The Elisa guidebook

Contents

¡¡

Page i

The ELISA Guidebook

Page ii

METHODS IN MOLECULAR BIOLOGYTM

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

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 BIOLOGYTM

Page iv

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Methods in molecular biologyTM.

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 616.07'56¡ªdc21

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

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-