes, 20 Vincent Avenue, Crownhill Business Centre, Crownhill, Milton Keynes MK8 0AB, UK. Tel: 01908 265744. Fax: 01908 265956

Wallac Inc., 9238 Gaither Road, Gaithersburg, MD 20877, USA. Tel: 301 963 3200. Fax: 301 963 7780

Sigma Chemical Company, 1001 West St Paul Avenue, Milwaukee, WI 53233, USA. Tel: 314 771 5765. Fax: 314 771 5757

APPENDIX

2

Molecular weights

DNA

Proteins

The repeating nucleotide units have molecular weights as follows:

Streptavidin Avidin HRP Alkaline phosphatase (calf intestinal) I]-Galactosidase Bovine serum albumin Immunoglobulin G Fab' fragment Phycobiliproteins

dA: dT: dC: dG:

313 304 289 329

The termini, H - and H O - , require addition of 18 (water), and if there is no terminal phosphate, then 80 (PO3H) needs to be subtracted. The formula for a normal (unphosphorylated) oligonucleotide is therefore:

66 000 60 240 44 000 140 000 465 000 67 000 150 000 46 000 see Table 4.2

MW = 313a + 304t + 289c + 329g - 62 where a, t, c, g represent the number of each of the four nucleotides present in the sequence. As an approximation, i~he molecular weight of single-stranded DNA (sodium salt) is 325n, where n is the number of nucleotides.

General: the average amino acid in a protein has a molecular weight of 110. Hence, as a rule of thumb, the molecular weight of an unglycosylated protein is 110n, where n is the number of amino acids.

116

N o n - r a d i o a c t i v e labelling

Some protein chemistry reagents Biotin-LC-NHS Carboxy-fluorescein-NHS EDC Ellman' s reagent FITC 2-Iminothiolane MBS 2-Mercaptoethanol MHS SATA SIAB SMCC SMPB Sulpho-SMCC SPDP LC-SPDP Sulpho-LC-SPDP

556.58 473 191.7 396.4 389 137.63 314.2 78.13 (neat is 14.2 M) 308.29 231.22 402.15 334.33 356.3 436.37 312.4 425.5 527.6

APPENDIX 3

Extinction coefficients for protein

conjugation

,

,

,

,,,,

|,,,

,,,

|,

Protein

A2so(0.1%, approx.)

Alkaline phosphatase I]-Galactosidase Bovine serum albumin Horseradish peroxidase Immunoglobulin G Streptavidin

0.96 2.09 0.66 2.5 (at 403 nm) 1.4-1.5 2.9 .........

,

i

,i

i,

,,

,

i,

Released groups

Wavelength (nm)

Extinction (M-1 cm -11

5-Thio-2-nitrobenzoate (reduced Ellman' s reagent) p-Nitrophenol (PNP) Pyridine-2-thione Pyridine-2-thione N-Hydroxysuccinimide

412

14 150

405 343 280 260

18 8 5 8

i

i

i

i

i

800 080 100 200

APPENDIX 4

Excitation and emission wavelengths of chemilumophores and some fiuorophores used for DNA labelling

Chemilumophores Aequorin Isoluminol Firefly luciferase Dioxetane (aqueous) Acridinium ester Ru(bpy)33§ (ECL)

Emission 469 460 562 477 429 620

For data on other fluorophores, see Table 4.1.

Fluorophores FAM TET JOE HEX TAMRA ROX

Excitation (nm)

Emission (nm)

500 520 525 540 555 585

525 545 555 565 580 610

APPENDIX 5

Determination of thiol groups using Ellman's reagent

This simple procedure, based on Ellman (1959) and Riddles et al. (1983), may be used to determine the concentration of free thiols on a biomolecule, whether added by means of chemical reagents or generated by reduction of protein disulphides (see Section 3.3). Buried protein thiols will not be detected since they require a denaturant (but are in any case not available for conjugation). The sample must be thoroughly free from reagents (by efficient de-salting (Appendix 6) or by dialysis). The following protocol is for a single beam spectrophotometer, using a 1 ml (reduced volume), 1 cm pathlength cuvette, but may be adapted to suit local conditions.

2. 3. 4.

Solutions

1. Buffer: 0.1 M NaHzPO4/Na2HPO4, pH 7.4, or PBS. 2. Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid), DTNB): 100 mM in DMSO. This stock solution may be stored at -20~ for several years. 3. Ellman's reagent: 2 mM in the above buffer. This working solution may be stored at -20~ for several months or until blank values become too high. Method

1. Dilute protein in phosphate buffer (0.98 ml) into a plastic disposable cuvette (e.g. Starstedt cat. no. 67.740). The precise dilution will depend on con-

5.

centration and availability of the sample. A suitable final thiol concentration to aim for is 10 laM (this will give an OD increase of 0.141), but, with care, useful estimates may be obtained as low as 0.5 ~tM. Zero the spectrophotometer at 412 nm and start recording. Add 20 ~tl of 2 mM Ellman's reagent and stir thoroughly (Starstedt disposable stirring rods, cat. no. 81.970, are useful). Continue recording. When the absorbance has reached a plateau, add a further 20 ktl aliquot of 2 mM Ellman's reagent and continue recording. This provides a useful check that the Ellman's reagent was in excess over the thiol (if the absorbance slowly rises to a new plateau, it indicates that the thiol was in excess over the Ellman's reagent) but, more usually, the small step increase obtained (typically in the order of 0.002 to 0.005) gives a reagent blank value that should be subtracted from the initial rise to give the true absorbance change, A. Calculate the thiol concentration in the cuvette, T: T = A / 1 4 150.

REFERENCES Ellman, G.L. (1959). Tissue sulphydryl groups. Arch Biochem. Biophys. 82, 70-77. Riddes, P.W., Blakeley, R.L & Zerner, B. (1983). Reassessment of Ellman's reagment. Meth. Enzymol. 91, 41-60.

APPENDIX

6

Use of disposable gel filtration columns

These are exceedingly useful devices for the rapid separation of a labelled macromolecule from excess labelling reagents and other low molecular weight components. They require no equipment to run, relying on gravity flow, and cannot run dry. The comments in this section apply specifically to the Pharmacia series of columns, but similar products are available from other manufacturers (e.g. Pierce, Biorad). Instructions for the use of these columns are supplied by the manufacturer but it is worthwhile noting the following. There is generally no need to collect and analyse many fractions. Provided that the labelled macromolecule is excluded from the gel filtration matrix and is freely soluble in the column buffer, then the elution volume will be consistent from column to column and the product may be collected 'blind'. (ii) The columns were originally intended mainly for de-salting purposes and the

(i)

sample volumes suggested by the manufacturers reflect this. For labelling purposes, more rigorous exclusion of reactants is generally necessary, and therefore the sample and collection volumes should be reduced and the columns used more cautiously. A guide to suitable volumes to use is given in the table below. (iii) It is safer not to attempt to reuse columns that have been used to separate reaction mixtures. Extensive washing would be necessary where large excesses of reagents have been used and many reagents, in particular aromatic reagents, stick to gel filtration matrices and are difficult to wash off. (iv) For proteins, particularly when used at lower concentrations (1 mg ml -~ or less), priming the column (with e.g. BSA) is useful for increasing recoveries. Use 1-2 sample volume equivalents of 5-10 mg ml -~ BSA, followed by 2 column volumes of running buffer. (v) For more dilute proteins, or proteins that

Appendix

6 - D i s p o s a b l e gel f i l t r a t i o n c o l u m n s

are 'sticky', use of 1 mg m1-1 BSA in the running buffer should be considered. Some post-column analyses will obviously not be possible, but for example spectral analysis is successful provided that column buffer blanks are carefully used. It is also necessary to kill off any reagent present before applying the reaction mixture to the column; this will eliminate any possibility of modified BSA appearing in the product. For NHS esters and most other amine-directed reagents, addition of 1/10th volume of 0.1 M ethanolamine, pH 8.5, followed by

121

10 min incubation at room temperature is suggested to achieve this. (vi) When adding small sample volumes, ensure that the solution covers the frit evenly. For the NAP-5 T M column, sample volumes less than 0.10 ml should be diluted to 0.10 ml before addition. (vii) For sample volumes larger than the suggested limit for the PD-10, the use of several columns run in parallel should be considered. This may seem extravagant, but it compares favourably to the time required for packing, equilibrating and running a larger gel filtration column.

Recommended volumes for use of Pharmacia disposable columns for removal of excess reagents Column

Total column volume

Maximum sample volume

PD-10 or NAP-25 T M

9 ml

1.2 ml

2.8 ml

1.6 ml

1 ml sample 1.8 ml buffer 1.6 ml product

NAP- 10T M

3 ml

0.4 ml

1.0 ml

0.7 ml

0.3 ml sample 0.7 ml buffer 0.7 ml product

NAP-5 T M

1.5 ml

0.2 ml

0.5 ml

0.4 ml

0.15 ml product 0.35 ml buffer 0.4 ml product

Exclusion volume Maximum product Typical volumes (start of product collection volume to be applied collection)

APPENDIX 7

Purity check for N-hydroxysuccinimide esters

All reagents incorporating N-hydroxysuccinimide (HOSu) esters are water sensitive and hydrolyse easily if not transported or stored correctly. This simple check (based on M~rtensson et al., 1984) may be used if there is any doubt about a reagent, e.g. if a reaction has failed. It is based on the absorbance at 260 nm of HOSu (Abdella et al., 1979). Since usually other parts of the reagent structure contribute to the absorbance at 260 nm, it is best not to attempt to measure that which has already hydrolysed off the reagent, but rather to hydrolyse and determine the remainder, as follows.

4. When the absorbance has reached at plateau (this should be in the order of 10 min), record the final reading. 5. The extinction coefficient of the HOSu is 8200, hence a 100% pure reagent (at 0.1 mM) should give an absorbance increase of 0.820. The purity is therefore the observed increase divided by 0.820. Note: a low purity does not mean the reagent is unusable, since the hydrolysed reagent will not in general interfere. Even a reagent that is only, say, 20% pure on this analysis may usually be used provided that account is taken of the purity in calculating reagent concentrations.

REFERENCES Method 1. Prepare a 10 mM solution of the reagent in DMSO or other water-miscible, aprotic solvent. 2. Set the spectrophotometer to 260 nm. Add 10 ~tl of this solution to 0.99 ml 10 mM sodium phosphate buffer, pH 7.0 (or other weak, nonnucleophilic, neutral buffer) in a cuvette (1 cm pathlength). Zero the absorbance immediately. 3. Add to the cuvette 20 ml of 1 M Na2CO3 and monitor the absorbance.

Abdella, P.M., Smith, P.K. & Royer, G.P. (1979). A new cleavable reagent for cross-linking and reversible immobilization of proteins. Biochem. Biophys. Res Comm. 87, 734-742. M~rtensson, A.M., Gustafsson, J.-G. & Ljunggren, H.D. (1984). A modified, highly sensitive enzyme-linked immunosorbent assay (ELISA) for Rhizobium meliloti strain identification. J. Gen. Microbol. 130, 247-253.

APPENDIX 8

V a l u e s of groups

Amino acid

C terminus Asp Glu His N terminus Lys Cys Tyr Arg

Group

-COOH -COOH -COOH Imidazole -NH2 -NH2 -SH -OH Guanidine

Data from Fersht (1985).

pKa for

a m i n o acid f u n c t i o n a l

Model Usualrange compounds in proteins (small peptides) 3.6 4.0 4.5 6.4 7.8 10.4 9.1 9.7 ~12

2-5.5 2-5.5 2-5.5 5-8 ~8 ~10 8-11 9-12

REFERENCE Fersht, A. (1985). Enzyme structure and mechanism, 2nd edn. W.H. Freeman & Co., New York.

APPENDIX

9

Signal enzyme detection

1. Detection in solution and microtitre plates

Colour measurement of alkaline phosphatase with PNPP (p-nitrophenylphosphate) Substrate: 1 mg m1-1 PNPP in 1 M diethanolamine, pH 9.8, containing 0.5 mM MgC12. Substrate must be freshly prepared. PNPP can be obtained in tablet form (Sigma): store at -20~ in the dark. Incubate with substrate conveniently at ambient temperature. Measure absorbance at 405 nm. Stop enzyme reaction, if required, with 3 M NaOH.

Fluorescence measurement of alkaline phosphatase with MUP (4-methyl umbelliferyl phosphate) 0.4 mM M U P i n 1 M Tris-HC1, pH 9.0, containing 2 mM MgC12 and 1 mM ZnSO4. Substrate may be stored for short periods at 4~ Incubate with enzyme at 25~ or ambient temperature,

measuring the fluorescence with excitation set at 360 nm and emission set at 450 nm.

Colour measurement of HRP with ABTS (2,2'azino-di-(3-ethylbenzthiazoline-sulphonic acid) Substrate: 0.1 mg m1-1 ABTS (5 mg tablets, Boehringer) in 0.2 M Na2HPO4, 0.1 M citric acid, pH 5.0 containing 0.03% sodium perborate (hydrogen peroxide substitute). Buffer capsules may be obtained from Sigma (P 4922). Stop the reaction with an equal volume of 2 M H2SO4 and measure the absorbance at 450 nm.

Colour measurement of HRP with TMB (3,3',5,5'-tetramethyl benzidine): Prepare a 5 mg ml -~ stock TMB solution in dimethylformamide. Prepare a 1:10 dilution of this in 0.2 M Na2HPO4, 0.1 M citric acid, pH 5.3 containing 0.1% H202. A ready-to-use formulation is available from Boehringer. Incubate with enzyme at ambient temperature. Stop reaction

Appendix with an equal v o l u m e of 2 M H2SO4, w h e r e u p o n the colour should change from blue to yellow. M e a s u r e the absorbance at 450 nm.

2. D e t e c t i o n / l o c a l i z a t i o n on filters etc.

Detection of alkaline phosphatase with BCIP/ NBT (5-bromo-4-chloro-indoyl phosphate with nitroblue tetrazolium) Solutions: 1. Tris buffer: 0.1 M Tris-HC1, 0.1 M NaC1, 50 mM MgC12, pH 9.5. 2. BCIP: 50 mg ml-~ BCIP in dimethylformamide. 3. NBT: 75 mg ml-1 NBT in 70% (v/v) dimethylformamide. 4. BCIP/NBT substrate solution: to 10 ml of Tris buffer, add 35 ~tl BCIP solution and 45 ~tl NBT solution.

Method: 1. Incubate the filters in the Tris buffer for 5 min. 2. Transfer filter to substrate solution and incubate until desired reaction is obtained, typically overnight. Dry the filter for storage (NB: colours fade somewhat upon storage).

Detection of alkaline phosphatase with chemiluminescent dioxetane substrates

9 - Signal enzyme

detection

125

1. Mix equal volumes of the two detection reagents (one containing the peracid salt and the other containing the luminol/enhancer system; Amersham). Use the freshly prepared substrate within 30 min of mixing. Discard unused substrate. 2. Drain the membrane after the final wash step. Lay the filter out on a clean surface, DNA/protein side up. 3. Cover with the freshly mixed ECL detection reagents (0.125 ml cm -2 of membrane). Leave for precisely 1 min at room temperature. 4. Drain the blots and place DNA/protein side down onto a clean sheet of Saran Wrap. Wrap the blots, gently smoothing out air pockets. Ensure that the detection reagents cannot come into contact with the X-ray film. 5. Place the wrapped blots DNA/protein side up in a film cassette. 6. Switch off the light. 7. Place piece of X-ray film onto the blots and close the casette. 8. Remove the film after 1 min and replace with a second piece of film. Begin timer. 9. Develop first film and, on basis of the signal and background, decide on the exposure time required for second (or subsequent) films.

Note: HRP activity is not prolonged and it is necessary to work without delay once the enzyme has been exposed to the detection reagents. Do not allow filters to dry out at any stage.

See Protocols 6.4 and 7.1. REFERENCE

Detection of HRP by enhanced chemiluminescence (ECL) This procedure (Durrant, 1992) applies to protein and nucleic acid blots.

Durrant, I. (1992). Detection of horseradish peroxidase by enhanced chemiluminescence. In Nonisotopic DNA probe techniques (ed. L.J. Kricka), pp. 167-183. Academic Press, London.

A P P E N D I X 10

Reagents for changing functional groups

of interest cannot

of one functional group to another can solve this

easily be linked together because the functional

problem. The following table gives some general

groups available are incompatible. 'Conversion'

approaches that may be considered.

Frequently

the molecules

To provide Reaction at

Amino

Carboxyi

Thioi

Thiol-reactive group

Amino

m

Succinic anhydride a

Iminothiolane and other reagents b

SMCC-like reagents b

Carboxyl

Carbodi-imide and (e.g.) ethylene diamine r

Thiol

Ethylene imine a Iodoacetic acid g (NB: carcinogen) or 2-bromoethylamine, e aminoethyl-8 f

Thiol-reactive group

Cysteamine

Gounaris & Perlman (1967). b See Chapter 3. r Kurzer & Douraghi-Zadeh (1967). d Raftery & Cole (1963). e Lindley (1956). f Schwartz et al. (1980). g Gurd (1967). a

Carbodi-imide and cysteamine, then reduction

Mercaptoacetic acid

2,2'-Dithiodipyridine

Dithiothreitol

Appendix

10 - R e a g e n t s for c h a n g i n g f u n c t i o n a l g r o u p s

REFERENCES Gounaris, A.D. & Perlman, G.E. (1967). Succinylation of pepsinogen. J. Biol. Chem. 242, 2739. Gurd, F.R.N. (1967). Carboxymethylation. Meth. Enzymol. 11, 532. Kurzer, F. & Douraghi-Zadeh, K. (1967). Advances in the chemistry of carbodi-imides. Chem. Rev. 67, 107. Lindley, H. (1956). A new synthetic substrate for

127

trypsin and its application to the determination of the amino-acid sequence of proteins. Nature 178, 647. Raftery, M.A. & Cole, R.D. (1963). Tryptic cleavage at cysteinyl peptide bonds. Biochem. Biophys. Res. Commun. 10, 967. Schwartz, W.E., Smith, P.K. & Royer, G.P. (1980). N-(13-Iodoethyl)trifluroacetamide: a new reagent for the aminoethylation of thiol groups in proteins. Biochemistry 106, 43-48.

APPENDIX

11

Protein-protein conjugation: worked example

Labelling of a monoclonal antibody with alkaline phosphatase using SMCC and 2-iminothiolane Proteins to be conjugated The antibody was an anti-AFP (alpha foetal protein) monoclonal antibody, supplied as a 1.0 mg m1-1 solution in 0.15 M NaC1. The calf intestinal alkaline phosphatase was from Boehringer (immunoassay grade).

Preparation of maleimido alkaline phosphatase To the alkaline phosphatase (10 mg ml-1 0.15 ml) was added 0.1 M triethanolamine.HC1, pH 7.4, containing 1 mM MgCI2 and 1 mM ZnSO4 (0.60 ml). A stock solution of 20 mM (6.7 mg ml -~) SMCC (Pierce) in DMF was prepared (fresh) and 22 ~1 added to the protein with gentle stirring and the reaction allowed to proceed at room temperature for 30 min. Meanwhile a Sephadex T M G25M column (Pharmacia PD-10)

was equilibrated with PBS (Difco haemaglutination buffer) and primed with 1 ml BSA (5 mg m1-1) followed by a column volume of PBS. The reaction mixture was applied and allowed to absorb onto the column, followed by 2.15 ml buffer. 1.2 ml was then applied and the eluting product collected. The product was kept on ice until required. Meanwhile it was analysed as follows.

Protein determination: 50 ktl was diluted 1:20 and the absorbance at 280 nm determined. This was found to be 0.040. Using an extinction coefficient of 0.96 for 1 mg m1-1 solution, the protein concentration is therefore 0.042 mg m1-1 i.e. 0.42 ~M, i.e. 8.4 t.tM for the original solution (P). Maleimido concentration: To 150 ktl product was added 25 ktl of 0.5 mM 2-mercaptoethanol. 150 ktl of buffer was treated likewise as a control, and the samples incubated at 37~ for

A p p e n d i x 11 - P r o t e i n - p r o t e i n c o n j u g a t i o n 20 min. Each was then diluted with PBS (1.2 ml) in a cuvette, the spectrophotometer zeroed at 412 nm and 20 gl 1.0 mM Ellman's reagent (DTNB) in PBS added, recording the absorbance at 412 nm until it had reached a plateau. The change in absorbance was found to be 0.054 (control) and 0.019 (product). The overall dilution factor of the product is 9.3, hence the concentration of maleimido groups in the (undiluted) product is: M = (0.054 - 0.019) • 9.3 = 23 gM 14 150

is therefore 0.041 mg m1-1 i.e. 0.27 ~M, i.e. P = 0.82 ~l,M for the original solution.

Thiol determination: Using the above solution, the spectrophotometer was zeroed at 412 nm, and 25 gl 1 mM Ellman's reagent (DTNB) added. The increase in absorbance (corrected for Ellman's reagent blank) was 0.009. Using the molar extinction coefficient of 14 150, the thiol concentration is therefore 0.64 gM, i.e. S = 1.92 gM for the original undiluted solution. Estimate of degree of modification:

Estimation of degree of substitution" M/P, i.e. 2.7 mol m o l - 1

This is

Thiolation of antibody using 2-iminothiolane Trial reaction:

To 100 gl of antibody was added 10 gl 0.1 M sodium phosphate buffer, pH 7.4. A solution of 2-iminothiolane (Pierce, 0.92 mg m1-1) in PBS was freshly prepared and 10 gl of this added to the antibody. The reaction was allowed to proceed at 37~ for 30 min. Meanwhile a Sephadex T M G25M column (Pharmacia NAP-5) was equilibrated with de-gassed PBS (Difco haemagglutination buffer) and primed with 0.1 ml BSA (5 mg m1-1) followed by a column volume of PBS. The reaction mixture was applied and allowed to absorb onto the column, followed by 380 gl buffer. 600 gl was then applied and the eluting product collected. The product was analysed as follows.

Protein determination" 250 gl was diluted 1"3 and the absorbance at 280 nm found to be 0.062. Using an extinction coefficient of 1.5 for 1 mg m1-1 solution, the protein concentration

129

This is S/P,

i.e. 2.3 mol mol-1.

Preparative reaction and conjugation The above trial thiolation reaction was repeated but with all volumes increased by a factor of 5. The reaction was passed through a Pharmacia PD-10 column (0.6 ml sample, followed by 2.1 ml buffer), then collecting 1.2 ml product (ca 2.5 nmol). This was immediately (without analysis) added to 0.87 ml of maleimido alkaline phosphatase (6.0 nmol). The reaction mixture was concentrated to ca 0.5 ml using a centrifugal microconcentrator (Centricon) and the conjugation allowed to proceed at room temperature for 2.5 h.

Conjugate purification: The reaction was applied to a Superose 6 FPLC column, equilibrated in PBS containing 0.1% sodium azide at 0.25 ml min -1, collecting 0.5 ml fractions. Fractions 28-31, comprising essentially 1:1 and 2:1 components, were pooled.

APPENDIX

12

HPLC of peptides and oligonucleotides

In general, no change to standard HPLC protocols is required to analyse or separate labelled peptides and oligonucleotides. Here are some basic protocols which may be modified to suit individual needs.

1. Peptide purification

2. Oligonucleotide purification

Reverse phase The following system is recommended for oligonucleotides up to ca 40 nucleotides long and for oligonucleotides containing biotin, trityl groups, dyes etc. Column:

Reverse phase Column: Eluent A: Eluent B:

C18 silica (Vydac, Protein and Peptide C18 (218TP54)). 0.1% trifluoroacetic acid in water. 0.1% trifluoroacetic acid in aceto-

Eluent A: Eluent B: Gradient:

B.

nitrile. Gradient:

C18 silica (Waters Micro Bondapak). 0.1 M triethylammonium acetate, pH 7.0. acetonitrile. linear, 30 min, up to 80% or 100%

linear, 60 min, 5-90% B.

Ion exchange Column: E l u e n t A: a

Eluent B :a a

Partisphere 5-SAX (Whatman). 1 mM potassium phosphate, pH 6.3. 300 mM potassium phosphate, pH 6.3.

Both A and B containing 60% formamide.

Index

Absorbance spectrum, use of 56-7, 107 N-Acetylhomocysteine thiolactone 39 S-Acetylmercaptosuccinic acid anhydride 40 Acridinium ester chemiluminescence 7, 8 Acylation reactions 23 Aequorin 8 Affinity chromatography 47 Alkaline phosphatase 9, 10-14, 15 amino-oligonucleotides 79 conjugation protocol 80 hybridization/detection protocol 81-2 amplification systems 13 bioluminescence systems 13 chemiluminescence 8, 12-13 colour detection 11-12 conjugate purification 46-7 conjugate storage 47-8 fluorescence 5, 12 gene fusion conjugates 36 maleimido groups introduction using SMCC 42 monoclonal antibody labelling 128-9 nucleic acid probe labelling 90 filter hybridization detection system 92 oligonucleotide labelling 73 substrates 5, 9, 11 thiolation-based conjugation kits 38 thyroxine T4 coupling 107, 108, 109 Amerlite system 10 Amine-directed reagents chemistry 23-6 changing groups 127-8 fluorophore reagents 53, 54

handling 31-32 tagging reagents 67-8 Amino acid analysis 107-8 Amino acid functional groups 21-3 pKa values 123 Amino group reactions 23-6 acylation 23 buffers 25 degree of modification control 24, 26 with imidoesters 24 with isothiocyanates 24 minimal modification 22-3 molar ratios 26 nucleic acid probes labelling 92 oligonucleotide labelling s e e Oligonucleotide labelling protein concentration 25-6 reaction temperature 26 reaction time 26 reagent concentrations 24, 26 side chain reactivity 21-2 small biomolecules labelling 104 specificity 24 with sulphonyl halides 24 Aminolink-2 77, 78 Ammonium sulphate precipitates 25 Ampicillin labelling 109 Antibodies anti-hapten tag 66-7 buffers 25 pyridyldithio groups introduction with SPDP 44 thiolation 37 TM

132

Index

Antibody-enzyme conjugates 33-49 gene fusion conjugates 36 minimal modification 22-3 periodate conjugation method 48-9 purification 46-7 thiolation-based schemes 36-7 disulphides reduction 38 2-iminothiolane (Traut's reagent) 39 Antigenic fusion protein tags 70-1 Arginine reactions 29-30 ATP assay 8 Attophos ~' 12 Avidin 64, 66 biotin binding affinity 66

BCIP/NBT 11 labelled nucleic acids filter~in situ hybridization 92 Beads as labels 15 Benzodiazepine labelling 110 I]-Galactosidase 9, 13-14 gene fusion conjugates 36 substrates 9, 13-14 fluorescent 5 Bicarbonate buffers 25 Bilin prosthetic groups 53-4 Biocytin 68 Bioluminescence 6 alkaline phosphatase system 13 definition 2 proteins/enzymes 8 Biotin 2, 64, 65, 66, 67 avidin/streptavidin binding affinity 66 fusion protein tags 70 glycoprotein carbohydrate tagging protocol 69 hydrazide tagging reagents 68, 69 nucleic acid probes enzymic labelling 90, 93 nick translation DNA labelling 93 RNA labelling 97 using PCR 96 nucleic acid probes photolabelling 100 nucleoside triphosphates 90, 91 detection 92 oligonucleotides 74 primary amino-oligonucleotide tagging 82, 83 peptide tagging 70 phosphoramidites 74-5 protein amino group coupling 67 with biotin-LC-NHS 67 protein carboxyl group coupling 68 protein thiol group coupling 68 tagged proteins characterization 69-70

tyrosine/histidine coupling 69 BODIPY FL 52 TM

c-myc tag 70 Carbodi-imide 28-9, 35, 106-7, 128 Carbohydrate group protein conjugation 30, 48-9 tagging methods 69 Carboxyl-directed reagents peptide tagging 70 protein tagging 68 small biomolecules labelling 10, 104 protocol 106 Carboxylic acid group reactions 28-9 Carrier conjugation methods 104 Cascade Blue TM53 CDP-Star 12, 92 CEDIA (cloned enzyme donor immunoassay) 13 Chemifluorescence 2 Chemiluminescence 2, 4, 6-9 alkaline phosphatase detection 12-13 electrochemiluminescence 8-9 enhanced 10 enzyme-generated 8 flash kinetics 8 horseradish peroxidase (HRP) detection 10 sensitivity 7, 8 Chemilumophore emission wavelengths 118 Chemiprobe nucleic acid labelling 98-9 Colour detection 125-6 alkaline phosphatase 11-12 horseradish peroxidase (HRP) 9-10 Confocal fluorescence microscopy 4 Coumarin derivatives 53 Cross-linking reagents 33-4, 40-1 CSPD ~' 12, 92 Cy-3 dye 52 nucleic acid probes labelling 92 Cy-5 dye 52, 53 nucleic acid probes labelling 92 R-phycoerythrin conjugate 55 Cy-7 dye 52 Cyanine (Cy) dyes 52-3 nucleic acid probes labelling 92 nick translation DNA labelling 93 phosphoramidite oligonucleotide labelling 76 Cyberfluor system 6 TM

DELFIA~ system 5, 6 amino-oligonucleotide labelling 82 labelling reagent (DTTA) 60 labelling method 60

Index p-Diazobenzoyl biocytin 69 Dicarboxylic acid cross-linking agents 36 Dichlorotriazine derivatives 55 Digoxigenin (DIG) 64, 66, 67 glycoprotein carbohydrate tagging 69 maleimido derivatives 68 NHS ester reagent tagging method 67-8 nucleic acid probes enzymic labelling 90, 91, 92, 93 detection 92 hybridization/chemiluminescent detection protocol 94 labelled RNA preparation protocol 98 nick translation DNA labelling 93 random primer labelling protocol 95-6 using PCR 96, 97 nucleic acid probes photolabelling 100 3' oligonucleotide tailing 85 primary amino-oligonucleotide tagging 82 protein carboxyl group coupling 68 small biomolecules labelling 109 Dimethoxyfluorescein 52, 62 Dioxetane-based chemiluminescent substrates 12-13, 14 labelled nucleic acids filter hybridization 92 mechanism of light generation 11 oligonucleotide labelling 73 sensitivity 13 Disulphide conjugates 27 using SPDP and thiols 36-48 DNA s e e Nucleic acid probes a n d Oligonucleotide labelling DNP (dinitrophenyl) oligonucleotide labelling 75 phosphoramidite multi-addition reagent 75-6 tagging 68 DTNB s e e Ellman's reagent Dysprosium chelate labels 5

ECL

Electrochemiluminescence a n d Renz/ ECL nucleic acid labelling EDTA, in buffers 28 Electrochemiluminescence 8-9 Ellman's reagent 27, 28, 44, 58, 59, 68 determination of thiol groups 119 Endothelin- 1 biotinylation 70 Energy transfer assays 5 fluorophores 52 protease substrates fluorescent labelling 61-3 Enhanced chemiluminescence 10 Enzymatic labelling 9-14, 15 nucleic acid probes s e e Nucleic acid probes oligonucleotide labelling 84-6 see

133

3' end tailing 84-6 5' end 86 Enzyme-amplified lanthanide luminescence (EALL) 12 Enzyme-generated chemiluminescence 8 Enzyme-generated fluorescence 5 Eosin 52, 62 Erythrocin 52 ES mass spectrometry 58 Europium chelate labelling 5, 6 nucleic acid probes 92 primary amino-oligonucleotides 82 proteins 60-1 small biomolecules 110 Extinction coefficients 117

Fab' fragments enzyme conjugation to thiols 38 fluorescent labelling 59 preparation protocol 38 FAM oligonucleotide labelling 75, 76 Fast protein liquid chromatography (FPLC) 46 Fc portion conjugation via carbohydrate groups 48-9 thiols introduction 40 Firefly luciferin alkaline phosphatase 13 enhanced chemiluminescence 10 FLAG peptide tag 70 Flow cytometry fluorophores 52, 54, 55 multi-label 55 Fluorescein 12, 52, 55, 67 aminolinked oligonucleotide tagging 82, 83 FRET protease substrate preparation 62 glycoprotein carbohydrate tagging 69 iodoacetyl derivatives 68 maleimido derivatives 68 NHS ester reagent protein labelling 59 tagging method 67-8 nucleic acid probes labelling 66, 90, 91, 92, 93 oligonucleotide labelling 76 3' end tailing 85 protein carboxyl group coupling 68 self-quenching (inner filter effect) 56, 76 small biomolecules labelling 109 Fluorescein dichlorotriazide (DTAF) 55 Fluorescein isothiocyanate (FITC) 55 protein labelling 57, 68 Fluorescence 2, 4-5, 57 enzyme-generated 5 alkaline phosphatase 12

134

Index

Fluorescence ( c o n t i n u e d ) horseradish peroxidase (HRP) 10 Fluorescence correlation spectroscopy 4, 5 Fluorescence in situ hybridization (FISH) 91, 92 Fluorescence polarization 5 Fluorescence-activated cell sorting (FACS) 4 fluorophores 54 Fluorescent deoxynucleoside triphosphates 90 Fluorescent label 2, 4-5, 15 noise avoidance 4 oligonucleotide labelling 72 sensitivity 4 Fluorescent labelling 51-63 characterization of product 56-8 absorbance spectrum 56-7 fluorescence characterization 57 mass spectrometry 58 reactive group analysis 58 degree of substitution 55-6 with europium chelates 60-1 fluorophore selection 51-5 labelled proteins storage 60 linkage chemistry 55 nucleic acids enzymic labelling 92 using PCR 96 with organic fluorophores 58-60 practical aspects 59-60 peptides 61-3 primary amino derivatized oligonucleotides 79, 82 self-quenching (inner filter effect) 56, 57 Fluorescent tags 66 Fluorophores 4 fluorescent properties 53 light sensitivity 59 oligonucleotide labelling 76 organic 52-3 protein labelling 58-60 phycobiliproteins 53-5 physicochemical properties 52 purity of reagents 59 selection 51-5 sensitivity 51 small biomolecules labelling 109 stability 51-2 Stokes' shift 52 time-resolved see Lanthanide chelates wavelengths 52, 118 Folate labelling 109 FRET (fluorescence resonance energy transfer) 61 Functional group analysis 107, 108-9 Functional groups conversion 126-7 Fusion protein tags 70-1

Gel filtration 120-1 conjugate purification 46-7 fluorescent labelled proteins separation 59 labelled peptides 61 Gene fusion protein-protein conjugates 36 Glucose-6-phosphate dehydrogenase 14 Gluteraldehyde 35-6 Glycoproteins 21 carbohydrate biotinylation protocol 69 protein conjugation via carbohydrate groups 35 tagging see Tagging methods Gold labelling 14 Green fluorescent protein (GFP) 4 chemiluminescent energy transfer 7

Haloacetyl reagents 27-8 Hapten carrier conjugates 29, 104 conjugate characterization 107, 108 Hapten tags 64, 66 anti-hapten binding affinity 66 anti-tag antibodies 66-7 hydroxyl groups labelling 106 nucleic acid probes enzymic labelling 91 detection 92 RNA labelling 97 HEPES buffer 25 Heterobifunctional reagents, protein-protein conjugation 35 schemes involving thiolation 36-41 HEX oligonucleotide labelling 75, 76 6-His tags 70-1 Histidine reactions 29 Homobifunctional reagents 34-5 Homogeneous enzyme immunoassays 14 Horseradish peroxidase (HRP) 9-10, 15 amplification systems 10 chemiluminescence 8, 10 colour detection 9-10 conjugate purification 46 conjugate storage 47-8 conjugation via carbohydrate groups (periodate method) 48-9 fluorescence detection 10 maleimido groups introduction using SMCC 43 nucleic acid labelling incorporation of tags 90 Renz/ECL method 97-8, 100 primary (aliphatic) amines coupling 104 Ni chelate conjugate preparation protocol 105

Index primary amino-oligonucleotide labelling protocol 80-1 substrates 9, 10, 125-6 thiolation-based conjugation kits 38 HPLC 130 labelled oligonucleotide 76 labelled peptides 61 small biomolecule conjugates 110 Hydrazide tagging reagents 68, 69 Hydroxyl groups labelling 106-7 N-Hydroxysuccinimide esters s e e NHS esters

Imidoester amino group reactions 24 2-Iminothiolane (Traut's reagent) 39 Immunoassay 7, 8, 11, 12, 13 enhanced chemiluminescence 10 fluorophores 53, 54 lanthanide chelate labels 6 protein-protein conjugates 34 small biomolecules labelling 104 Immunochemistry, fluorophores 52, 53, 54 Immuno-PCR 14 Intermediates characterization protein-protein conjugates 34, 35 thiol-reactive groups introduction 45 Iodoacetyl derivatives 28 biotinylated proteins 68 characterization 43 fluorescent labelling 55, 58-9 light sensitivity 43 specificity 43 stability 43 thiols introduction into proteins 41 Iodophenol 10 Isoluminol system 7 Isothiocyanates 25 amino group reactions 24 fluorescent labelling 55, 58 JOE oligonucleotide labelling 76

Labels definition 1-2 selection 2-4, 15 sensitivity 3-4, 15 Lanthanide chelates 5-6, 55 small biomolecule labelling 110 LC-SPDP 39 Lucifer Yellow TM 53 Luciferases 8 Luminescent signals 2

135

classification 3 Luminol system 7 horseradish peroxidase (HRP) detection 10 Lysozyme immunoassay 14

Malate dehydrogenase immunoassays 14 MALDI mass spectrometry 58 Maleimido derivatives biotinylated proteins 68 fluorescent labelling 55, 58-9 stability 44 thiol-reactive groups introduction into proteins 40-1 characterization 43 into alkaline phosphatase 42 into horseradish peroxidase (HRP) 43 protocols 42, 43 Maleimido reagents 28 Mass spectrometry 58, 107 MBS 40, 107 4-Methylumbelliferyl phosphate 12, 124 Midivariant RNA reporter system 14 Minimal modification principle 22-3 protein-protein conjugation 34, 35 Molecular weights 115-16

Neutravidin 66 NHS (N-hydroxysuccinimide) esters 23, 24, 25, 26 biotinylation peptide amino groups 70 protein amino groups 67 cyanine (Cy) dyes 53 fluorescent labelling 58, 59 primary amino-oligonucleotide tagging 82, 83 purity/active ester content 59, 122 thiol-reactive groups introduction into proteins 43 iodoacetyl groups 41 maleimido groups 40 pyridyldithio groups 41, 43-4 Nick translation DNA labelling 89, 93 Nomenclature 1-2 Nucleic acid probes 12, 14, 88-100 s e e a l s o Oligonucleotide labelling chemical modification 88, 97-100 photolabelling 99-100 Renz/ECL method 97-8, 100 sulphonation (Chemiprobe TM system) 98-9 direct/indirect labelling 89 enzymic labelling 88, 89-97 chemical handles 92

136

Index

Nucleic acid probes ( c o n t i n u e d ) fluorescent deoxynucleoside triphosphates 9O fluorescent groups labelling 92 incorporation rate 90 nick translation labelling 93 practical aspects 92-3 random primer labelling 93 RNA labelling 96-7 tag detection 92 tag/anti-tag systems 90 tags incorporation 90-2 using PCR 93, 96, 97

Oligonucleotide labelling 72-86 enzymatic labelling 84-6 3' end tailing 84-6 5' end 86 fluorophores 52 introduced primary amino groups 77-83 3' terminus 78 5' terminus 77, 78 enzyme labelling 78, 79, 80-2 fluorescent labelling 79, 82 labelling/tagging options 76 reagents 77 tagging 82-3 within sequence 77-9 introduced thiols 84 3' end 84, 86 5' end 84 attachment of labels 84 phosphodiester backbone 84 phosphoramidite reagents 73-7 biotin phosphoramidites 74-5 purification 76-7 sensitivity 72

Particles as labels 15 PCR nucleic acid labelling 89, 93, 96 with modified primers 96 with nucleoside triphosphate analogues 96 PCR products detection 9, 72 Peptide energy transfer substrates 61-3 Peptide labelling 20, 30-1 amino acid side chain reactivity 31 fluorescent 61-3 minimal modification 23 solubility 30-1 tagging s e e Tagging methods Periodate conjugation method 48-9 Phenanthroline dicarboxylic acid chelate 6

Phosphate buffers 25 Phosphate-buffered saline (PBS) 25 Phosphodiester backbone, oligonucleotide labelling 84 Phosphoramidite reagents amino group introduction 77, 78 multi-addition 74 oligonucleotide labelling 73-7 biotin phosphoramidites 74-5 DNP phosphoramidite 75-6 fluorophore phosphoramidites 76 stability/storage 74 thiols introduction 84 Phosphorescence 2 Phthalocyanine derivatives 53 Phycobiliproteins 4, 53-5 bilin prosthetic groups 53-4 conjugation/conjugates 55 properties 55 Phycoerythrin 55 PinPoint~ system 70 Protein A gene fusion conjugates 36 Protein modification 20-32 amino acid functional groups 21-3 amino group reactions 23-6 arginine reactions 29-30 carbohydrate group reactions 30, 48-9 carboxylic acid group reactions 28-9 histidine reactions 29 minimal modification 22-3 reagents 31-2 thiol group reactions 26-8 tyrosine phenol acylation 29 Protein tagging s e e Tagging methods Protein-protein conjugates 33-50 conjugation schemes 34-6 gene fusion conjugates 36 heterobifunctional reagents 35 homobifunctional reagents 34-5 zero-length cross-linking agents 35-6 ideal properties 34 intermediates characterization 34, 35 minimal modification 34, 35 sensitivity 34 Pyridyldisulphide-activated phycobiliproteins 55 Pyridyldithio groups introduction characterization 43 quantitation 44 specificity 43 thiol-reactive groups introduction into proteins 41, 43-4 protocol using SPDP 44

Index Radioactive tracers 107 Random primer DNA labelling 89, 93 protocol 95-6 Reaction vessels 32 Reagents, practical points 31-2 Renz/ECL nucleic acid labelling 97-8 protocol 100 Rhodamines 52 RNA labelling 96-7 digoxigenin (DIG) labelling protocol 97 ROX oligonucleotide labelling 76 Ruthenium complex labels 8

Samarium chelate labels 5 SATA, thiols introduction into proteins 39-40 SIAB 41, 43 Signals definition 1-2 dynamic range 4 relative sensitivities 3 selection 2-4 Small biomolecules labelling 103-10 enzyme/protein labels 103-9 carboxyls 106, 107 conjugate characterization 107-9 coupling schemes 105 hydroxyl groups 106-7 primary (aliphatic) amines 104-6 thiol-based couplings 107 with low molecular weight labels/tags 109-10 SMCC, maleimido groups introduction into proteins 40, 41 into alkaline phosphatase 42 into horseradish peroxidase (HRP) 43 protocol 42, 43 SMPB, maleimido groups introduction into proteins 40 SMPT 39 SNAP alkaline phosphatase-labelled probes 79 Solubility of reagents 32 Southern blotting 72, 73 SPDP pyridyldithio groups introduction into proteins 41, 43-4 protocol 44 stability of conjugates 44 thiols introduction into proteins 39 Stirring-induced changes 32 Storage of reagents 31-2 Strep-Tag 70 Streptavidin 64, 65, 66 biotin binding affinity 66

137

biotin-labelled nucleic acid probes detection 92 Succinic anhydride 23 Sulpho-LC-SPDP 39 Sulpho-NHS analogues 23 thiol-reactive groups introduction into proteins 43 iodoacetyl groups 41 maleimido groups 40 Sulpho-SPDP 39 Sulphonation (Chemiprobe system), nucleic acid labelling 98-9 Sulphonyl halides amino group reactions 24 fluorophore linkage 55 Suppliers 111-14 Surface plasmon resonance 14-15

Tagging methods 67-70 amino-directed agents 67-8 carbohydrate-directed reagents 69 carboxyl-directed reagents 68 nucleic acids 90-2, 95, 97 oligonucleotides 74-6, 82-5 peptides 70 primary amino-oligonucleotides 82-3 small biomolecules 109 thiol-directed reagents 68 tyrosine-directed reagents 69 Tags 2, 64-71 advantages/disadvantages 64-5 binding agents 66-7 fusion proteins 70-1 nucleic acid probes 90 oligonucleotides 72 selection 66 sensitivity 65 size 65 solution phase assay 65 stability 64-5 TAMRA oligonucleotide labelling 76 TaqmanTM PCR products detection 72 Terbium chelate labels 5 Terminal deoxynucleotidyl transferase, oligonucleotide labelling 84-6 TET oligonucleotide labelling 75, 76 Tetramethylrhodamine 52 FRET protease substrate preparation 62 Texas Red TM52 Thioether conjugates 27-8 Thiol group reactions 26-8 biotin introduction to proteins 68 buffers 28

138

Index

Thiol group reactions (continued) disulphide exchange 27 fluorescent labelling 58-9 fluorophore linkage 55 haloacetyl reagent alkylations 27-8 maleimido reagent alkylations 28 oligonucleotide labelling 84, 86 peptide tagging 70 reactions conditions 28 small biomolecules labelling 107 specificity 28 tagging methods 68 thiols as thiol-reactive groups 43 Thiol-reactive groups introduction into proteins 40-5 characterization 43-4 of intermediates 44 iodoacetyl groups introduction 41 light sensitivity 43 maleimido groups introduction 40-1 optimal degree of modification 44 pyridyldithio groups introduction 41, 43-4 quantitation 45 reagent selection 43 reagents 40, 41 spacer groups 43 specificity 43 stability of conjugate 43, 45 thiols as thiol-reactive groups 43 Thiolation-based protein conjugation schemes 36-7 conjugate purification 46-7 FPLC protocol 46 conjugate storage 47-8 protein-protein conjugation 44-8 blocking reactive groups 45

molar ratio 45 protocol 46 thiol-reactive groups introduction into proteins 40-5 thiols introduction into proteins 37, 38-40 Thiols introduction into proteins 37, 38-40 N-acetylhomocysteine thiolactone 39 S-acetylmercaptosuccinic acid anhydride 40 disulphides reduction 38-9 Fc portion of immunoglobulin 40 2-iminothiolane (Traut's reagent) 39 optimal degree of modification 44 SATA 39-40 SPDP/SPDP analogues 39 thiol content quantification 44 Thyroxine-alkaline phosphatase conjugate 107, 108 preparation protocol 109 Time-resolved fluorescence 4, 5-6, 15 alkaline phosphatase detection system 12 sensitivity 5 TNBS (trinitrobenzene sulphonic acid) 58 Traut's reagent see 2-Iminothiolane Triethanolamine buffers 25 Tris buffer, reactivity 25 Tris-bipyridine europium cryptate 6 TSA system 10 Tyrosine phenol acylation reaction 29 Tyrosine-directed tagging methods 69

Western blotting 10, 12

Zero-length cross-linking agents 35-6