1、鄭重該書來源于，由網(wǎng)友搜集整理并提供，學(xué)習(xí)參考用，請勿用作商業(yè)目的。生物服務(wù)網(wǎng)只是提供地址，如果有任何不妥或者版在 2 個工作日內(nèi)刪除鏈權(quán)，請立即通知生物服務(wù)網(wǎng)，接。生物服務(wù)網(wǎng)，服務(wù)生物人！MSN: .¡¡Page iThe ELISA GuidebookPage iiMETHODS IN MOLECULAR BIOLOGYTMJohn M. Walker, Series Editor170. DNA Arrays: Methods and Protocols, edited by Jang B. Rampal, 2001169. Neurotrophin Protocol

2、s, edited by Robert A. Rush, 2001168. Protein Structure, Stability, and Folding, edited by Kenneth P. Murphy, 2001167. DNA Sequencing Protocols, Second Edition, edited by Colin A. Graham and Alison J. M. Hill, 2001166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001165. SV40 Protoc

3、ols, edited by Leda Raptis, 2001164. Kinesin Protocols, edited by Isabelle Vernos, 2001163. Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R. Mitchelson and Jing Cheng. 2001162. Capillary Electrophoresis of Nucleic Acids, Vo

4、lume 1: The Capillary Electrophoresis System as an Analytical Tool, edited by Keith R. Mitchelson and Jing Cheng, 2001161. Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001160. Nuclease Methods and Protocols, edited by Catherine H. Schein, 2000159. Amino Acid Analysis Protocols, edite

5、d by Catherine Cooper, Nicole Packer, and Keith Williams, 2000158. Gene Knockoout Protocols, edited by Martin J. Tymms and Ismail Kola, 2000157. Mycotoxin Protocols, edited by Mary W. Trucksess and Albert E. Pohland, 2000156. Antigen Processing and Presentation Protocols, edited by Joyce C. Solheim,

6、 2000155. Adipose Tissue Protocols, edited by G¨¦rard Ailhaud, 2000154. Connexin Methods and Protocols, edited by Roberto Bruzzone and Christian Giaume, 2000153. Neuropeptide Y Protocols, edited by Ambikaipakan Balasubramaniam, 2000152. DNA Repair Protocols: Prokaryotic Systems, edited by

7、Pat Vaughan, 2000151. Matrix Mloproteinase Protocols, edited by Ian M. Clark, 2000150. Complement Methods and Protocols, edited by B. Paul Morgan, 2000149. The ELISA Guidebook, edited by John R. Crowther, 2000148. DNA¨CProtein Interactions: Principles and Protocols (2nd ed.), edited by Tom Moss

8、. 2000147. AffiChromatography: Methods and Protocols, edited by Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, and Wolfgang Berthold, 2000146. Mass Spectrometry of Proteins and Peptides, edited by John R. Chapman, 2000145. Bacterial Toxins: Methods and Protocols, edited by Otto Hoist, 2000144. Cal

9、pain Methods and Protocols, edited by John S. Elce, 2000143. Protein Structure Prediction: Methods and Protocols, edited by David Webster, 2000142. Transforming Growth Factor-Beta Protocols, edited by Philip H. Howe, 2000141. Plant Hormone Protocols, edited by Gregory A. Tucker and Jeremy A. Roberts

10、, 2000140. Chaperonin Protocols, edited by Christine Schneider, 2000139. Extracellular Matrix Protocols, edited by Charles Streuli and Michael Grant, 2000138. Chemokine Protocols, edited by Amanda E. I. Proudfoot, Timothy N. C. Wells, and Christine Power, 2000137. Developmental Biology Protocols, Vo

11、lume III, edited by Rocky S. Tuan and Cecilia W. Lo, 2000136. Developmental Biology Protocols, Volume II, edited by Rocky S. Tuan and Cecilia W. Lo, 2000135. Developmental Biology Protocols, Volume I, edited by Rocky S. Tuan and Cecilia W. Lo, 2000134. T Cell Protocols: Development and Activation, e

12、dited by Kelly P. Kearse, 2000133. Gene Targeting Protocols, edited by Eric B. Kmiec. 2000132. Bioinformatics Methods and Protocols, edited by Stephen Misener and Stephen A. Krawetz, 2000131. Flavoprotein Protocols, edited by S. K. Chapman and G. A. Reid, 1999130. Transcription Factor Protocols, edi

13、ted by Martin J. Tymms, 2000129. Integrin Protocols, edited by Anthony Howlett, 1999128. NMDA Protocols, edited by Min Li, 1999127. Molecular Methods in Developmental Biology: Xenopus and Zebrafish, edited by Matthew Guille, 1999126. Adrenergic Receptor Protocols, edited by Curtis A. Machida, 200012

14、5. Glycoprotein Methods and Protocols: The Mucins, edited by Anthony P. Corfield, 2000124. Protein Kinase Protocols, edited by Alastair D. Reith, 2000123. In Situ Hybridization Protocols (2nd ed.), edited by Ian A. Darby, 2000122. Confocal Microscopy Methods and Protocols, edited by Stephen W. Paddo

15、ck, 1999121. Natural Killer Cell Protocols: Cellular and Molecular Methods, edited by Kerry S. Campbell and Marco Colonna, 2000120. Eicosanoid Protocols, edited by Elias A. Lianos, 1999119. Chromatin Protocols, edited by Peter B. Becker, 1999118. RNA¨CProtein Interaction Protocols, edited by Su

16、san R. Haynes, 1999117. Electron Microscopy Methods and Protocols, edited by M. A. Nasser Hajibagheri, 1999116. Protein Lipidation Protocols, edited by Michael H. Gelb, 1999115. Immunocytochemical Methods and Protocols (2nd ed.), edited by Lorette C. Javois, 1999114. Calcium Signaling Protocols, edi

17、ted by David G. Lambert, 1999113. DNA Repair Protocols: Eukaryotic Systems, edited by Daryl S. Henderson, 1999112. 2-D Proteome Analysis Protocols, edited by Andrew J. Link, 1999111. Plant Cell Culture Protocols, edited by Robert D. Hall, 1999110. Lipoprotein Protocols, edited by Jose M. Ordovas, 19

18、98109. Lipase and Phospholipase Protocols, edited by Mark H. Doolittle and Karen Reue, 1999108. Free Radical and Antioxidant Protocols, edited by Donald Armstrong, 1998Page iiiThe ELISA GuidebookByJohn R. CrowtherThe International Atomic Energy Agency, Vienna, AustriaMETHODS IN MOLECULAR BIOLOGYTMCo

19、ntentsPrefacev1Overview of ELISA in Relation to Other Disciplines12Systems in ELISA93Stages in ELISA454Titration of Reagents835Theoretical Considerations1156Practical Exercises1537Monoclonal Antibodies2338Validation of Diagnostic Tests for Infectious Diseases3019Charting Methods for Internal Quality

20、 Control34710Immunochemical Techniques39511Test Questions407415IndexPage iv© 2001 Humana Press Inc.999 Riverview Drive, Suite 208 Totowa, New Jersey 07512s. No part of this book may be reproduced, stored in a retrieval system, or transmittedin any form or by any means, electronic, mechanical, p

21、hotocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular BiologyTM is a trademark of The Humana Press Inc.All authored papers, comments, opinions,s, or recommendations are those of the author(s),and do not necessarily reflect the views of

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25、 Humana Press Inc. The fee code for users of the Transactional Reporting Service is: 0-89603-728-2/01 $10.00 + $00.25.Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1Library of Congress Cataloging in Publication DataMain entry under title:Methods in molecular biologyTM.The ELISA guidebo

26、ok/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. biology (Totowa, NJ); v. 149. QP519.9E48 E452001 616.07'56¡

27、70;dc21I. Crowther, J. R.II. Series: Methods in molecular99-087692CIPPage vPrefaceThe 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 ser

28、ies 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 r

29、eorganization 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,

30、 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

31、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 as

32、says 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 consti

33、tutes 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 systemsin commercial applications of ELISA; however, there is stiljor use for more manual t

34、echniques inthe development of assays, and for routine use in laboratories with lesser facilities. A thorough understand-Page viing 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 protein

35、s produced in animals inresponse to antigenic stimuli. Antibodies are specific chemicals that bind to theused for theirproduction; thus, they can be used to detect particularif binding can be demonstrated.Conversely, specific antibodies can be measured by the use of defined of many assays in diagnos

36、tic biology., and this forms the basisBesides 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 ex

37、amination 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

38、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 fromi

39、nvestigations of the ability of enzyme-labeled antibodies (1¨C3) to identifyin tissue. Themethods 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 wel

40、l 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 r

41、eviews 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 viiAdvantages of ELISA(a) Reagents added in small volumes(b) Separation of bound and free reactant

42、s is made by simple washing procedures(c) Passive adsorption of proteins to plastic is easy(d) Specialized equipment readily available1. Simplicity2. 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

43、 in RIA)(b) Multichannel spectrophotometers quantify results that can be examined statistically3. Rapidity(a) Tests can be performed in a few hours(b) Spectrophotometric reading of results is rapid (96 wells read in 5 s)4. SensitivityDetection levels of 0.01 to 1 µg/mL are easily and consistent

44、ly achievable. These levels are ideal for most diagnostic purposes5. ReagentsCommercially available reagents offer great flexibility in ELISA design and achievement of specific assays6. AdaptabilityDifferent configurations allow different methods to be examined to solve problems. This is useful in d

45、eveloping tests and research science7. Cost(a) Startup costs are low(b) Reagent costs are low8. AcceptabilityFully standardized ELISAs in many fields are now accepted as "gold- standard" assays9. SafetySafe nonmutagenic reagents are available. Disposal of waste poses no problem (unlike rad

46、ioactivity)10. AvailabilityELISAs can be performed anywhere, even in laboratories where facilities are less than state of the art11. KitsELISA kits are widespread and successful12. StandardizationQuantification of data allows easier standardizationAll the key elements listed will be examined in deta

47、il 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 sever

48、al excellent textbooks, including Roitt and colleagues (22), that should be read. Immunochemical methods are also important, e.g., in purifying andexploitingand antibodies, and for conjugat-Page viiiing proteins. An excellent manual covering all aspects of immunochemistry is available Harlow and Lan

49、e (23), which also outlines many relevant laboratory practices.JOHN R. CROWTHER, PHDReferences1. Avrameas, S. (1966) Methode de marquage d'antigenes et d'anticorps avec des enzymes et son application en immunodiffusion. Comptes Rendus Hendomadaires des Seances de l'Acadamie des Sciences:

50、 D: Sciences naturelles (Paris), 262, 2543¨C2545.2. Nakane, P. K. and Pierce, G. B. (1966) Enzyme-labelled antibodies: preparation and application forthe localization of. J. Histochem. Cytochem. 14, 929¨C931.3. Avrameas, S. (1969) Coupling of enzymes to proteins with gluteraldehyde. Use of

51、 the conjugates forthe detection ofand antibodies. Immunochemistry 6, 43¨C52.4. Avrameas, S. and Guilbert, B. (1971) Dosage enzymo immunologique de proteines a l'aide d'immunosadorbants et d'antigenes marques aux enzymes. Comptes Rendus Hendomadaires des Seances de l'Acadamie de

52、s Sciences: D: Sciences naturelles (Paris), 273, 2705¨C2707.5. Engvall, E. and Perlman, P. (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871¨C874.6. Van Weeman, B. K. and Schuurs, A. H. W. M. (1971) Immunoassay using antigen-en

53、zyme conjugates. FEBS Lett. 15, 232¨C236.7. Voller, A., Bidwell, D. E., Huldt, G., and Engvall, E. (1974) A microplate method of enzyme linked immunosorbent assay and its application to malaria. Bull. World Health Organ. 51, 209.8. Burgess, G. W. (ed.) (1988) ELISA technology in diagnosis and r

54、esearch. Graduate School of Tropical Veterinary Science, James Cook University of North Queensland, Townsville, Australia.9. Collins, W. P. (1985) Alternative Immunoassays. Wiley, Chichester, UK.10. Collins, W. P. (1985) Complimentary Immunoassays. Wiley, Chichester, UK.11. Crowther, J. R. (1995) EL

55、ISA Theory and Practice. Humana Press, Totowa, NJ.12. Ishikawa, E., Kawia, T., and Miyai, K. (1981)Enzyme Immunoassay. Igaku-Shoin, Tokyo, Japan.13. Kemeny, D. M. and Challacombe, S. J. (1988) ELISA and Other Solid-Phase Immunoassays. Theoretical and Practical Aspects. Wiley, Chichester, UK.14. Magg

56、io, T. (1979) The Enzyme Immunoassay. CRC, New York.15. Ngo, T. T. and Leshoff, H. M. (1985) Enzyme-Mediated Immunoassay. Plenum, New York.16. Voller, A., Bidwell, D. E., and Bartlett, A. (1979) The Enzyme-Linked Immunosorbent Assay (ELISA). Dynatech Europe, UK.17. Avrameas, S., Ternynck, T., and Gu

57、esdon, J. L. (1978) Coupling of enzymes to antibodies and. Scand. J. Immunol. 8(Suppl 7), 7¨C23.18. Blake, C. and Gould, B. J. (1984) Use of enzymes in immunoassay techniques. A review. Analyst109, 533.Page ix19. Guilbault, G. G. (1968) Use of enzymes in analytical chemistry. Anal. Chem. 40, 45

58、9.20. Kemeny, D. M. and Challacombe, S. J. (1986) Advances in ELISA and other solid-phase immunoassays. Immunol. Today 7, 67.21. Voller, A., Bartlett, A., and Bidwell, D. E. (1981) Immunoassays for the 80s. MTP Press, Lancaster, UK, pp. 457¨C479.22. Roitt, I. M., Brostoff, J., and Male, D. K. (1993) Immunology, 3rd ed. Mosby, St. Louis, MO.23. Harlow, E. and Lane, D. (eds.) (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Page 11¡ªOverview of