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 (