The Elisa Guidebook

446 Pages·2000·6.014 MB·English

by  John R. Crowther (auth.)

¡¡
The ELISA Guidebook
By
John R. Crowther
The International Atomic Energy Agency, Vienna, Austria
METHODS IN MOLECULAR BIOLOGYTM
¡¡
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
415
Index
¡¡

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
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 BIOLOGYTM
Page iv
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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-