THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Lassen - Exhibit 1041, p. 1
`
`

`

`immuno
`iologvye
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Charles A. Janeway, Jr.
`Yale University School of Medicine
`@
`Paul Travers
`
`Anthony Nolan Research Institute, London
`CJ
`Mark Walport
`Imperial College School of Medicine, London
`@
`Mark J. Shlomchik
`
`Yale University School of Medicine
`
`p Pug>
`
`ep
`
`Lassen - Exhibit 1041, p. 2
`
`Lassen - Exhibit 1041, p. 2
`
`

`

`Vice President:
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`
`® 2001 by Garland Pubtishing.
`All rights reserved. Nopart of this publication may be reproduced, stored in a retrieval
`system or transmitted in any form or by any means—electronic, mechanical, photocopying,
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`
`Distributors:
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`inside Japan. Nankodo Co.Lid., 42-6, Hongo 3-Chrome, Bunkyo-ku,
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`
`ISBN 0 6153 3642 X (paperback) Garland
`ISBN 0 4430 7698 9 (paperback) Churchill Livingstone
`ISBN 0 4430 7099 7 (paperback)International Student Edition
`
`Library of Congress Cataloging-in-Publication Data
`Immunobiology : the immune system In health and disease / Charles A. Janeway, Jr....
`[et al.].-- Sth ed.
`p. cm.
`Includes bibliographical references and index.
`ISBN 0-8153-3642-X (pbk.)
`1. Immunology. 2. Immunity,
`
`|. Janeway, Charles. |I. Title,
`
`QR181 1454 2001
`616.07'9--de21
`
`2001016039
`
`This book was produced using QuarkXpress 4.11 and AdobeIllustrator 9.0
`
`Published by Garland Publishing, a memberof the Taylor & Francis Group,
`29 West 35th Street, New York, NY 10001-2299,
`
`Printed in the United States of America.
`168 141312111098765432
`
`Lassen - Exhibit 1041, p. 3
`
`Lassen - Exhibit 1041, p. 3
`
`

`

` CONTENTS
`
`
`
`
`PART fl]|THE DEVELOPMENT OF MATURE LYMPHOCYTE RECEPTOR
`
`Chapter6
`Signaling Through Immune System Receptors
`187
`
`Chapter 7
`The Development and Survival of Lymphocytes
`221
` PARTIV|THE ADAPTIVE IMMUNE RESPONSE
`Chapter8
`—T Cell-Meciated immunity
`295
`Chapter9
`The Humoral Immune Response
`341
`Chapter 10 Adaptive Immunity to Infection
`381
`
`PARTI|AN INTRODUCTION TO IMMUNOBIOLOGY AND INNATE IMMUNITY
`
`1
`35
`
`93
`
`123
`155
`
`Chapter1
`Chapter 2
`
`Basic Concepts In Immunology
`Innate Immunity
`
`THE RECOGNITION OF ANTIGEN
`
`Chapter3
`
`Chapter4
`Chapter5
`
`Antigen Recognition by B-cell andT-cell Receptors
`
`The Generation of Lymphocyte Antigen Receptors
`Antigen Presentation to T Lymphocytes
`
`REPERTOIRES
`
`|
`
`| |
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`|
`
`i
`
`
`
`
`Afterword Evolution of the Immune System:Past, Present, and Future,
`by Charles A. Janeway,Jr.
`
`Appendix!
`
`Immunologists’ Toolbox
`
`Appendix Il CD Antigens
`
`Appendix Ill Cytokines and their Receptors
`Appendix IV Chemokines andthelr Receptors
`Appendix V Immunological Constants
`
`Biographies
`
`Glossary
`
`425
`471
`504
`553
`
`597
`
`613
`
`661
`
`677
`680
`681
`
`682
`
`Failures of Host Defense Mechanisms
`Chapter 11
`Chapter 12 Allergy and Hypersensitivity
`Chapter 13 Autolmmunlty and Transplantation
`Chapter 14 Manipulation of the Immune Response
`
`
`
`
`
`
`
`
`683 Index
`
`Lassen - Exhibit 1041, p. 4
`
`Lassen - Exhibit 1041, p. 4
`
`

`

`APPENDIX|
`
`
`
`Immunologists’ Toolbox
`
`Immunization.
`
`Natural adaptive immuneresponsesare normally directed at antigens borne
`by pathogenic microorganisms. The immune system can, however, also be
`induced to respond to simple nonliving antigens, and experimental immuno-
`logists have focused on the responsesto these simple antigens in developing
`our understanding of the immuneresponse. The deliberate induction of an
`immuneresponse is known as immunization, Experimental immunizations
`are routinely carried out by injecting the test antigen into the animal or
`human subject. The route, dose, and form in which antigen is administered
`can profoundly affect whether a response occurs and the type of response
`that is produced, and are considered in Sections A-1-A-4, The induction of
`protective immune responses against common microbial pathogens in
`humansis often called vaccination, although this term is correctly only
`applied to the induction of immuneresponsesagainst smallpox by immunizing
`with the cross-reactive cowpox virus, vaccinia (sec Chapter14).
`
`To determine whether an immuneresponsehas occurred and to follow its
`course,
`the immunized individual
`is monitored for the appearance of
`immune reactants directed at the specific antigen. Immune responses to
`most antigenselicit the production of both specific antibodies and specific
`effector T cells, Monitoring the antibody response usually involves the analysis
`ofrelatively crude preparations of antiserum (plural: antisera). The serum is
`the fluid phase of clotted blood, which,if taken from an immunizedindividual,
`is called antiserum becauseit contains specific antibodies against the immun-
`izing antigen as well as other soluble serum proteins. To study immune
`responses mediated by T cells, blood lymphocytesorcells from lymphoid
`organs such as the spleen are tested; T-cell responses are more commonly
`studied in experimental animals than in humans.
`
`Any substance that can elicit an immuneresponseis said to be immunogenic
`and is called an immunogen. There is a clear operational distinction
`between an immunogen and an antigen. An antigen is defined as any
`sustance that can bind to a specific antibody. All antigens therefore have the
`potentialto elicit specific antibodics, but some need to be attached to an
`immunogenin orderto do so. This means that althoughall immunogens are
`antigens, not all antigens are immunogenic. The antigens used most
`frequently in experimental immunology are proteins, and antibodies to
`proteins are of enormousutility in experimental biology and medicine.
`Purified proteins are, however, not always highly immunogenic and to
`provoke an immuneresponse have (o be administered with an adjuvant(see
`Section A-4), Carbohydrates, nucleic acids, and other types of molecule are
`all potential antigens, but will often only induce an immuneresponse if
`attached to a protein carrier. Thus, the immunogenicity of protein antigens
`determines the outcomeofvirtually every immuneresponse.
`
`
`
`Lassen - Exhibit 1041, p. 5
`
`Lassen - Exhibit 1041, p. 5
`
`

`

`
`
`
`
`QnSe
`a=
`
`27
`
`units) S2
`Antibody{arbitraryresponse
`
`
`
`to’
`
`10% 10?
`
`104
`
`107 10°
`y0°
`10°
`Antigen dose
`
`Antigen dose given in primary !mmunization
`
`Antisera generated by immunization with even the simplest antigen wil}
`contain manydifferent antibody molecules that bind to the immunogen jn
`slightly different ways. Some of the antibodies in an antiserum are cross.
`reactive. A cross-reaction is defined as the binding ofan antibodyto an antigen
`other than the immunogen; mostantibodies cross-react with closely related
`antigensbut, on occasion, somebindantigens having no clear relationship to
`the immunogen. These cross-reacting antibodies can create problems when
`the antiserum is used to detect a specific antigen. They can be removedfrom
`an antiserum by absorption with the cross-reactive antigen, leaving behind
`the antibodies that bind only to the immunogen. Absorption can be
`performedbyaffinity chromatography using immobilized antigen, a technique
`thatis also used for purification of antibodies or antigens (see Section A-5),
`Most problems of cross-reactivity can be avoided, however, by making
`monoclonal antibodies(see Section A-12).
`Although almostany structure can be recognized by antibody as an antigen,
`usually only proteins elicit fully developed adaptive immuneresponses. This
`is because proteins have theability to engage T cells, which contribute to
`inducing most antibody responses and are required for immunological
`memory, Proteins engage T cells because the T cells recognize antigens as
`peptide fragments of proteins bound to major histocompatibility complex
`(MHC) molecules (see Section 3-11), An adaptive immune response that
`includes immunological memory can be induced by nonpeptide antigens
`only when they are attached to a protein carrier that can engage the necessary
`T cells (see Section 9-2 and Fig.9.4).
`Immunological memory is produced as a result of the initial or primary
`immunization, which evokes the primary immuneresponse. This is also
`knownas priming, as the animal or person is now ‘primed’like a pumpto
`mount a more potent response to subsequent challenges with the same
`antigen. The response to each immunizationis increasingly intense, so that
`secondary,tertiary, and subsequentresponsesare of increasing magnitude
`(Fig. A.1). Repetitive challenge with antigen to achieve a heightened state of
`immunity is known as hyperimmunization.
`Certain properties of a protein thatfavor the primingof an adaptive immune
`response have beendefined by studying antibody responses to simple natural
`proteinslike hen egg-white lysozyme andto synthetic polypeptide antigens
`(Fig. A.2). The larger and more complex a protein, and the more distantits
`relationship to self proteins, the morelikelyit is to elicit a response. Thisis
`because such responses dependonthe proteins being degraded into peptides
`that can bind to MHCmolecules, and on the subsequent recognition of these
`peptide:MHC complexesby T cells. The larger and moredistinct the protein
`antigen, the more likelyit is to contain such peptides. Particulate or aggregated
`antigens are more immunogenic because they are taken up more efficiently
`by the specialized antigen-presenting cells responsible for initiating a
`response. Indeed small soluble proteins are unable to induce a response
`unless they are madeto aggregate in some way. Manyvaccines,for example,
`use aggregated protein antigens to potentiate the immune response,
`
`
`
`
`
`
`
`Amtibedyresponse(arbitraryunits}
`
`
`
`high-zonetolerance
`
`1
`
`10"
`
`10?
`
`10% 104
`
`10°
`
`408
`
`107 40°
`
`Fig. A.1 The dose of antigen used
`in an Initial immunization affects
`the primary and secondary antibody
`response. The typical antigen
`dose-response curve shown here
`iustrates the influence of dose on both
`a primary antibody response (amounts
`of antibody produced expressed in
`arbitrary units) and the effect of the
`dose used for priming on a secondary
`antibody responseelicited by a dose of
`antigen of 10° arbitrary mass units. Very
`low dosesof antigen do not cause an
`A-1—Haptens.,
`immune responseatall. Slightly higher
`doses appearto inhibit specific antibody
`production, an effect known as low-zone
`tolerance. Above these dosesthere is a
`steady increase in the response with
`antigen dose to reach a broad optimum.
`Very high dosesof antigen also Inhibit
`immune responsiveness to a subsequent
`challenge, a phenomenon known as
`high-zone tolerance.
`
`
`
`
`Small organic molecules of simple structure, such as phenyl arsonates and
`nitrophenyls, do not provoke antibodies when injected by themselves.
`However, antibodies can be raised against themif the moleculeis attached
`covalently, by simple chemical reactions, to a protein carrier. Such small
`molecules were termed haptens (from the Greek haptein, to fasten) by the
`immunologist Karl Landsteiner, whofirst studied them in the early 1900s. He
`found that animals immunized with a hapten-carrier conjugate produced
`
`Lassen - Exhibit 1041, p. 6
`
`Lassen - Exhibit 1041, p. 6
`
`

`

`
`Immunization|615
`
`"i
`
`th
`
`gen
`
`-Increaged immunogenicity
`
`= a
`
`
`
`:
`
`
`
`
`
`
`ve
`
`Decreased Immunogenicity
`
`Fig. A.2 Intrinsic properties and
`extrinsic factors that affect the
`immunogenicity of proteins.
`
`
`
`
`
`
`
`[
`
`tn
`
`oneal
`
`> intravenous or intragastric
`
` Form
`
`Similarity to self protein
`
` Adjuvants
`
`
`
`Few diffarances
`
`
`Rapid release
`
`
`
`
`
`
`
`
`
`
`
`
`
`Route
`
`Interaction with host MHC
`
`Effective
`
`Ineffective
`
`
`
`conjugate-specific antibody.
`
`three distinct sets of antibodies (Fig. A.3). One set comprised hapten-specific
`antibodies that reacted with the same hapten on anycarrier, as well as with
`free hapten. The secondset of antibodies was specific for the carrier protein,
`as shownbytheirability to bind both the hapten-modified and unmodified
`carrier protein. Finally, some antibodies reacted only with the specific conjugate
`of haptenandcarrier used for immunization. Landsteiner studied mainly the
`antibody responseto the hapten,as these small molecules could be synthesized
`in manyclosely related forms. He observed that antibodies raised against a
`particular hapten bind that hapten but, in general, fail to bind even very
`closely related chemical structures. The binding of haptens by anti-hapten
`antibodies has played an importantpart in defining the precision of antigen
`binding by antibody molecules. Anti-hapten antibodies are also important
`medically as they mediate allergic reactions to penicillin and other
`compoundsthatelicit antibody responses whentheyattachto self proteins
`(see Section 12-10).
`
`Fig. A.3 Antlbodies can be elicited by
`small chemical groups called haptens
`only when the haptenie linked to an
`immunogenic protein carrier. Three
`types of antibodies are produced. One
`set (blue) binds the carrier protein alone
`and |s called carrier-specific. One set
`(red) binds to the hapten on any carrier
`or to free hapten In solution and |s called
`hapten-specific. One set (purple) only
`binds the specific conjugate of hapten
`
`and carrier used for immunization,
`apparently binding to sites at which
`the hapten joins the carrler, andis
`called conjugate-specific. The amount
`of antibody of each type in this serum
`is shown schematically in the graphs at
`the bottom; note that the original antigen
`binds more antibody than the sum of
`anti-hapten and anti-carrier antibodies
`owing to the additional binding of
`
`Binding to
`hapten-carrler
`conjugate
`
`
`
`Antibodybound
`
`Antigen
`
`Lassen - Exhibit 1041, p. 7
`
`Lassen - Exhibit 1041, p. 7
`
`

`

`
`
`Appendix I: Immunologists’ Toolbox
`TT$$LR————
`
`A-2
`
`Routes of immunization.
`
`The route by which antigen is administered affects both the magnitude anq
`the type of response obtained. The most commonroutesby which antigenis
`introduced experimentally or as a vaccine into the badyare injection into
`tissue by subcutaneous(s.c.) injection between the epidermis and dermal]
`layers, or by intradermal (i.d.) injection, or intramuscular(i,m.) injection; by
`intravenous(i.v.) injection or transfusion directly into the bloodstream; into
`the gastrointestinal tract by oral administration; into the respiratory tract by
`intranasal (i.n.) administration orinhalation.
`
`Antigens injected subcutaneously generally elicit the strongest responses,
`most probably because the antigen is taken up by Langerhans’cells and
`efficiently presented in local lymph nodes, and sothis is the method most
`commonly used when the object of the experimentis to elicit specific anti-
`bodies or‘I’ cells against a given antigen. Antigens injected or transfused
`directly into the bloodstream tend to induce immune unresponsivenessor
`tolerance unless they bind to hostcells or are in the form of aggregates that
`are readily taken up by antigen-presentingcells.
`Antigen administration via the gastrointestinal tract is used mostly in the
`studyofallergy.It has distinctive effects, frequentlyeliciting a local antibody
`responsein the intestinal lamina propria, while producing a systemic state of
`tolerance that manifests as a diminished response to the same antigen if
`subsequently administered in immunogenic form elsewhere in the body.
`This‘split tolerance’ may be importantin avoidingallergy to antigens in food,
`as the local response prevents food antigens from entering the body, while
`the inhibition of systemic immunity helps to prevent the formation of IgE
`antibodies, which are the causeof suchallergies (see Chapter 12).
`Introduction of antigen into the respiratory tract is also used mainlyin the
`study of allergy. Protein antigens that enter the body throughthe respiratory
`epithelium tendtoelicit allergic responses, for reasonsthat are not clear,
`
`A-3
`
`Effects of antigen dose.
`
`The magnitude ofthe immuneresponse dependsonthe dose of immunogen
`administered. Below a certain threshold dose, mostproteins do notelicit any
`immuneresponse. Above the threshold dose, there is a gradualincreasein
`
`the responseas the doseof antigenis increased, until a broad plateaulevelis
`reached, followed by a decline at very high antigen doses (see Fig. A.1). As
`mostinfectious agents enter the body in small numbers, immuneresponses
`are generally elicited only by pathogens that multiply to a level sufficient to
`exceed the antigen dose threshold. The broad response optimumallows the
`system to respond to infectious agents across a wide range of doses, At very
`high antigen dosesthe immuneresponseis inhibited, which may be important
`in maintaining tolerance to abundantself proteins such as plasmaproteins.
`In general, secondary and subsequent immuneresponses occur at lower
`antigen doses and achieve higher plateau values, whichis a sign of immuno-
`logical memory. However, under some conditions, very low or very high
`doses of antigen may induce specific unresponsivestates, knownrespectively
`as acquired low-zoneorhigh-zone tolerance,
`
`A-4 Adjuvants.
`
`Mostproteins are poorly immunogenic or nonimmunogenic when adminis-
`tered by themselves. Strong adaptive immune responsesto protein antigens
`almost always require that the antigen be injected in a mixture knownas an
`
`!
`
`ve
`|
`
`|
`
`I
`
`|
`
`|
`
`|
`
`'
`
`|
`
`
`
`Lassen - Exhibit 1041, p. 8
`
`Lassen - Exhibit 1041, p. 8
`
`

`

`Immunization|617—_—sese
`
`adjuvant. An adjuvant is any substance that enhances the immunogenicity of
`substances mixed withit. Adjuvants differ from protein carriers in that they
`do notform stable linkages with the immunogen. Furthermore, adjuvants are
`needed primarily for initial immunizations, whereas carriers are required to
`elicit not only primary but also subsequent responses to haptens. Commonly
`used adjuvantsare listed in Fig, A.4.
`
`Adjuvants can enhance immunogenicity in twodifferent ways. First, adjuvants
`convert soluble protein antigens into particulate material, which is more
`readily ingested by antigen-presenting cells such as macrophages. For
`example, the antigen can be adsorbed on particles of the adjuvant (such as
`alum), made particulate by emulsification in mineral oils, or incorporated
`into the colloidal particles of ISCOMs. This enhances immunogenicity some-
`what, but such adjuvants are relatively weak unless they also contain bacteria
`or bacterial products. Such microbial constituents are the second means by
`which adjuvants enhance immunogenicity, and although their exact contri-
`bution to enhancing immunogenicity is unknown,theyare clearly the more
`important component of an adjuvant. Microbial products may signal
`macrophagesor dendritic cells to become moreeffective antigen-presenting
`cells (see Chapier 2). One of their effects is to induce the production of
`inflammatory cytokines and potentlocal inflammatory responses; this effect
`is probably intrinsic to their activity in enhancing responses, but precludes
`their use in humans.
`
`Nevertheless, some human vaccines contain microbial antigensthat can also
`act as effective adjuvants. For example, purified constituents of the bacterium
`Bordetella pertussis, whichis the causal agent of whooping cough,are used as
`both antigen and adjuvantin the triplex DPT (diphtheria, pertussis, tetanus)
`vaccine against these diseases.
`
`Composition
`
`Mechanieam of action
`
`Incomplete Freund's adjuvant
`
`Oil-in-water emulsion
`
`Delayed release of antigen;
`enhanced uplake by
`macrophages
`
`
`
`
`
`Oil-in-water emulsion
`with dead mycobacteria
`
`Delayed release of antigen;
`enhanced uptake by
`macrophages; induction of
`co-slimulators in macrophages
`
`Complete Freund's adjuvant Oil-in-water emulsion with
`Freund's adjuvant
`
`Freund's adjuvant with MDP
`
`muramyldipeptide (MDP),
`a constituent of mycobacteria
`
`Similar to complete
`
`.
`:
`Alum (aluminum hydroxide)
`
`‘
`1
`Aluminum hydroxide get
`
`Delayed release of antigen,
`enhanced macrophage uptake
`
`Alum plus
`Bordefella pertussis
`
`Delayed release of antigen;
`Aluminum hydroxide gel
`enhanced uplake by
`
`with killed 8. pertussis
`macrophages;
`
`induction of co-stimulators
`
`
`Delivers antigen to cytosol;
`
`Immune stimulatory
`Matrlx of Quill A
`allows induction of
`
`
`
`complexes (ISCOMs)} containing viral proteins
`cytotoxic T calls
`
`
`Fig. A.4 Common adjuvants and their
`use. Adjuvants are mixed with the
`antigen and usually renderit particulate,
`which helps to retain the antigen in the
`body and promotes uptake by
`macrophages.Most adjuvants include
`bacteria or bacterial components that
`stimulate macrophages, aiding in the
`induction of the immune response.
`ISCOMs (immune stimulatory
`complexes) are small micelles of the
`detergent Quil A; when viral proteins
`are placed in these micelles, they
`apparently fuse with the antigen-
`presenting cell, allowing the antigen
`to enter the cytosol. Thus, the antigen-
`presenting cell can stimulate a response
`to the viral protein, much as a virus
`infecting these cells would stimulate
`an anti-viral response.
`
`Lassen - Exhibit 1041, p. 9
`
`Lassen - Exhibit 1041, p. 9
`
`

`

`618
`
`Appendix |: Immunologists’ Toolbox
`
`
`The detection, measurement, and characterization
`of antibodies and their use as research and
`diagnostic tools.
`
`B cells contribute to adaptive immunity by secreting antibodies, and the
`response of B cells to an injected immunogenis usually measured by analyzing
`the specific antibody produced in a humoral immuneresponse.This is most
`conveniently achieved by assaying the antibody that accumulatesin the fluid
`phaseofthe blood or plasma; such antibodies are knownascirculating anti-
`bodies. Circulating antibody is usually measured by collecting blood,allowing
`it to clot, and then isolating the serum from the clotted blood. The amount
`and characteristics of the antibody in the resulting antiserum are then
`determined using the assays we will describe in Sections A-5-A-11.
`
`The most important characteristics of an antibody responseare the specificity,
`amount, isotype or class, and affinity of the antibodies produced. The
`specificity determinestheability of the antibodyto distinguish the immunogen
`from other antigens. The amount of antibody can be determined in many
`different ways andis a function of the numberof respondingB cells, their rate
`of antibody synthesis, and the persistence of the antibody after production,
`The persistence of an antibody in the plasma andextracellularfluid bathing
`the tissues is determined mainly byits isotype (see Sections 4-15 and 9-12);
`each isotype has a different half-life in vivo. The isotypic composition of an
`antibody response also determinesthe biological functions these antibodies
`can perform and the sites in which antibody will be found. Finally, the
`strength of binding of the antibodyto its antigen in terms of a single antigen-
`binding site binding to a monovalent antigenis termed its affinity (the total
`binding strength of a molecule with more than onebindingsite is called its
`avidity). Binding strength is important, since the higher the affinity of the
`antibodyforits antigen,the less antibodyis required to eliminate the antigen,
`as antibodies with higheraffinity will bind at lower antigen concentrations.
`All these parameters of the humoral immuneresponse help to determine the
`capacity of that response to protect the host from infection.
`
`Antibody moleculesare highly specific for their corresponding antigen, being
`able to detect one moleculeof a protein antigen out of more than 10® similar
`molecules. This makes antibodies both easy to isolate and study, and
`invaluable as probes of biological processes. Whereas standard chemistry
`would havegreatdifficulty in distinguishing between two suchclosely related
`proteins as human andpig insulin, or two such closely related structures as
`ortho- and para-nitrophenyl, antibodies can be made that discriminate
`between these two structures absolutely. The value of antibodics as molecular
`probeshas stimulated the development of manysensitive and highly specific
`techniques to measure their presence, to determine their specificity and
`affinity for a range of antigens, and to ascertain their functional capabilities.
`Many standard techniques used throughout biology exploit the specificity
`andstability of antigen binding by antibodies. Comprehensive guides to the
`conductof these antibody assays are available in many books on immuno-
`logical methodology, wewill illustrate here only the most important tech-
`niques, especially those used in studying the immuneresponseitself.
`
`Some assays for antibody measure the direct binding of the antibodytoits
`antigen, Such assays are based on primary interactions. Others determine
`the amountof antibody present by the changesit inducesin the physicalstate
`
`
`
`Lassen - Exhibit 1041, p. 10
`
`Lassen - Exhibit 1041, p. 10
`
`

`

`
`
`
`
`The detection, measurement, and characterization of antibodies and their use as research and diagnostic tools|619 o® depleted of
`
`Piet Puritted
`
`
`®—antigenA of antigen A
`Fig. A.S Affinity chromatography uses antigen-antibody
`passed overthe matrix. The specific antibady binds the antigen
`binding to purify antigens or antibodles. To purify a specific
`of interest; other molecules are washed away. Specific antigen
`antigen from a complex mixture of molecules, a monoclonal
`is then eluted byaltering the pH, which can usually disrupt
`antibodyis attached to an insoluble matrix, such as
`antibody-antigen bonds, Antibodies can be purified in the
`chromatography beads, and the mixture of molecules is
`same way on beads coupled to antigen (not shown).
`
`of the antigen, such asthe precipitation of soluble antigen or the clumping
`of antigenic particles; these are called secondary interactions. Both types of
`assay can be used to measure the amountand specificity of the antibodies
`produced after immunization, and both can be applied to a wide range of
`other biological questions.
`
`As assays for antibody were originally conducted with antisera from immune
`individuals, they are commonlyreferred to as serological assays, and the use
`of antibodies is often called serology. The amount of antibody is usually
`determined by antigen-binding assaysafter titration of the antiserum by
`serial dilution, and the pointat which bindingfalls to 50% of the maximum is
`usually referredto as the titer of an antiserum,
`
`A-5
`
`Affinity chromatography.
`
`Specific antibody can be isolated from an antiserum by affinity
`chromatography, whichexploits the specific binding of antibodyto antigen
`held onasolid matrix (Fig. A.5). Antigen is boundcovalently to small, chemi-
`cally reactive beads, which are loaded into a column, and the antiserum is
`allowed to pass over the beads. The specific antibodies bind, while all the
`other proteinsin the serum,including antibodies to other substances, can be
`washed away. Thespecific antibodiesare theneluted,typically by lowering the
`pH to2.5 or raisingit to greater than 11. Antibodies bind stably under physio-
`logical conditionsof salt concentration, temperature, and pH,butthe binding
`is reversible as the bondsare noncovalent. Affinity chromatography can also be
`used to purify antigens from complex mixtures by using beads coated with
`specific antibody. The technique is known asaffinity chromatography because
`it separates molecules on the basis of their affinity for one another.
`
`A-6
`
`Radioimmunoassay(RIA), enzyme-linked immunosorbent assay
`(ELISA), and competitive inhibition assay.
`
`
`
`Radioimmunoassay(RIA) and enzyme-linked immunosorbentassay (ELISA)
`are direct binding assays for antibody (or antigen) and both work on the same
`principle, but
`the means of detecting specific binding is different.
`Radioimmunoassays are commonly used to measure the levels of hormones
`in blood and tissue fluids, while ELISA assays are frequently used in viral
`diagnostics, for example in detecting cases of HIV infection. For both these
`
`Lassen - Exhibit 1041, p. 11
`
`Lassen - Exhibit 1041, p. 11
`
`

`

`covalently linked to enzyme
`dlWashawayunboundantibody|J
` Enzyme makes colored
`colorleas substrate Messure absorbance ofilght
`
`Add anti-A antibody
`
`
`
`1
`Sample 2
`
`
`Gaines 8)
`Sample
`(antigen A)
`
`
`
`product from added
`
`by colored product
`
` Appendix |: Immunologists’ Toolbox
`
`
`
`methods one needs a pure preparation of a knownantigenor antibody, oy
`bath,in order to standardize the assay. We will describe the assay witha sample
`of pure antibody, which is the more usual case, but the principleis similarif
`pure antigen is used instead. In RIA for an antigen, pure antibody against that
`antigen is radioactively labeled, usually with '*I; for the ELISA, an enzymeig
`linked chemically to the antibody. The unlabeled component, which in this
`case would beantigen,is attached to a solid support, such as the wells of a
`plastic multiwell plate, whichwill adsorb a certain amountof anyprotein.
`The labeled antibodyis allowed to bind to the unlabeled antigen, under
`conditions where nonspecific adsorption is blocked, and any unboundanti-
`body and otherproteins are washed away. Antibody bindingin RIA is measured
`directly in terms of the amountofradioactivity retained by the coated wells,
`whereasin ELISA, binding is detected by a reaction that converts a colorless
`substrate into a colored reaction product(Fig. A.6), The color change can be
`read directly in the reaction tray, making data collection very easy, and ELISA
`also avoids the hazards ofradioactivity. This makes ELISA the preferred
`method for most direct-binding assays, Labeled anti-immunoglobulin anti-
`bodies(see Section A-10) canalso be used in RIA or ELISA to detect binding
`of unlabeled antibody to unlabeled antigen-coated plates. In this case, the
`labeled anti-immunoglobulin antibody is used in what is termed a ‘second
`layer.’ The use of such a second layer also amplifies the signal, as at least two
`moleculesof the labeled anti-immunoglobulin antibody are able to bind to
`eachunlabeled antibody. RIA and ELISA canalsobecarried out with unlabeled
`antibody stuck to the plates and labeled antigen added.
`A modification of ELISA knownas a capture or sandwich ELISA (or more
`generally as an antigen-capture assay) can be used to detect secreted products
`such as cytokines. Rather than the antigen beingdirectly attachedtoa plastic
`plate, antigen-specific antibodies are bound to the plate. These are able to
`bind antigen with highaffinity, and thus concentrate it on the surface of the
`plate, even with antigens that are present in very low concentrationsin the
`initial mixture. A separate labeled antibody that recognizesa different epitope
`Fig. A.6 The principle of the enzyme-
`to the immobilizedfirst antibodyis then usedto detect the bound antigen.
`linked Immunosorbent assay (ELISA).
`Theseassaysillustrate two crucial aspectsofall serological assays. First, at least
`To detect antigen A, purified antibody
`specific for antigen A is linked
`one of the reagents mustbe available in a pure, detectable form in orderto
`chemleally to an enzyme. The samples
`obtain quantitative information. Second, there must be a meansofseparating
`to be tasted are coated onto the surface
`the boundfraction of the labeled reagent from the unbound,free fraction so
`of plastic wells to which they bind
`that the percentageofspecific binding can be determined, Normally,this sep-
`nonspecifically; residual sticky sites on
`aration is achieved by having the unlabeled partner trapped onasolid support.
`the plastic are blocked by adding
`Labeled molecules that do notbind can then be washed away, leavingjustthe
`Irrelevant proteins (not shown). The
`labeled antibody is then added to the
`labeled partnerthat has bound.In Fig. A.G, the unlabeled antigenis attached to
`wells under conditions where nonspecific
`the well and the labeled antibodyis trapped by bindingtoit. The separationof
`binding is pravented, so that only
`boundfrom free is an essential step in every assay that uses antibodies.
`binding to antigen A causesthe labeled
`antibody to be retained on the surface.
`RIA and ELISA donotallow oneto measuredirectly the amountofantigen or
`Unbound labeled antibody is removed
`antibodyin a sample of unknown composition, as both dependon the binding
`from all wells by washing, and bound
`of a pure labeled antigen or antibody, There are various ways aroundthis
`antibody is detected by an enzyme-
`problem, one of whichis to use a competitive inhibition assay, as shown in
`dependent color-change reaction. This
`Fig. A.7. Inthis type of assay, the presence and amou