Antigen & antibody lecture 5

Antibody

Antigens & AntibodiesBy Dr Tabassum Mahmood1

Antigen

Any substance which when introduced into body, is capable of provoking an immune response(antibodies production ) 2

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Antigens CharacteristicsForeignness: Molecules recognized as self are not immunogenicMolecular Size: Small foreign molecule with molecular weight below 10,000 (hapten ) weakly immunogenic & must be coupled to carrier molecule to be antigenicOnce antibodies are formed they recognize hapten3

Antigen CharacteristicsChemicalStructural Complexity: Amino acidhomopolymers less immunogenic than heteropolymers (containing two or three different amino acids)4

Antigens CharacteristicsAntigenic Determinants (Epitopes) :Small chemical groups on antigen molecule that can elicit immunological response & react with antibodyDosage , Route & Timing of Antigen administration: These factors affect immunogenicity

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AdjuvantsEnhance immune response to antigenChemically unrelated to antigen Differ from a carrier protein :Not covalently bound to antigen like carrier protein Cause slow release of antigen, prolonging stimulus

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AdjuvantsStimulate Toll-like receptors at surface of macrophages, cytokine production enhances response of T cells and B cells to antigenSome vaccines contain adjuvants i.e. aluminum hydroxide or lipids

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EpitopeSmall part of antigen that interacts with an antibodyAny given antigen may have several epitopesEach epitope is recognized by a different antibody

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Super antigenThey are able to bind to MHC molecules outside the peptide binding cleft They cause upto 10% of T cells to be nonspecifically stimulated/ activated9

Epitopes: Antigen Regions Interact with Antibodies10

Antibody Gamma globulin proteins that react specifically with antigen that stimulated their production 20% of plasma proteinFive classes of antibodies: IgG, IgM, IgA, IgD, and IgE (based on differences in heavy chains)11

Functions of antibodiesNeutralize toxins and virusesOpsonize microbes to be easily phagocytosedActivate Complement, and prevent attachment of microbes to mucosal surfacesCatalytic :Antibody can act as an enzyme to catalyze synthesis of ozone (O3) that has microbicidal activity. 12

Antibody can take singlet oxygen produced by neutrophils and react it with water to produce hydrogen peroxide and O3. The O3 generated can kill Escherichia coli. The catalytic function of antibodies is independent of their antigen specificity and of requirement to bind to any antigen.12

Antigen-Antibody Reactions

AntibodiesAfter stimulation, B cells differentiate into plasma cells, & secrete antibodies,(immunoglobulins) ,mediators of humoral immunity

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Antibodies Characteristics Diversity Respond to different antigensLong memory Respond many years after initial exposure due to memory T cells and B cells 15

Antibodies Characteristics Specificity Actions specifically directed against antigen that initiated responseInflammatory response : Combined effect of cells (e.g., T cells, B cells, macrophages & neutrophils) & proteins (e.g., interleukins, antibodies & complement)

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Structure of immunoglobulinSimplest antibody molecule has Y shape Consists of four polypeptide chains: Two H chains and two L chainsFour chains linked by disulfide bondsAntibody molecule always consists of identical H chains and identical L chains17

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Structure of immunoglobulinL and H chains subdivided into variable and constant regions.Regions composed of three-dimensionally folded, repeating segments called domainsEach domain is about 110 amino acids long globular in shape stabilized by intrachain disulphide bondsAntigen binding sites located in variable domains

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Structure of immunoglobulin20

Structure of immunoglobulinVariable regions of light and heavy chain responsible for antigen-bindingConstant region of heavy chain responsible for biologic functions (e.g., complement activation and binding to cell surface receptors)21

Structure of immunoglobulinLight chain : One variable region and one constant regionL chain attached to H chain by disulphide & non- covalent bonds22

Structure of immunoglobulinL chains belong to one of two types, (kappa) or (lambda), due to amino acid differences in their constant regions Both types occur in all classes of immunoglobulins (IgG, IgM, etc.), but any one immunoglobulin molecule contains only one type of L chain23

Structure of immunoglobulinHeavy chain consists of a variable region and a constant region divided into three domains: CH1, CH2, and CH3Each domain 110 amino acids long CH2 domain contains complement-binding siteCH3 domain is site of attachment of IgG to receptors on neutrophils and macrophagesH chains structurally & antigenically distinct for each classH chains are distinct for each of five immunoglobulin classes and are designated , , , , and

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Structure of immunoglobulinVariable regions of L and H chains have three extremely variable(hypervariable) amino acid sequences at amino-terminal end that form antigen-binding site Specificity of antibodies is due to hypervariable regions25

Structure of immunoglobulinAmino-terminal portion of each L & H chain participates in antigen-binding siteCarboxy terminal forms Fc fragment, which has biologic activities26

Heavy chainH chain designated by Greek letter.

Ig classH chainIg GIg MIg AIg DIg E

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Structure of immunoglobulinAntibody molecule treated with a proteolytic enzyme s papain,break peptide bonds in hinge region , producing two identical Fab fragments, which carry antigen-binding sites, and one Fc fragment, involved in placental transfer, complement fixation, attachment site for various cells,& other biologic activities28

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Antigen-Antibody Responses

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Classification of antibodies Immunoglobulin A (IgA)Immunoglobulin G (IgG)Immunoglobulin M (IgM)Immunoglobulin D (IgD)Immunoglobulin E (IgE)Based on structural differences in constant regions of heavy chainsClasses have specialized effector functions31

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Ig M5-8 % of serum immunoglobulinsShort lived Pentameric structurePredominant Ab in primary immune response.Earliest Ab synthesized by fetusConfined to intravascular pool due to large sizeNot transported across placentaPresence of IgM in newborn indicates intra uterine infectionUseful in the diagnosis of congenital infections like syphilis, rubella, HIV, toxoplasmosis etc.

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DistributionClass of Immuno-globulin (Antibody)IgM(pentamer)J chain

First Ig classproduced afterinitial exposure toantigen; then itsconcentration inthe blood declinesPromotes neutraliza-tion and cross-linking of antigens;very effective incomplement systemactivationFunction33

33Figure 43.20 The five antibody, or immunoglobulin (Ig), classes

75% of total immunoglobulins4 subclasses IgG1, IgG2, IgG3 & IgG4 Each having a distinct type of gamma chainMajor Antibody of secondary response, found in serum & body fluidsOnly maternal Ig to be transported across placenta natural passive immunity in newbornParticipates in complement fixation, precipitation & neutralization of viruses & toxinsIgG

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DistributionFunctionClass of Immuno-globulin (Antibody)IgG(monomer)Most abundant Igclass in blood;also present intissue fluidsPromotes opsoniza-tion, neutralization,and cross-linking ofantigens; less effec-tive in activation ofcomplement systemthan IgMOnly Ig class thatcrosses placenta,thus conferringpassive immunityon fetus35


Ig A2nd most abundant 10-13 %Major Ig in colostrum, saliva, tears & other body fluids.Two forms : IgA1 & IgA2.Secretory IgA in dimeric form composed of 2 basic chain units, a J chain & the secretory component.Secretory component helps to transport dimer from submucosa to mucosal cell surface.Secretory component protects IgA from proteolytic digestion and denaturation.36

DistributionFunctionClass of Immuno-globulin (Antibody)IgA(dimer)J chain

Secretorycomponent

Present insecretions suchas tears, saliva,mucus, andbreast milkProvides localizeddefense of mucousmembranes bycross-linking andneutralization ofantigensPresence in breastmilk conferspassive immunityon nursing infant37


Ig ELow levels in serumOn surface of mast cells & basophils which have specific receptors for Fc portion of IgEProduced in linings of respiratory & intestinal tractsCauses anaphylactic type of hypersensitivityDefense against parasitic infections 38

DistributionFunctionClass of Immuno-globulin (Antibody)IgE(monomer)Present in bloodat low concen-trationsTriggers release frommast cells andbasophils of hista-mine and otherchemicals that causeallergic reactions39


Ig DResembles Ig G structurallyPresent with Ig M on B cell surfaceSusceptible to proteolytic attack

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DistributionFunctionClass of Immuno-globulin (Antibody)IgD(monomer)Trans-membraneregionPresent primarilyon surface ofB cells that havenot been exposedto antigensActs as antigenreceptor in theantigen-stimulatedproliferation anddifferentiation ofB cells (clonalselection)

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IsotypesAntigenic (amino acid) differences in constant regions of heavy chainsIgG and IgM are different isotypes; constant region of their H chains ( and ) is different antigenically 42

AllotypeAdditional antigenic features of immunoglobulins that vary among individualsGenes coding for L and H chains are polymorphic, and individuals can have different alleles.e.g. H chain contains an allotype called Gm, which is due to a one or twoamino acid difference that provides a different antigenecity to the moleculeEach individual inherits different allelic genes that code for one or another amino acid at the Gm site43

IdiotypeAntigenic determinants formed by specific amino acids in hypervariable regionEach idiotype is unique for immunoglobulin produced by a specific clone of antibody-producing cellsAnti-idiotype antibody reacts only with hypervariable region of specific immunoglobulin molecule that induced it44

Immunoglobulin class switching(isotype switching)Initially, all B cells carry IgM specific for an antigen and produce IgM antibody in response to exposure to that antigenLater, gene rearrangement permits elaboration of antibodies of same antigenic specificity but of different immunoglobulin classes 45

Immunoglobulin class switching(isotype switchingAntigenic specificity remains same for lifetime of B cell and plasma cell because specificity is determined by variable region genes (V, D, and J genes on the heavy chain and V and J genes on the light chain) no matter which heavy-chain constant region is utilized

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Immunoglobulin class switching(isotype switchingSame assembled VH gene can sequentially associate with different CH genesImmunoglobulins produced (IgG, IgA, or IgE) are specific for same antigen as original IgM but have different biologic characteristics47

Immunoglobulin class switching(isotype switchingMature B cells can express both IgM and IgDOnce a B cell has class switched past a certain H chain gene, it can no longer make that class of H chain because intervening DNA is excised and discarded. . 48

Immunoglobulin class switching(isotype switchingClass switching occurs only with heavy chains; light chains do not undergo class switching. Switch recombinase enzyme catalyzes rearrangement of VDJ genes during class switching.Control of class switching is dependent on 1.concentration of various interleukins(IL)-4 enhances production of IgE,IL-5 increases IgA2.Interaction of CD40 protein on B cell with CD40 ligand protein on the helper T cell49

Primary & Secondary antibody responsePrimary ResponseFollowing exposure to an antigen, there is a slow rise in IgM followed by a slow rise in IgG

Secondary ResponseFollowing exposure to previously encountered antigen, there is a rapid rise in IgG and slow or no rise in IgMMemory or anamnestic response50

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Abnormal Immunoglobulins

Bence Jones protein in multiple myeloma light chains of Immunoglobulins

Cryoglobulinemia formation of gel or precipitate on cooling serum which redissolves on warming in myelomas, SLE53

Monoclonal AntibodiesAntibodies produced in response to antigens are heterogeneous, formed by different clones of plasma cells ( polyclonal)Antibodies that arise from a single clone of cells (e.g., in a plasma cell tumor [myeloma])are homogeneous (monoclonal) 54

HybridomaMonoclonal antibodies made in laboratory by fusing a myeloma cell with an antibody-producing cell are called hybridoma

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HybridomaHybridoma cells made as following (1) A mouse immunized with antigen of interest(2) Spleen cells from this mouse grown in a culture dish in the presence of mouse myeloma cellsMyeloma cells grow indefinitely in culture, & do not produce immunoglobulins

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Hybridoma3)Fusion of cells by adding certain chemicals (e.g., polyethylene glycol) (4) Cells grown in a special culture medium(HAT medium) that supports growth of fused, hybrid cells but not of parental cells (5) Resulting clones of cells screened for production of antibody to antigen of interest

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Production of monoclonal antibodies

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Go & have some tea & snacks please

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Education

Antigen & antibody lecture 5