Immunochemical Protocols

329 Pages·2005·4.31 MB·English

by  Robert Burns (auth.)

MMEETTHHOODDSS IINN MMOOLLEECCUULLAARR BBIIOOLLOOGGYYTTMM
Volume 295
IImmmmuunnoocchheemmiiccaall
PPrroottooccoollss
TThhiirrdd EEddiittiioonn
EEddiitteedd bbyy
RRoobbeerrtt BBuurrnnss
Burns_PCS 1 11/16/04, 11:37 AM
Immunochemical Protocols
M E T H O D S I N M O L E C U L A R B I O L O G Y™
John M. Walker, SERIES EDITOR
309. RNA Silencing: Methods and Protocols, edited by 285. Cell Cycle Control and Dysregulation Protocols:
Gordon Carmichael, 2005 Cyclins, Cyclin-Dependent Kinases, and Other
Factors, edited by Antonio Giordano and Gaetano
308. Therapeutic Proteins: Methods and Protocols,
Romano, 2004
edited by C. Mark Smales and David C. James, 2005
284. Signal Transduction Protocols, Second
307. Phosphodiesterase Methods and Protocols, Edition, edited by Robert C. Dickson and
edited by Claire Lugnier, 2005 Michael D. Mendenhall, 2004
306. Receptor Binding Techniques: Second Edition, 283. Bioconjugation Protocols, edited by Christof M.
Niemeyer, 2004
edited by Anthony P. Davenport, 2005
282. Apoptosis Methods and Protocols, edited by
305. Protein–Ligand Interactions: Methods and Hugh J. M. Brady, 2004
Protocols, edited by G. Ulrich Nienhaus, 2005 281. Checkpoint Controls and Cancer, Volume 2:
304. Human Retrovirus Protocols: Virology and Activation and Regulation Protocols, edited by
Molecular Biology, edited by Tuofu Zhu, 2005 Axel H. Schönthal, 2004
280. Checkpoint Controls and Cancer, Volume 1:
303. NanoBiotechnology Protocols, edited by Sandra
Reviews and Model Systems, edited by Axel H.
J. Rosenthal and David W. Wright, 2005
Schönthal, 2004
302. Handbook of ELISPOT: Methods and 279. Nitric Oxide Protocols, Second Edition, edited by
Protocols, edited by Alexander E. Kalyuzhny, Aviv Hassid, 2004
2005 278. Protein NMR Techniques, Second Edition,
301. Ubiquitin–Proteasome Protocols, edited by edited by A. Kristina Downing, 2004
Cam Patterson and Douglas M. Cyr, 2005 277. Trinucleotide Repeat Protocols, edited by
300. Protein Nanotechnology: Protocols, Yoshinori Kohwi, 2004
Instrumentation, and Applications, edited by 276. Capillary Electrophoresis of Proteins
Tuan Vo-Dinh, 2005 and Peptides, edited by Mark A. Strege
299. Amyloid Proteins: Methods and Protocols, and Avinash L. Lagu, 2004
edited by Einar M. Sigurdsson, 2005 275. Chemoinformatics, edited by Jürgen Bajorath,
2004
298. Peptide Synthesis and Application, edited by
274. Photosynthesis Research Protocols, edited by
John Howl, 2005
Robert Carpentier, 2004
297. Forensic DNA Typing Protocols, edited by
273. Platelets and Megakaryocytes, Volume 2:
Angel Carracedo, 2005 Perspectives and Techniques, edited by
296. Cell Cycle Protocols, edited by Tim Humphrey and Jonathan M. Gibbins and Martyn P. Mahaut-
Gavin Brooks, 2005 Smith, 2004
272. Platelets and Megakaryocytes, Volume 1:
295. Immunochemical Protocols, Third Edition,
Functional Assays, edited by Jonathan M.
edited by Robert Burns, 2005
Gibbins and Martyn P. Mahaut-Smith, 2004
294. Cell Migration: Developmental Methods and
271. B Cell Protocols, edited by Hua Gu and Klaus
Protocols, edited by Jun-Lin Guan, 2005 Rajewsky, 2004
293. Laser Capture Microdissection: Methods and 270. Parasite Genomics Protocols, edited by Sara
Protocols, edited by Graeme I. Murray and E. Melville, 2004
Stephanie Curran, 2005 269. Vaccina Virus and Poxvirology: Methods
292. DNA Viruses: Methods and Protocols, edited by and Protocols,edited by Stuart N. Isaacs, 2004
Paul M. Lieberman, 2005 268. Public Health Microbiology: Methods and
291. Molecular Toxicology Protocols, edited by Protocols, edited by John F. T. Spencer and
Alicia L. Ragout de Spencer, 2004
Phouthone Keohavong and Stephen G. Grant, 2005
267. Recombinant Gene Expression: Reviews and
290. Basic Cell Culture, Third Edition, edited by
Protocols, Second Edition, edited by Paulina
Cheryl D. Helgason and Cindy Miller, 2005 Balbas and Argelia Johnson, 2004
289. Epidermal Cells, Methods and Applications, 266. Genomics, Proteomics, and Clinical
edited by Kursad Turksen, 2004 Bacteriology: Methods and Reviews, edited
288. Oligonucleotide Synthesis, Methods and by Neil Woodford and Alan Johnson, 2004
Applications, edited by Piet Herdewijn, 2004 265. RNA Interference, Editing, and
287. Epigenetics Protocols, edited by Trygve O. Modification: Methods and Protocols, edited
Tollefsbol, 2004 by Jonatha M. Gott, 2004
286. Transgenic Plants: Methods and Protocols, 264. Protein Arrays: Methods and Protocols,
edited by Leandro Peña, 2004 edited by Eric Fung, 2004
M E T H O D S I N M O L E C U L A R B I O L O G Y™
Immunochemical
Protocols
Third Edition
Edited by
Robert Burns
Antibody Unit, Scottish Agricultural Science Agency,
Edinburgh, Scotland
© 2005 Humana Press Inc.
999 Riverview Drive, Suite 208
Totowa, New Jersey 07512
www.humanapress.com
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 BiologyTM is a trademark of The
Humana Press Inc.
All 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. ∞
ANSI Z39.48-1984 (American Standards Institute)
Permanence of Paper for Printed Library Materials.
Cover illustration: Immunonosorbent electron migrograph (ISEM) of potato latent virus. Cover art courtesy
of R. Burns, SASA, Edinburgh, Scotland.
Production Editor: C. Tirpak
Cover design by Patricia F. Cleary
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact
Humana at the above address or at any of the following numbers: Tel: [phone]; Fax: [phone];
E-mail: [email protected]; or visit our Website: www.humanapress.com
Photocopy Authorization Policy:
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific
clients, is granted by Humana Press Inc., provided that the base fee of US $25.00 per copy is paid directly
to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations
that have been granted a photocopy license from the CCC, a separate system of payment has been arranged
and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is:
[1-58829-274-6/05 $25.00 ].
eISBN: 1-59259-873-0
ISSN 1064-3745
Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
Library of Congress Cataloging-in-Publication Data
Immunochemical protocols. – 3rd ed. / edited by Robert Burns.
p. ; cm. -- (Methods in molecular biology ; 295)
Includes bibliographical references and index.
ISBN 1-58829-274-6 (alk. paper)
1. Immunochemistry--Laboratory manuals. I. Burns, Robert, 1959-.
II. Series: Methods in molecular biology (Clifton,N.J.) ; v. 295.
[DNLM: 1. Antibodies--isolation & purification--Laboratory
Manuals. 2. Immunochemistry--methods--Laboratory Manuals.
W1 ME9616J v.279 2004 / QW 25 I334 2005]
QR183.6.I43 2005
616.07'9--dc22
2004009405
Preface
Immunochemical techniques have been in use for many years with early
examples of bacterial strain typing dating back to the 1940s. The basis for
the science is the exquisite elegance of the mammalian immune system with
its ability to recognize foreign proteins and to manufacture antibody mol-
ecules that strongly bind to the substances that elicited them. Not only are
potentially harmful pathogens and toxins recognized by the immune system,
but the system can be persuaded to manufacture antibodies to an astonishing
array of substances.
In the early days of this science, all antibodies for investigative work were
produced by immunizing mammals with the substance of interest, followed by
regular donor bleeds that yielded antisera. Serum produced in this way yields
heterogenic populations of antibody molecules recognizing different
epitopes on the target protein, which may be adequate for its intended pur-
poses, but can also cause problems of crossreactivity. In 1975, Kohler and
Milstein reported that spleen cells from immune donor animals could be
immortalized, cloned from single cells, and grown in continuous culture.
This original work described the method for the production of monoclonal
antibodies.
The development of techniques based on antibodies has increased across the
years and the routine use of them is now commonplace in many kinds
of diagnostic and other investigative work. Workers new to the science may
find the array of techniques and reagents bewildering, and this third edition
of Immunochemical Protocols seeks to provide both the basic methods
for producing and using antibodies along with some advanced protocols
that will prove especially valuable to the more experienced worker. It pro-
vides full details of methods for antigen selection and preparation, antibody
production, reagent manufacture, as well as protocols covering many of the
areas where immunochemical techniques are used. Each protocol has been
written by an investigator who has precise, practical knowledge and hands-
on experience of the individual technique. Chapters contain detailed back-
ground to the use of the method, step-by-step instructions and a Notes section
based on the authors’ practical experience.
Immunochemical Protocols, Third Edition should provide workers
with limited experience of antibody technology a broad array of protocols
immediately of use in the laboratory. Experienced workers will find details of
v
vi Preface
alternative methods to those being currently used by them, and should also find
new techniques that they may wish to use for their investigative work.
I would like to thank Dr. Gerry Saddler and the members of Diagnostics, and
Molecular Biology at SASA for their encouragement and support throughout
this project. A special thanks goes to Dr. Alex Reid of SASA for his help in
dealing with the graphics and some of the more unusual file formats.
Robert Burns
Contents
Preface. .............................................................................................................v
Contributors .....................................................................................................ix
1. Immunization Strategies for Antibody Production
Robert Burns.........................................................................................1
2. Synthetic Peptides as Antigens for Antibody Production
David C. Hancock and Nicola J. O’Reilly..........................................13
3. Production of Polyclonal Antibodies in Rabbits
David C. Hancock and Nicola J. O’Reilly..........................................27
4. Making Hybridomas
Robert Burns.......................................................................................41
5. Growing Hybridomas
Gary Entrican and Gareth Young.......................................................55
6. Phage Display Vectors for the In Vitro Generation of Human
Antibody Fragments
Michael Hust and Stefan Dübel..........................................................71
7. Purification of Antibodies and Preparation of Antibody Fragments
Graham D. Kemp................................................................................97
8. Conjugation of Antibodies to Alkaline Phosphatase
G. Brian Wisdom..............................................................................123
9. Conjugation of Antibodies to Horseradish Peroxidase
G. Brian Wisdom..............................................................................127
10. Conjugation of Antibodies to Fluorescein or Rhodamine
G. Brian Wisdom..............................................................................131
11. Conjugation of Antibodies to Biotin
James D. Hirsch and Rosaria P. Haugland.......................................135
12. Preparation of Colloidal Gold Probes
David Hughes....................................................................................155
13. Immunogold Probes in Light Microscopy
David Hughes....................................................................................173
14. Immunoelectron Microscope Techniques in Plant Virus Diagnosis
Daphne M. Wright............................................................................193
15. Dual-Antibody Sandwich Enzyme-Linked Immunosorbent Assay
William Jordan..................................................................................207
vii
viii Contents
16. Competitive Enzyme-Linked Immunosorbent Assay
William Jordan..................................................................................215
17. Immunoblotting Techniques
Barbara Magi and Sabrina Liberatori...............................................227
18. Epitope Mapping
Glenn E. Morris.................................................................................255
19. Generation and Testing of Immunoaffinity Columns
Weilin L. Shelver...............................................................................269
20. Immunocapture-Polymerase Chain Reaction
Vincent Mulholland..........................................................................281
21. Magnetic Cell Sorting
Maciej Zborowski and Jeffrey J. Chalmers.......................................291
22. Cell Enrichment and Immunochemical Staining
Oscar R. Lara and Jeffrey J. Chalmers..............................................301
Index...............................................................................................................311
Contributors
ROBERT BURNS • Antibody Unit, Scottish Agricultural Science Agency,
Edinburgh, Scotland
JEFFREY J. CHALMERS • Department of Chemical Engineering, The Ohio
State University, Columbus, OH
STEFAN DÜBEL • Professor of Biotechnology, Institute of Biochemistry
and Biotechnology, Carolo-Wilhelmina Technical University,
Braunschweig, Germany
GARY ENTRICAN • Head of Immunology, Moredun Research Institute,
Bush Loan, Edinburgh, Scotland
DAVID C. HANCOCK • Signal Transduction Laboratory, Cancer
Research UK, London Research Institute Laboratories, London, UK
ROSARIA P. HAUGLAND • Molecular Probes Inc., Eugene, OR
JAMES D. HIRSCH • Molecular Probes Inc., Eugene, OR
DAVID HUGHES • Nuffield Department of Surgery, John Radcliffe Hospital,
Oxford, UK
MICHAEL HUST • Department of Biotechnology, Institute of Biochemistry
and Biotechnology, Carolo-Wilhelmina Technical University,
Braunschweig, Germany
WILLIAM JORDAN • Department of Immunology, University of Medicine
and Dentistry of New Jersey, Newark, NJ
GRAHAM D. KEMP • Centre for Biomolecular Sciences, University
of St. Andrews, St. Andrews, Scotland
OSCAR R. LARA • Department of Chemical Engineering, The Ohio State
University, Columbus, OH
SABRINA LIBERATORI • Department of Molecular Biology, University
of Siena, Siena, Italy
BARBARA MAGI • Department of Molecular Biology, University of Siena,
Siena, Italy
GLENN E. MORRIS • Biochemistry Group, North East Wales Institute,
Wrexham, UK
VINCENT MULHOLLAND • Diagnostics and Molecular Biology, Scottish
Agricultural Science Agency, Edinburgh, Scotland
NICOLA J. O’REILLY • Cancer Research UK, London Research Institute
Laboratories, London, UK
WEILIN L. SHELVER • Department of Agricultural Chemical Research,
USDA-ARS Biosciences Research Laboratory, Fargo, ND
ix