¡¡ The ELISA Guidebook By John R. Crowther The International Atomic Energy Agency, Vienna, Austria METHODS IN MOLECULAR BIOLOGY TM ¡¡ Contents Preface v 1 Overview of ELISA in Relation to Other Disciplines 1 2 Systems in ELISA 9 3 Stages in ELISA 45 4 Titration of Reagents 83 5 Theoretical Considerations 115 6 Practical Exercises 153 7 Monoclonal Antibodies 233 8 Validation of Diagnostic Tests for Infectious Diseases 301 9 Charting Methods for Internal Quality Control 347 10 Immunochemical Techniques 395 11 Test Questions 407 Index 415 ¡¡ Page i The ELISA Guidebook Page ii METHODS IN MOLECULAR BIOLOGY TM John M. Walker, Series Editor 170. DNA Arrays: Methods and Protocols, edited by Jang B. Rampal, 2001 169. Neurotrophin Protocols, edited by Robert A. Rush, 2001 168. Protein Structure, Stability, and Folding, edited by Kenneth P. Murphy, 2001 167. DNA Sequencing Protocols, Second Edition, edited by Colin A. Graham and Alison J. M. Hill, 2001 166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001 165. SV40 Protocols, edited by Leda Raptis, 2001 164. Kinesin Protocols, edited by Isabelle Vernos, 2001 163. Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R. Mitchelson and Jing Cheng. 2001 162. Capillary Electrophoresis of Nucleic Acids, Volume 1: The Capillary Electrophoresis System as an Analytical Tool, edited by Keith R. Mitchelson and Jing Cheng, 2001 161. Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001 160. Nuclease Methods and Protocols, edited by Catherine H. Schein, 2000 159. Amino Acid Analysis Protocols, edited by Catherine Cooper, Nicole Packer, and Keith Williams, 2000 158. Gene Knockoout Protocols, edited by Martin J. Tymms and Ismail Kola, 2000 157. Mycotoxin Protocols, edited by Mary W. Trucksess and Albert E. Pohland, 2000 156. Antigen Processing and Presentation Protocols, edited by Joyce C. Solheim, 2000 155. Adipose Tissue Protocols, edited by G¨¦rard Ailhaud, 2000 154. Connexin Methods and Protocols, edited by Roberto Bruzzone and Christian Giaume, 2000 153. Neuropeptide Y Protocols, edited by Ambikaipakan Balasubramaniam, 2000 152. DNA Repair Protocols: Prokaryotic Systems, edited by Pat Vaughan, 2000 151. Matrix Metalloproteinase Protocols, edited by Ian M. Clark, 2000 150. Complement Methods and Protocols, edited by B. Paul Morgan, 2000 149. The ELISA Guidebook, edited by John R. Crowther, 2000 148. DNA¨CProtein Interactions: Principles and Protocols (2nd ed.), edited by Tom Moss. 2000 147. Affinity Chromatography: Methods and Protocols, edited by Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, and Wolfgang Berthold, 2000 146. Mass Spectrometry of Proteins and Peptides, edited by John R. Chapman, 2000 145. Bacterial Toxins: Methods and Protocols, edited by Otto Hoist, 2000 144. Calpain Methods and Protocols, edited by John S. Elce, 2000 143. Protein Structure Prediction: Methods and Protocols, edited by David Webster, 2000 142. Transforming Growth Factor-Beta Protocols, edited by Philip H. Howe, 2000 141. Plant Hormone Protocols, edited by Gregory A. Tucker and Jeremy A. Roberts, 2000 140. Chaperonin Protocols, edited by Christine Schneider, 2000 139. Extracellular Matrix Protocols, edited by Charles Streuli and Michael Grant, 2000 138. Chemokine Protocols, edited by Amanda E. I. Proudfoot, Timothy N. C. Wells, and Christine Power, 2000 137. Developmental Biology Protocols, Volume III, edited by Rocky S. Tuan and Cecilia W. Lo, 2000 136. Developmental Biology Protocols, Volume II, edited by Rocky S. Tuan and Cecilia W. Lo, 2000 135. Developmental Biology Protocols, Volume I, edited by Rocky S. Tuan and Cecilia W. Lo, 2000 134. T Cell Protocols: Development and Activation, edited by Kelly P. Kearse, 2000 133. Gene Targeting Protocols, edited by Eric B. Kmiec. 2000 132. Bioinformatics Methods and Protocols, edited by Stephen Misener and Stephen A. Krawetz, 2000 131. Flavoprotein Protocols, edited by S. K. Chapman and G. A. Reid, 1999 130. Transcription Factor Protocols, edited by Martin J. Tymms, 2000 129. Integrin Protocols, edited by Anthony Howlett, 1999 128. NMDA Protocols, edited by Min Li, 1999 127. Molecular Methods in Developmental Biology: Xenopus and Zebrafish, edited by Matthew Guille, 1999 126. Adrenergic Receptor Protocols, edited by Curtis A. Machida, 2000 125. Glycoprotein Methods and Protocols: The Mucins, edited by Anthony P. Corfield, 2000 124. Protein Kinase Protocols, edited by Alastair D. Reith, 2000 123. In Situ Hybridization Protocols (2nd ed.), edited by Ian A. Darby, 2000 122. Confocal Microscopy Methods and Protocols, edited by Stephen W. Paddock, 1999 121. Natural Killer Cell Protocols: Cellular and Molecular Methods, edited by Kerry S. Campbell and Marco Colonna, 2000 120. Eicosanoid Protocols, edited by Elias A. Lianos, 1999 119. Chromatin Protocols, edited by Peter B. Becker, 1999 118. RNA¨CProtein Interaction Protocols, edited by Susan R. Haynes, 1999 117. Electron Microscopy Methods and Protocols, edited by M. A. Nasser Hajibagheri, 1999 116. Protein Lipidation Protocols, edited by Michael H. Gelb, 1999 115. Immunocytochemical Methods and Protocols (2nd ed.), edited by Lorette C. Javois, 1999 114. Calcium Signaling Protocols, edited by David G. Lambert, 1999 113. DNA Repair Protocols: Eukaryotic Systems, edited by Daryl S. Henderson, 1999 112. 2-D Proteome Analysis Protocols, edited by Andrew J. Link, 1999 111. Plant Cell Culture Protocols, edited by Robert D. Hall, 1999 110. Lipoprotein Protocols, edited by Jose M. Ordovas, 1998 109. Lipase and Phospholipase Protocols, edited by Mark H. Doolittle and Karen Reue, 1999 108. Free Radical and Antioxidant Protocols, edited by Donald Armstrong, 1998 Page iii The ELISA Guidebook By John R. Crowther The International Atomic Energy Agency, Vienna, Austria METHODS IN MOLECULAR BIOLOGY TM Page iv © 2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology TM is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. C0004- 01.gif ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Patricia F. Cleary. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Main entry under title: Methods in molecular biology TM . The ELISA guidebook/by John R. Crowther. p. cm.¡ª(Methods in molecular biology; 149) Includes bibliographical references and index. Comb: ISBN 0-89603-728-2 (alk. paper); hardcover: ISBN 0-89603-950-1. 1. Enzyme-linked immunosorbent assay. I. Crowther, J. R. II. Series: Methods in molecular biology (Totowa, NJ); v. 149. QP519.9E48 E45 2001 99-087692 616.07'56¡ªdc21 CIP Page v Preface The aim of The ELISA Guidebook is to expand the information concerning enzyme-linked immunosorbent assay (ELISA) published in ELISA: Theory and Practice by J. R. Crowther (1995), in the Methods in Molecular Biology series by Humana Press (vol. 42). The earlier book concentrated on the immunological background of the reagents exploited in such assays, and dealt practically with the various assays, through examples using noninfectious systems. This new volume is a major extension and updating of that book, with a reorganization of the chapters, and extra information dealing, in particular, with chessboard titration of reagents, quality control, monoclonal antibodies, validation of assays, statistics, and epidemiological considerations. Suitable for scientists with previous experience of the technique, it can, however, be used successfully by those with little experience, and as a teaching aid. The ELISA Guidebook deals with heterogeneous enzyme-linked immunosorbent assays. The abbreviation ELISA, or in the plural ELISAs, will be used from now on to denote this kind of assay. Besides the inherent feature of all ELISAs¡ªthat there is an enzyme linked to one of the reagents¡ªheterogeneous assays involve the attachment of one reagent to a solid phase and subsequent addition of reagents that bind. The separation of bound and free components is necessary through washing steps. Such assays must be distinguished from homogeneous ELISAs, in which reagents are added simultaneously. ELISAs remain the mainstay of testing in which the specificity inherent in antibodies is exploited. The technique is still expanding in all fields of pure and applied biology, and in particular, now constitutes a backbone diagnostic technique. Recent applications into quality assessment of foods for contaminants is testimony to the flexibility for these possible systems. There is an increasing use of automated systems in commercial applications of ELISA; however, there is still a major use for more manual techniques in the development of assays, and for routine use in laboratories with lesser facilities. A thorough understand- Page vi ing of the principles is vital to the proper use of ELISA, even where established kits are provided. The key to all ELISA systems is the use of antibodies. These are proteins produced in animals in response to antigenic stimuli. Antibodies are specific chemicals that bind to the antigens used for their production; thus, they can be used to detect particular antigens if binding can be demonstrated. Conversely, specific antibodies can be measured by the use of defined antigens, and this forms the basis of many assays in diagnostic biology. Besides covering the various assay parameters, the basic reagents, and the skills needed to perform ELISA, The ELISA Guidebook introduces these increasingly important topics: quality control of testing; kit production; validation; statistical requirements for examination of data and for epidemiological studies; equipment choice, care, and calibration; technology transfer; and monoclonal antibodies. Wherever possible, explanations are provided in diagrammatic, as well as written, form. The text may, in places, seem repetitious. However, in the experience of the author, and through feedback from the previous publication, readers respond very differently to various approaches, so that conveying information by multiple exposures is considered pedagogically useful. Although often reviewed, it is worth considering the beginnings of ELISA, which stemmed from investigations of the ability of enzyme-labeled antibodies (1¨C3) to identify antigens in tissue. The methods of conjugation were exploited to measure serum components in the first "true" ELISAs (4¨C6). By far the most exploited ELISAs use plastic microtiter plates in an 8 ¡Á 12 well format as the solid phase (7). Such systems benefit from a large selection of specialized commercially available equipment including multichannel pipets for the easy simultaneous dispensing of reagents and multichannel spectrophotometers for rapid data capture. There are many books, manuals, and reviews of ELISA and associated subjects that may be examined for more practical details (8¨C21). The following table summarizes some of the features that make ELISA so sustainable a technique. Page vii Advantages of ELISA 1. Simplicity (a) Reagents added in small volumes (b) Separation of bound and free reactants is made by simple washing procedures (c) Passive adsorption of proteins to plastic is easy (d) Specialized equipment readily available 2. Reading (a) Colored end-product can be read by eye to assess whether tests have worked (avoiding waiting for results where machine reading essential as in RIA) (b) Multichannel spectrophotometers quantify results that can be examined statistically 3. Rapidity (a) Tests can be performed in a few hours (b) Spectrophotometric reading of results is rapid (96 wells read in 5 s) 4. Sensitivity Detection levels of 0.01 to 1 µg/mL are easily and consistently achievable. These levels are ideal for most diagnostic purposes 5. Reagents Commercially available reagents offer great flexibility in ELISA design and achievement of specific assays 6. Adaptability Different configurations allow different methods to be examined to solve problems. This is useful in developing tests and research science 7. Cost (a) Startup costs are low (b) Reagent costs are low 8. Acceptability Fully standardized ELISAs in many fields are now accepted as "gold- standard" assays 9. Safety Safe nonmutagenic reagents are available. Disposal of waste poses no problem (unlike radioactivity) 10. Availability ELISAs can be performed anywhere, even in laboratories where facilities are less than state of the art 11. Kits ELISA kits are widespread and successful 12. Standardization Quantification of data allows easier standardization All the key elements listed will be examined in detail in this book. The background needed in immunologic/serologic aspects is not dealt with extensively as a discrete chapter, rather points are included at appropriate times. Scientists involved in developing and using ELISA should be familiar with the concepts inherent in immunology. There are several excellent textbooks, including Roitt and colleagues (22), that should be read. Immunochemical methods are also important, e.g., in purifying and exploiting antigens and antibodies, and for conjugat- [...]... inherent in the test design The knowledge and skills required to both perform ELISA and make use of the data have to be gained through a variety of sources, including textbooks As with all other techniques, the ultimate benefit is not the technique in itself, but the meaningful gathering and analysis of the data One factor not included in all these examples is that of common sense: the ability to really... systems have is the varying degree of nonspecific binding in individual sera This tends to widen the dispersion (variability) in assay results and, therefore, increases the need to process many sera to assess confidence 2.3¡ª Sandwich ELISA Sandwich ELISA can be divided into two systems, which have been named the direct sandwich ELISA and the indirect sandwich ELISA 2.3.1¡ª Direct Sandwich ELISA The direct... direct sandwich ELISA illustrated as follows and in Fig 3 The direct sandwich ELISA, involves the passive attachment of antibodies to the solid phase (stages i and ii) These antibodies (capture antibodies) then Page 18 Fig 3 Direct sandwich ELISA This system exploits antibodies attached to a solid phase to capture antigen The antigen is then detected using serum specific for the antigen The detecting... Direct ELISA Direct ELISA can be regarded as the simplest form of the ELISA, and is illustrated in Fig 1 and in the following diagram Page 13 Antigen is diluted in a buffer (stage i), commonly a high pH (9.6) carbonate/ bicarbonate buffer or neutral phosphate-buffered saline (PBS) The key is that the buffer contains no other proteins that might compete with the target antigen for attachment to the plastic... one of the reactants in the ELISA and one of the main reasons for its success Washing The simple flooding and emptying of the wells with a buffered solution to separate bound (reacted) from unbound (unreacted) reagents in the ELISA Again, this is a key element to the successful exploitation of the ELISA Antigens A protein or carbohydrate that when injected into animals elicits the production of antibodies... to all ELISAs: 1 Direct ELISA 2 Indirect ELISA 3 Sandwich ELISA All three systems can be used to form the basis of a group of assays called competition or inhibition ELISAs The systems (arrangement and use of reagents in the test), are illustrated herein through the use of symbols (as defined in Table 2), as well as in terms In this way, it is hoped that the reader will gain a clear idea of the various... considering then use of ELISA: 1 The ELISA is a tool to solve a problem 2 Any problem should be defined, as clearly as possible, with reference to all previous work defining the specific agent involved and related agents 3 Other methods for analyzing the problem should be reviewed, particularly when tests are already established This has implications if the ELISA is to replace existing tests 4 The capacity... (directly as a color change of the substrate or indirectly by its effect on another chemical) Chromophore A chemical that alters color as a result of an enzyme interaction with substrate Stopping The process of stopping the action of an enzyme on a substrate It has the effect of stopping any further change in color in the ELISA Reading Measurement of color produced in the ELISA This is quantified using... using the ELISA Attention to increasing knowledge in those areas highlighted is essential both in developmental work to produce a working ELISA and in the ultimate value of any test devised A good deal of attention should be directed at defining, as clearly as possible, the objectives for the ELISA The development of a diagnostic test for a specific disease requires that all other data pertaining to the. .. overlook the simplicity of what is needed by being blinded by the technology for its own sake Most problems are relatively simple to examine after some clear thought Thus, the good ELISA Page 8 person will consider the problem first, obtain the necessary technical skills and equipment to perform a test, and then obtain data that is from a planned perspective As much data from all other tests and the scientific . forms the basis of many assays in diagnostic biology. Besides covering the various assay parameters, the basic reagents, and the skills needed to perform ELISA, The ELISA Guidebook introduces these. ¡¡ The ELISA Guidebook By John R. Crowther The International Atomic Energy Agency, Vienna, Austria METHODS IN MOLECULAR BIOLOGY TM ¡¡ Contents Preface v 1 Overview of ELISA in Relation to Other. immunosorbent assays. The abbreviation ELISA, or in the plural ELISAs, will be used from now on to denote this kind of assay. Besides the inherent feature of all ELISAs¡ªthat there is an enzyme