Antibody Techniques

350 Pages·1994·4.98 MB·English

by  Vedpal S. Malik and Erik P. Lillehoj (Eds.)

ANTIBODY TECHNIQUES
EDITED BY
Vedpal S. Malik
U.S. Department of Agriculture, APHIS
Hyattsville, Maryland
Erik P. Lillehoj
Department of Biochemistry and Molecular Biology
The George Washington University
School of Medicine and Health Sciences
Washington, D.C.
Academic Press
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Copyright © 1994 by ACADEMIC PRESS, INC.
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Library of Congress Cataloging-in-Publication Data
Antibody techniques / edited by Vedpal S. Malik, Erik P. Lillehoj.
p. cm.
Includes bibliographical references and index.
ISBN 0-12-466460-1
1. Immunoassay-Laboratory manuals. 2. Immunoglobulins-
-Laboratory manuals. 3. Monoclonal antibodies—Laboratory manuals.
4. Immunochemistry-Laboratory manuals. I. Malik, Vedpal S.
II. Lillehoj, Erik P.
QP519.9.I42A56 1994
574.2'93-dc20 94-16438
CIP
PRINTED IN THE UNITED STATES OF AMERICA
94 95 96 97 98 99 EB 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
NUMBERS IN PARENTHESES INDICATE THE PAGES ON WHICH THE AUTHORS'
CONTRIBUTIONS BEGIN.
PATRICIA C. ALLEN (115) U. S. Department of Agriculture, Agricultural
Research Service, Livestock and Poultry Sciences Institute, Belts-
ville, Maryland 20705
ANTHONY C. ALLISON (141) Syntex Research, Palo Alto, California
94304
ROBERT F. BALINT (103) Palo Alto Institute for Molecular Medicine,
Mountain View, California 94043
MICHAEL D. P. BOYLE (177) Department of Microbiology, Medical
College of Ohio, Toledo, Ohio 43699
NOELENE E. BYARS (141) Syntex Research, Palo Alto, California 94304
YONGOCK CHO (259) Department of Urology, College of Medicine,
University of Illinois at Chicago, Chicago, Illinois 60680
BYUNG-KIL CHOE (211, 259) Department of Immunology, Kiemyung
University, School of Medicine, 194 Dongsan Dong, Taegu, Korea
HANNE FLYGE (227) Department of Chemistry, The Royal Veterinary
and Agricultural University, DK-1871Frederiksberg C, Copenhagen,
Denmark
xiii
xiv Contributors
CRISTINA GLAD (153) Biolnvent International AB, S-223 70 Lund, Sweden
CATHY HEFFNER BARRETT (71) Frederick Research Center, Frederick,
Maryland 21701
ARNE HOLM (227) Department of Chemistry, The Royal Veterinary and
Agricultural University, DK-1871 Frederiksberg C, Copenhagen,
Denmark
JAMES W. LARRICK (103) Palo Alto Institute for Molecular Medicine,
Mountain View, California 94043
EDGAR LAURITZEN (227) Department of Virology, Statens Seruminstitute,
and The Royal Veterinary and Agricultural University, DK-1871 Fred-
eriksberg C, Copenhagen, Denmark
ERIK P. LILLEHOJ (273) Department of Biochemistry and Molecular Biol-
ogy, The George Washington University School of Medicine and
Health Sciences, Washington, D.C. 20037
HYUN S. LILLEHOJ (291) Protozoan Diseases Laboratory, Livestock and
Poultry Sciences Institute, U.S. Department of Agriculture, Agricul-
tural Research Service, Beltsville, Maryland 20705
ALISON G. MARTIN (291) Protozoan Diseases Laboratory, Livestock and
Poultry Sciences Institute, U.S. Department of Agriculture, Agricul-
tural Research Service, Beltsville, Maryland 20705
DENNIS W. METZGER (177) Department of Microbiology, Medical College
of Ohio, Toledo, Ohio 43699
INGE NILSSON (153) Biolnvent International AB, S-223 70 Lund, Sweden
WILLIAM J. PATRIE (307) Department of Biology, Shippensburg Univer-
sity, Shippensburg, Pennsylvania 17257
DENIS P. SNIDER (49) Department of Pathology, Health Sciences Center,
McMaster University, Hamilton, Ontario, Canada L8N 3Z5
JOHN THARAKAN (327) Department of Chemical Engineering, Howard
University, Washington, D.C. 20059
IAN M. ZITRON1 (1) Department of General Pediatrics, Children's Hospital
of Michigan, Detroit, Michigan 48201
1 Present address: Division of Gastroenterology-Hepatology, Department of Internal
Medicine, William Beaumont Hospital, Royal Oak, Michigan 48073
PREFACE
This manual is intended to serve as a guide for those who have little
or no practical laboratory experience in techniques using antibodies.
The primary purpose is to provide a step-by-step, bench-top reference
detailing the protocols and recipes of common laboratory procedures
that use antibodies as reagents for research in all areas of biological
science. Concurrently, an effort has been made, where appropriate,
to provide a theoretical background to the procedures described. This
book is not intended to provide a survey of all methods and techniques
available because readers are probably better served by a critical evalu-
ation of a number of procedures rather than a mere description of all
of them. We hope that this book will provide, in one volume, a
common consensus of antibody-related protocols currently in practice
in research and clinical biological sciences.
The first half of this book encompasses subjects relevant to how
antibodies are produced in research laboratories. The introductory
chapter provides a theoretical overview of how the immune system
works, particularly regarding antigen processing and presentation
to T and B cells, and subsequent immunoglobulin gene expression.
Chapters 2 through 7 furnish procedures for production of polyclonal,
monoclonal, and recombinant antibodies; proper methods for use and
care of laboratory animals; recent developments in adjuvant research;
and large-scale production and purification of antibodies. The second
xv
xvi Preface
half of the book (Chapters 8 through 15) describes particular techniques
that use antibodies as immunoreagents in both research and clinical
settings. These include the use of antibody-binding bacterial proteins,
immunoaffinity purification of antigens, preparation of antibody hetero-
conjugates, immunostaining, cell sorting, immunoassays, and immuno-
screening of expression libraries.
As the editors, we extend our appreciation to the individual contribu-
tors who have provided their expertise, time, and effort on this project.
Finally, this guide could not have been written without the encourage-
ment and guidance of Dr. Lorraine Lica and the editorial staff at Academic
Press.
ERIK P. LILLEHOJ
VEDPAL S. MALIK
1
ANTIBODY MOLECULES AND
THE IMMUNE RESPONSE:
THEORETICAL BACKGROUND
IAN M. ZITRON
I. THE ORGANIZATION OF IMMUNE PROTECTION
The normal function of the immune system is to provide protection
against invasion by pathogens. It is, unfortunately, also the source
of less welcome events, such as graft rejection and autoimmune dis-
eases, in which the system reacts against normal body constituents.
An immune response, whether it be beneficial or harmful, is a multi-
layered phenomenon. The purpose of this chapter is twofold. The
first is to provide an introductory description of the various layers,
in conjunction with an indication of some of the interactions that
occur both between the layers and within them. The second is a more
detailed consideration of those aspects of the system that are of most
interest and importance to scientists who wish simply to use immuno-
logical techniques, particularly antibodies, in their research. The em-
phasis is on the genetic, cellular, and molecular events that give rise
to the production of antibodies, rather than on immunochemistry per
se. Nonimmunologists frequently complain that the language and
terminology used in immunology present major barriers to an appreci-
ation of the system, obscuring rather than clarifying. Although this
chapter is in no sense a replacement for a comprehensive dictionary,
it may make some aspects of the immune system more understand-
able.
ANTIBODY TECHNIQUES
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved. 1
2 Ian M. Zitron
II. INNATE AND ACQUIRED IMMUNITY
The first broad separation is the discrimination between innate and
acquired immunity. Innate immunity is, as its name suggests, present
at birth and persists throughout life. It represents the first line of defense
against insult (the word "insult" is used broadly in immunology and
refers to invasion by pathogenic microorganisms, parasites, and, in some
instances, tumors) and is composed of a number of physical, cellular,
and chemical barriers. Skin and mucous membranes physically impede
invasion. Chemical barriers include gastric pH, enzymes such as lyso-
zyme in tears and saliva, and other biologically active molecules such
as the interferons and the proteins of the complement system. Cells,
such as polymorphonuclear leukocytes (PMNs) and natural killers (NKs)
are also integral components of this aspect of protection. Thus at least
three layers of protection exist in innate immunity. Although each is
mechanistically quite different in the way in which protection is effected,
there are characteristics that all layers have in common. Each layer is
either continuously present, or very rapid in response to insult. The
layers show no specificity vis a vis the insult: they are incapable of discrim-
inating between different pathogens. Also, they show no memory of
prior insults: neither the quality nor the quantity of innate immunity is
increased by a second exposure to the same pathogen. Acquired immu-
nity differs from innate immunity in each of these characteristics. Table
I shows a comparison between innate and acquired immunity. The ele-
ments of innate immunity provide a significant degree of protection; the
severity of immune deficiency syndromes that reflect a lack of individual
components of this system provides ample evidence of its importance.
TABLE I Differences between Innate and Acquired Immunity
Property Innate immunity Acquired immunity
Components Physical barriers, e.g., skin; Lymphocytes and accessory
chemical barriers, e.g., cells
lysozyme, interferons;
cellular components,
e.g., NK cells
Specificity None Specific for insult or
pathogen
Presence at birth Yes Some elements; others
develop postnatally
Effect of exposure None; no memory Effector function and
to insult generated memory elicited
1 Antibody Molecules and the Immune Response 3
In contrast, acquired immunity is adaptive, since it responds to the
particular insult with effector functions that act only on that insult.
Adaptation shows itself in two characteristics, specificity and memory,
which combine to give the system its efficiency. Figure 1 shows the time
courses and magnitudes of (antibody) responses to two immunologically
unrelated substances, designated A and B. The figure illustrates the
difference in response subsequent to first (primary) and second (second-
ary) exposure to a foreign substance; this behavior reflects immunological
memory. Specificity is the ability to discriminate between different in-
sults; in the figure, the primary immunization with A elicits antibodies
that recognize A only; the memory that this response generates is specific
for A. Specificity may be maintained even when the substances are
closely related biologically and chemically. One of the best known exam-
ples of the system's ability to distinguish subtle differences is the human
ABO blood group system (Landsteiner, 1946). Perhaps an even more
striking example of the level of discrimination possible is that antibody
populations may be identified that are capable of discriminating between
closely related nitrophenyl groups (Little and Eisen, 1969). Immunologi-
cal memory is very much like that observed in the functioning of the
nervous system: the cells have the ability to recognize, and respond to,
the second (or third, and so on) exposure to a given insult in a way that
is both quantitatively and qualitatively different from the response to
the first, or primary, exposure. Moreover, memory is itself specific be-
cause the enhanced response subsequent to re-exposure is directed only
toward the foreign substance used for initial immunization.
The acquired immune response shows an additional characteristic that
is of considerable significance for homeostasis, the ability to discriminate
between self and nonself. The phenomenon was first shown in the
19th century by Paul Ehrlich (1900), who gave it the name "Horror
Autotoxicus." In its simplest terms, the observation is that, although
vertebrates may be immunized with foreign (i.e., nonself) material and
be shown to mount an immune response, spontaneous responses to the
animal's own molecules leading to autoimmune disease (i.e., anti-self)
are rare. The process by which recognition of self is minimized is referred
to as self-tolerance. Self-tolerance is not simply a failure to recognize
self components, but is an active process involving the regulation of
lymphocyte survival and function (see subsequent text and Figures 7
and 8; Nossal, 1994).
The three defining characteristics of the acquired response—specifi-
city, memory, and self/nonself discrimination—reflect the properties
and activities of lymphocytes. These cells are directly responsible for the
acquired immune response, although in many instances the response
4 Ian M. Zitron
Antibody
titer I
100 J secondary
Anti-A antibody
Anti-B antibody
primary
0 7 14 "o 7 14 21
time after immunization (days)
t t
Immunize Boost with A
with A Immunize
with B
FIGURE 1 Kinetic and quantitative differences between primary and secondary humoral
(antibody) responses. The primary response to immunization with A shows a lag period
of ~7 days, followed by a logarithmic increase, a relatively low plateau titer, and a rapid
decay. In contrast, secondary immunization with A gives rise to a response with a shorter
lag period, a steeper logarithmic increase reaching a significantly higher plateau (note the
logarithmic scale of the ordinate), and a slow decay phase. The difference between primary
and secondary responses reflects immunological memory, which has been generated as
a consequence of primary immunization, but which requires rechallenge to be expressed.
The secondary immunization with A is accompanied by primary immunization with an
immunologically distinct substance, B. The indicated response to B is characteristic of a
primary response, indicating that memory itself is specific.
also requires the involvement of cells of the monocyte/macrophage lin-
eage (Unanue and Allen, 1987). For the investigator interested in
applying immunological techniques to his or her work, the acquired
immune response and the functions and interactions of lymphocytes,
both with other lymphocytes and with other cell types, are of paramount