THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`immuno
`bi OIOgy@
`
`CHARLES A JA'NEWAY PAUL
`
`Lassen - Exhibit 1041, p. 1
`
`

`

`immuno
`iologye
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Charles A. Janaway, Jr.
`
`Yale University School of Medicine
`
`Paul Travers
`
`Anthony Nolan Research Institute. London
`
`I
`
`Mark Walpurt
`
`Imperial College School of Medicine, London
`
`I
`
`Mark J. Shlamchik
`
`Yale University School of Medicine
`
`
`
`Lassen — Exhibit 1041, p. 2
`
`Lassen - Exhibit 1041, p. 2
`
`

`

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`
`© 2001 by Garland Publishing.
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`
`iSBN 0 8153 3642 X (paperback) Garland
`ISBN 0 4430 7098 9 (paperback) Churchill Livingstone
`iSBN O 4430 7099 7 (paperback) International Student Edition
`
`Library at Congress Cataloging-in-Pubiication Date
`immunobiology : the immune system In health and disease / Charles A. Janeway. Jr.
`[et al.].-- 5th ed.
`p. cm.
`includes bibliographical references and index.
`iSBN 0-8153-3642-X (pbk)
`1. immunology. 2. Immunity.
`
`I. Janeway. Charles. ll. Title.
`
`QR1B1 .1454 2001
`616.07‘9--d021
`
`2001016039
`
`This book was produced using QuerkXpress 4.11 and Adobe illustrator 9.0
`
`Published by Garland Publishing. a member of the Taylor & Francis Group.
`29 West 35th Street, New York. NY 10001-2299.
`
`Printed in the United States of America.
`15141312111098765432
`
`Lassen — Exhibit 1041, p. 3
`
`Lassen - Exhibit 1041, p. 3
`
`

`

` CONTENT-S
`
`PART I
`
`AN INTRODUCTION TO IMMUNOBIOLOGY AND INNATE IMMUNITY
`
`Chapter 1
`
`Basic Concepts In Immunology
`
`Chapter 2
`
`Innate Immunity
`
`THE RECOGNITION OF ANTIGEN
`
`Chapter 3
`
`Antigen Recognition by Beell and T-cell Receptors
`
`Chapter 4
`
`The Generation of Lymphocyte Antigen Receptors
`
`Chapter 5
`
`Antigen Presentation to T Lymphocytes
`
`PART III
`
`THE DEVELOPMENT OF MATURE LYMPHOCYTE RECEPTOR
`REPERTOIRES
`
`Chapter 6
`
`Signaling Through immune System Receptors
`
`Chapter 7
`
`The Development and Survival of Lymphocytes
`
`
`EARTH:
`Chapter 8
`
`THE ADAPTIVE IMMUNE RESPONSE
`
`T Cell-Mediated Immunity
`
`Chapter 9
`
`The Humoral Immune Response
`
`Chapter 10 Adaptive Immunity to Infection
`
`PART V
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Chapter 11
`
`Failures of Host Defense Mechanisms
`
`Chapter 12 Allergy and Hypersensitivity
`
`Chapter 13 Autoimmunity and Transplantatlon
`
`Chapter 14 Manipulation of the Immune Response
`
`Afterward Evolution of the Immune System: Past, Present, and Future,
`by Charles A. Janeway, Jr.
`
`Appendix I
`
`lmmunologists' Toolbox
`
`Appendix II CD Antigens
`
`Appendix III Cytokines and their Receptors
`
`Appendix IV Chemoklnes and their Receptors
`
`1
`
`35
`
`93
`
`1 23
`
`1 55
`
`187
`
`221
`
`295
`
`341
`
`381
`
`425
`
`471
`
`501
`
`553
`
`597
`
`613
`
`661
`
`677
`
`680
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Appendix V Immunological Constants
`
`Biographies
`
`Glossary
`Index
`
`
`
`
`
`681
`
`682
`
`683
`
`Lassen — Exhibit 1041, p. 4
`
`Lassen - Exhibit 1041, p. 4
`
`

`

`lmmunologists’Toolbox
`
`Immunization.
`
`Natural adaptive immune responses are 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 responses to these simple antigens in developing
`our understanding of the immune response. The deliberate induction of an
`immune response 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-l—A-4. The induction of
`protective immune responses against common microbial pathogens in
`humans is often called vaccination, although this term is correctly only
`applied to the induction of immune responses against smallpox by immunizing
`with the cross—reactive cowpox virus, vaccinia (see Chapter 14).
`
`To determine whether an immune response has 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 antigens elicit the production of both specific antibodies and specific
`effector T cells. Monitoring the antibody response usually involves the analysis
`of relatively crude preparations of antiserum (plural: antisera). The serum is
`the fluid phase of clotted blood, which, if taken from an immunized individual,
`is called antiserum because it contains specific antibodies against the immun-
`izing antigen as well as other soluble serum proteins. To study immune
`responses mediated by T cells, blood lymphocytes or cells 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 immune response is 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
`potential to elicit specific antibodies, but some need to be attached to an
`immunogen in order to do so. This means that although all immunogens are
`antigens, not all antigens are immunogenic. The antigens used most
`frequently in experimental immunology are proteins, and antibodies to
`proteins are of enormous utility in experimental biology and medicine.
`Purified proteins are, however, not always highly immunogenic and to
`provoke an immune response have to 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 immune response if
`attached to a protein carrier. Thus, the immunogenicity of protein antigens
`determines the outcome of virtually every immune response.
`
`APPENDIX l
`
`
`
`
`
`Lassen — Exhibit 1041, p. 5
`
`Lassen - Exhibit 1041, p. 5
`
`

`

`
`
`
`Antisera generated by immunization with even the simplest antigen will
`
`contain many dliiercnt antibody molecules that bind to the immunogen in
`
`slightly didcrent ways. Some of the antibodies in an antiserum are cross-
`
`reactive. it cross-reaction is defined as the binding ofan antibody to an antigen
`
`other than the Immunogen; most antibodies cross—react with closely related
`
`antigens but. on occasion. some bind antigens having no clear relationship to
`the immunogen. These cross-reacting antibodies can create problems when
`the antiserum is used to detest a specific antigen. They can be removed from
`an antiserum by absorption with the cross-reactive antigen. leaving behind
`the antibodies that bind only to the Immunogen. Absorption can be
`performed by affinity chromatography using immobilized antigen. a technique
`that is also used for purification of antibodies or antigens {see Section tit-5].
`Most problems of cross-reactivity can be avoided. however. by making
`monoclonal antibodies [see SectiouA-iZJ.
`
`
`
`Although almost any structure can be recognized by antibody as an antigen.
`usually only proteins elicit fully developed adaptive immune responses. This
`is because proleins have the ability 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 histocontpatibiilty complex
`(Mi-[Ci 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 immune response. This is also
`knoum as priming. as the animal or person is now 'primed’ like a pump to
`mount a more potent response to subsequent challenges with the same
`antigen. The response to each immunization is increasingly intense. so that
`secondary. tertiary. and subsequent responses are of increasing magnitude
`(Fig. M). Repetitive challenge with antigen to achieve a heightened state of
`immunity is known as hyperimmunization.
`
`Certain properties of a protein that favor the priming of an adaptive immune
`response have been defined by studying antibody responses to simple natural
`proteins like hen egg-white iysozyme and to synthetic polypeptide antigens
`(Fig. 21.2}. The larger and more complex a protein. and the more distant its
`relationship to self proteins. the more likely it is to elicit a response. This is
`because such responses depend on the proteins being degraded into peptides
`that can bind to MHC molecules. and on the subsequent recognition of these
`peptldo:Mi-IC compiettcs by T cells. The larger and more distinct the protein
`antigen. the more likely it 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 made to aggregate in some way. Many vaccines. for example.
`use aggregated protein antigens to potentiatc the immune response.
`
`Lassen — Exhibit 1041, p. 6
`
`
`
`gS
`E‘
`
`E5L
`
`EaE E 5
`
`
`
`
`
`10‘
`
`to2 to” 10‘
`
`1o“
`107
`10°
`105
`Antigen dose
`
`
`
`high-zonetolerance
`
`
`
`Antibodyresponse(arbitraryunis)8‘6‘68u.:xu.
`
`10“
`107
`10°
`105
`to3 10"
`102
`10‘
`1
`Antigen dose given in primary immunization
`
`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
`Illustrates 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 response elicited by a dose of
`antigen of 103 arbitrary mass units. Very
`low doses of antigen do not cause an
`immune response at all, Slightly higher
`doses appear to inhibit specific antibody
`production. an effect known as low-zone
`tolerance. Above these doses there is a
`steady increase in the response with
`antigen dose to reach a broad optimum.
`Very high doses of antigen also Inhibit
`immune responsiveness to a subsequent
`challenge. a phenomenon known as
`high~zone tolerance.
`
`
`
`
`A-t
`
`Haptens.
`
`Small organic molecules of simple structure, such as phenyl arsnnates and
`nitrophenyls. do not provoke antibodies when injected by themselves.
`However. antibodies can be raised againar them if the molecule is attached
`covalently. by simple chemical reactions. to a protein carrier. Such small
`molecules were termed haptens {from the Greek tropism. to fasten) by the
`lmmunoiogist Karl Landsteinsr. who first studied them in the early 19005. He
`found that animals immunized with a human—carrier conjugate produced
`
`Lassen - Exhibit 1041, p. 6
`
`

`

`
`
`Immunization 615
`
`i
`
`:..
`
`.,
`
`_
`
`I
`
`.
`
`I'
`
`Damned lmmunogenlclty
`
`II.
`
`I
`
`- 3.
`
`> intravenous or lnlregestrlc
`
`Fig. A.2 Intrinsic properties and
`extrinsic factors that affect the
`immunogenicity of proteins.
`
`I
`
`
`
`
`
`Small (MW<2500)
`
`
`
`
`
`
`
`
`
`
`
`
`Form
`
`
`Similarity to self protein
`
`
`Few differences
`
`
`Rapid misses
`
`
`
`
`, _
`
`Adjuvants
`
`
`
`
`
`Interaction with host MHC
`
`
`:_-.
`
`.
`
`.
`
`HI“ L_
`
`Eliectlve
`
`
`
`
`lnelfective
`
`
`
`three distinct sets of antibodies (Fig. A.3). One set comprised hapten-specific
`antibodies that reacted with the same hapten on any carrier, as well as with
`free hapten. The second set of antibodies was specific for the carrier protein,
`as shown by their ability to bind both the hapten-modified and unmodified
`carrier protein. Finally. some antibodies reacted only with the specific conjugate
`of hapten and carrier used for immunization. Landsteiner studied mainly the
`antibody response to the hapten, as these small molecules could be synthesized
`in many closely 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 important part 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
`compounds that elicit antibody responses when they attach to self proteins
`(see Section 12-10).
`
`Fig. A.3 Antibodies can be elicited by
`small chemical groups called haptens
`only when the hapten is linked to an
`immunogenic protein carrier. Three
`types of antibodies are produced. One
`set (blue) binds the carrier protein alone
`and Is called carrier-specific. One set
`(red) binds to the hapten on any carrier
`or to free hapten In solution and Is called
`hapten-speciflc. 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 carrier, and is
`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
`conjugate-specific antibody.
`
`
`
`Antigen
`
`Lassen — Exhibit 1041, p. 7
`
`Lassen - Exhibit 1041, p. 7
`
`

`

`
`
`Appendix I: Immunologists’ Toolbox
`———-——————.—.—_—_—_____
`
`A-2
`
`Routes of immunization.
`
`The route by which antigen is administered affects both the magnitude and
`the type of response obtained. The most common routes by which antigen is
`introduced experimentally or as a vaccine into the body are injection into
`tissue by subcutaneous (5.0.) injection between the epidermis and dermal
`layers. or by intradermal (i.d.) injection. or intramuscular [Ln-i.) 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 or inhalation.
`
`.
`
`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 so this is the method most
`commonly used when the object of the experiment is to elicit specific anti-
`bodies or '1' cells against a given antigen. Antigens injected or transfused
`directly into the bloodstream tend to induce immune unresponsiveness 0r
`tolerance unless they bind to host cells or are in the form of aggregates that
`are readily taken up by antigen-presenting cells.
`
`Antigen administration via the gastrointestinal tract is used mostly in the
`study of allergy. It has distinctive effects. frequently eliciting a local antibody
`response in 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 important in avoiding allergy 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 cause of such allergies (see Chapter 12).
`
`Introduction of antigen into the respiratory tract is also used mainly in the
`study of allergy. Protein antigens that enter the body through the respiratory
`epithelium tend to elicit allergic responses. for reasons that are not clear.
`
`A-3
`
`Effects of antigen dose.
`
`The magnitude ofthc immune response depends on the dose ofimmnnogen
`administered. Below a certain threshold dose. most proteins do not elicit any
`immune response. Above the threshold dose. there is a gradual increase in
`the response as the dose of antigen is increased. until a broad plateau level is
`reached. followed by a decline at very high antigen doses (see Fig. A.1). As
`most infectious agents enter the body in small numbers, immune responses
`are generally elicited only by pathogens that multiply to a level sufficient to
`exceed the antigen dose threshold. The broad response optimum allows the
`system to respond to infectious agents across a wide range of doses. At very
`high antigen doses the immune response is inhibited, which may be important
`in maintaining tolerance to abundant self proteins such as plasma proteins.
`In general. secondary and subsequent immune responses occur at lower
`antigen doses and achieve higher plateau values. which is a sign of immune-
`logical memory. However. under some conditions. very low or very high
`doses of antigen may induce specific unresponsive states. known respectively
`as acquired low-zone or high -zone tolerance.
`
`A-4 Adjuvants.
`
`Most proteins are poorly immunogenic or nonimmunogenic when adminis-
`tered by themselves. Strong adaptive immune responses to protein antigens
`almost always require that the antigen be injected in a mixture known as an
`
`d
`
`J
`I
`|
`'
`
`_
`
`'
`
`-
`
`i
`[
`|
`
`i
`
`_
`
`'.
`
`'
`
`
`
`Lassen — Exhibit 1041, p. 8
`
`Lassen - Exhibit 1041, p. 8
`
`

`

`immunization
`———-————-—————_—_———___—_
`
`61'!
`
`adjuvant. An adjuvant is any substance that enhances the immunogenicity of
`substances mixed with it. Adjuvants differ from protein carriers in that they
`do not form 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 adjuvants are listed in Fig. A.4.
`
`Adjuvants can enhance immunogenicity in two different 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 adj uvants 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, they are clearly the more
`important component of an adjuvant. Microbial products may signal
`macrophages or dendritic cells to become more effective antigen-presenting
`cells (see Chapter 2). One of their effects is to induce the production of
`inflammatory cytokines and potent local 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 antigens that can also
`act as effective adjuvants. For example, purified constituents of the bacterium
`Bordetella pertussis, which is the causal agent of whooping cough, are used as
`both antigen and adjuvant in the triplex DPT (diphtheria, pertussis, tetanus)
`vaccine against these diseases.
`
`Composition
`
`Mechanism oi action
`
`
`Delayed release oi antigen;
`Oil-in-water emulsion
`Incomplete Freund's adiuvanl
`enhanced uptake by
`macrophages
`
`
`Delayed release of antigen;
`Oil-in-waier emulsion
`enhanced uptake by
`
`macrophages; induction oi
`Complete Freund's adJuvant
`cosiimulalors in macrophages
`
`Freund's adiuvant with MDP
`
`murarnyldipepiide (MDP),
`a constituent oi mycobacterla
`
`Similar to complete
`Freund‘s adiuvant
`
`with dead mycobaoteiia Oil-in-waier emulsion with
`
`
`.
`.
`Alum (aluminum hydroxtde)
`
`.
`,
`Alum‘num hydmx'de 99'
`
`Delayed release of antigen;
`enhanced macrOphage uptake
`
`Delayed release oi antigen;
`Alum plus
` Aluminum hydroxide gel
`enhanced uptake by
`with killed B. pertussis
`Bordeieiia pertussis
`macrophages;
`induction oi co-slimuiatcis
` Delivers antigen to cylosoi;
`
`
`Matrix oi Ouil A
`Immune stimulatory
`allows induction oi
`complexes (iSCOMs)
`containing viral proteins
`cytotoxic T cells
`
`
`
`Fig. A.4 Common adjuvants and their
`use. Adjuvanis are mixed with the
`antigen and usually render it 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 oi the
`detergent Quii 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
`
`

`

`ll
`
`|
`|
`
`313
`
`Appendix I: 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 immunogen is usually measured by analyzing
`the specific antibody produced in a humoral immune response. This is most
`conveniently achieved by assaying the antibody that accumulates in the fluid
`phase of the blood or plasma; such antibodies are known as circulating 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-l 1.
`
`The most important characteristics of an antibody response are the specificity,
`amount, isotype or class, and affinity of the antibodies produced. The
`specificity determines the ability of the antibody to distinguish the immunogen
`from other antigens. The amount of antibody can be determined in many
`different ways and is a function of the number of responding B cells, their rate
`of antibody synthesis, and the persistence of the antibody after production.
`The persistence of an antibody in the plasma and extracellular fluid bathing
`the tissues is determined mainly by its isotype (see Sections 4-15 and 9—12);
`each isotype has a different half-life in viva. The isotypic composition of an
`antibody response also determines the biological functions these antibodies
`can perform and the sites in which antibody will be found. Finally, the
`strength of binding of the antibody to its antigen in terms of a single antigen-
`binding site binding to a monovalent antigen is termed its affinity (the total
`binding strength of a molecule with more than one binding site is called its
`avidity). Binding strength is important, since the higher the affinity of the
`antibody for its antigen, the less antibody is required to eliminate the antigen,
`as antibodies with higher affinity will bind at lower antigen concentrations.
`All these parameters of the humoral immune response help to determine the
`capacity of that response to protect the host from infection.
`
`Antibody molecules are highly specific for their corresponding antigen, being
`able to detect one molecule of a protein antigen out of more than 108 similar
`molecules. This makes antibodies both easy to isolate and study, and
`invaluable as probes of biological processes. Whereas standard chemistry
`would have great difficulty in distinguishing between two such closely related
`proteins as human and pig insulin, or two such closely related structures as
`0rtho~ and para—nitrophenyl, antibodies can be made that discriminate
`between these two structures absolutely. The value of antibodies as molecular
`probes has stimulated the development of many sensitive 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
`and stability of antigen binding by antibodies. Comprehensive guides to the
`conduct of these antibody assays are available in many books on immuno-
`logical methodology; we will illustrate here only the most important tech-
`niques, especially those used in studying the immune response itself.
`
`Some assays for antibody measure the direct binding of the antibody to its
`antigen. Such assays are based on primary interactions. Others determine
`the amount of antibody present by the changes it induces in the physical state
`
`,
`
`'
`
`'
`-
`
`,
`
`I
`
`
`
`Lassen — Exhibit 1041, p. 10
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`Lassen - Exhibit 1041, p. 10
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`The detection, measurement, and characterization of antibodies and their use as research and diagnostic tools
`619
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` O O
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`dfifilfllfid 0i
`antigen A
`
`I It
`
`... PliilllEd
`: antigen A
`
`Fig. A.5 Affinity chromatography ueee antigen—antibody
`binding to purify antigens or antibodies. To purity a specific
`antigen from a complex mixture oi molecules, a monoclonal
`antibody is attached to an insoluble matrix, such as
`chromatography beads, and the mixture oi molecules is
`
`passed over the matrix. The specific antibody binds the antigen
`of interest; other molecules are washed away. Speciilc antigen
`is then eluted by altering the pH, which can usually disrupt
`antibody—antigen bonds. Antibodies can be purified in the
`same way on beads coupled to antigen (not shown).
`
`of the antigen, such as the 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 amount and 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 commonly referred to as serologica] assays, and the use
`of antibodies is often called serology. The amount of antibody is usually
`determined by antigen-binding assays after titration of the antiserum by
`serial dilution, and the point at which binding falls to 50% of the maximum is
`usually referred to as the titer of an antiserum.
`
`A-5
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`Affinity chromatography.
`
`Specific antibody can be isolated from an antiserum by affinity
`chromatography, which exploits the specific binding of antibody to antigen
`held on a solid matrix (Fig. A5}. Antigen is bound covalently to small, chem [-
`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 proteins in the serum, including antibodies to other substances, can be
`washed away. The specific antibodies are then eluted, typically by lowering the
`pH to 2.5 or raising it to greater than ii. Antibodies bind stably under physio-
`logical conditions of salt concentration. temperature, and pH, but the binding
`is reversible as the bonds are noncovaient. Attittity chromatography can also he
`used to purify antigens from complex mixtures by using beads coated with
`specific antibody. The technique is known as affinity chromatography because
`it separates molecules on the basis of their affinity for one another.
`
`A-G
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`Radioimmunoassay (FllA), enzyme-linked immunosorbent assay
`(ELISA), and competitive inhibition essay.
`
`Radioimmunoassay (BIA) and enzyme-linked immunosorbent assay (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
`
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`Lassen — Exhibit 1041, p. 11
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`Lassen - Exhibit 1041, p. 11
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`methods one needs a pure preparation of a known antigen or antibody, 0.-
`both. in order to standardize the assay. We will describe the assay with a sample
`of pure antibody, which is the more usual case. but the principle is similar if
`
`pure antigen is used instead. in BIA for an antigen, pure antibody against that
`
`antigen is radioactively labeled. usually with 1251: for the ELISA. an enzyme is
`linked chemically to the antibody. The unlabeled component. which in this
`
`case would be antigen, is attached to a solid support, such as the wells of a
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`plastic multiwell plate. which will adsorb a certain amount of any protein.
`
`
`
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`I Wash away unboundantibody I
`
`
`
`Measure absorbarm at light
`by colored product
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`
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`Enzyme makes colored
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`
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`product from added
`colorless substrate
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`Fig. A.6 The principle of the enzyme-
`linked lmmunosorbent assay (ELISA).
`To detect antigen A, purified antibody
`specific for antigen A is linked
`chemically to an enzyme. The samples
`to be tested are coated onto the surface
`of plastic wells to which they bind
`nonspecifloally: residual sticky sites on
`the plastic are blocked by adding
`Irrelevant proteins (not shown). The
`labeled antibody is then added to the
`wells under conditions where nonspecific
`binding is prevented. so that only
`binding to antigen A causes the labeled
`antibody to be retained on the surface.
`Unbound labeled antibody is removed
`from all wells by washing, and bound
`antibody is detected by an enzyme-
`dependenl color-change reaction. This
`assay allows arrays of wells known as
`microtiter plates to be read in fiberoptic
`multichannel spectrometers. greatly
`speeding the assay. Modifications of this
`basic assay allow antibody or antigen in
`unknown samples to be measured as
`shown in Figs A.7 and A29 (see also
`Section A-10),
`
`The labeled antibody is allowed to bind to the unlabeled antigen. under-
`conditions where nonspecific adsorption is blocked. and any unbound anti-
`body and ether preteins are washed away. Antibody binding in Ria is measured
`directly in terms of the amount of radioactivity retained by the coated walls,
`whereas in ELISA. binding is detected by a reaction that converts a colorless
`substrate into a colored reaction product (Fig. A15]. The color change can be
`read directly in the reaction tray. making data collection very easy. and ELISA
`also avoids the hazards oi” radioactivity. This makes HI .1511 the preferred
`method for most direct-binding assays. Labeled anti-immunogiobulin anti-
`bodies {see Section A-lOl can also be used in [tin or ELISA to detect binding
`oi“ unlabeled antibody to unlabeled antigen-coated plates. In this case, the
`labeled anri-immunoglobulin antibody is used in what is termed a ‘sccond
`layer.‘ The use or such a second layer also amplifies the signal. as at least two
`molecules of the labeled anti-immunoglobulin antibody are able to bind to
`each unlabeled antibody. RIA and ELISA can also be carried out with unlabeled
`antibody stuck to the plates and labeled antigen added.
`
`A modification of ELISA known as a capture or sandwich ELISA (or more
`generally as an antigemcapture assay) can be used to detect secreted products
`such as cytokines. Rather than the antigen being directly attached to a plastic
`plate. antigen-Specific antibodies are bound to the plate. These are able to
`bind antigen with high affinity. and thus concentrate it on the surface of the
`plate. even with antigens that are present in very low concentrations in the
`initial mixture. A separate labeled antibody that recognizes a different epitope
`to the immobilized first antibody is then used to detect the bound antigen.
`
`These assays illustrate two crucial aspects of all serological assays. First. at least
`one of the reagents must be available in a pure, detectable form in order to
`obtain quantitative information. Second. there must be a means of separating
`the bound fraction of the labeled reagent from the unbound. free fraction so
`that the percentage of specific binding can be determined. Normally. this sep—
`aration is achieved by having the unlabeled partner trapped on a solid support.
`Labeled molecules t