1、Chapter 4 Antigens and AntibodiesOct 17,19&24,2006Complementarity of interacting surfaces of Ab and AgAbAg你需要學習的課題你需要學習的課題:1.好的免疫原(immunogen)有些什麼特質？2.什麼叫做抗原決定區(epitope)？3.B-cell epitope 有何特性？*4.抗體分子的基本構造及各部位的名稱。5.抗體的種類、特性及功能。6.單株抗體與多株抗體。Outline 1.Immunogenicity versus antigenicity 2.Epitopes*3.Basic
2、 structure of antibodies(Abs)4.Ab-binding site 5.Ab-mediated effector functions 6.Ab classes and biological activities 7.Antigenic determinants on immunoglobulins(Ig)8.The B-cell receptor 9.The Ig superfamily10.Monoclonal AbsImmunogenicity vs.AntigenicityImmunogenicity 免疫性:the ability to induce an A
3、b and/or cell-mediated immune responseAntigenicity 抗原性:the ability to combine specifically with Ab and/or cell-surface receptors(Ig/TCR)-Although a substance that induces a specific immune response is usually called an antigen,it is more appropriately called an immunogen.-Although all molecules that
4、 have the property of immunogenicity also have the property of antigenicity,the reverse is not true.-Some small molecules,called haptens,are antigenic but incapable,by themselves,of inducing a specific immune response.In other words,they lack immunogenicity.DNP:dinitrophenol(bovine serum albumin)Lan
5、dsteiners work demonstrated both the specificity of the immune system for small structural variations on haptens and the enormous diversity of epitopes that the immune system is capable of recognizing.Factors That Influence ImmunogenicityIntrinsic properties of an immunogen:-Foreignness-Molecular si
6、ze-Chemical composition and heterogeneity-Susceptibility to antigen processing and presentationThe biological system:-Genotype of the recipient animal-Immunogen dosage and route of administration-Adjuvants Foreignness-Generally,the greater the phylogenetic distance between two species,the greater th
7、e structural(and therefore the antigenic)disparity between them.-Some macromolecules(e.g.,collagen and cytochrome c)were highly conserved throughout evolution and therefore display very little immunogenicity across diverse species lines.-Conversely,some self-components(e.g.,corneal tissue and sperm)
8、are effectively sequestered from the immune system,so that if these tissues are injected even into the animal from which they originated,they will function as immunogens.Molecular size-There is a correlation between the size of a macromolecule and its immunogenicity.-The best immunogens tend to have
9、 a molecular mass 100,000 daltons(Da).-Generally,substances with a molecular mass less than 5,000 10,000 Da are poor immunogens.Chemical composition and complexity-Synthetic homopolymers tend to lack immunogenicity regardless of their size.-All 4 levels of protein organization primary,secondary,tert
10、iary and quaternary contribute to the structural complexity of a protein and hence affect its immunogenicity.Four levels of protein organizational structure For Ab(B cell)responses:Proteins are the most potent immunogens,with polysaccharides ranking second.Lipids and nucleic acids of an infectious a
11、gent generally do not serve as immunogens unless they are complexed with proteins or polysaccharides.For T cell responses:Only proteins and some lipids(glycolipids and phospholipids)serve as immunogens.Susceptibility to antigen processing and presentation-The development of both Ab-mediated and T-ce
12、ll-mediated immune responses requires interaction of T cells with Ag that has been processed and presented together with MHC molecules.-Large,insoluble macromolecules generally are more immunogenic than small,soluble ones because the larger molecules are more readily phagocytosed and processed.-Mole
13、cules that cannot be degraded(e.g.,polymers of D-amino acids)and/or cannot be presented with MHC molecules are poor immunogens.The biological system contributes to immunogenicity-Genotype of the recipient animal-Immunogen dosage and route of administration-Adjuvants Immunogen dosage and route of adm
14、inistrationDoses:too low no response,too high toleranceExposure:repeated administration(booster)over a period of time is usually more effectiveRoutes:orally(從口入的)parenterally(非從口入的)-intravenous(iv):into a vein -intradermal(id):into the skin -subcutaneous(sc):beneath the skin -intramuscular(im):into
15、a muscle -intraperitoneal(ip):into the peritoneal cavity為什麼 Ag 的量、接觸次數 及 路徑與免疫反應的強度有關？Adjuvants 佐劑佐劑-Latin adjuvare,to help-Substances that,when mixed with an antigen and injected with it,enhance the immunogenicity of that antigen.Effects of adjuvants1.antigen persistence is prolonged -slower releas
16、e of antigen at the injection site e.g.,alum 明礬 AlK(SO4)2,2.costimulatory signals(p.159)are enhanced -increased expression of B7 molecules on APC maximal activation of TH cells3.local inflammation is increased4.nonspecific proliferation of lymphocytes is stimulated -formation of a dense,macrophage-r
17、ich mass of cells called a granuloma 肉芽腫 e.g.,incomplete Freunds adjuvant(IFA),complete Freunds adjuvant(CFA)1.在實驗動物中製備抗體時，經常使用 CFA (complete Freunds adjuvant)，其作用機轉 為何？2.為何局部發炎反應能增強 Ab 反應？Epitopes-Lymphocytes do not interact with,or recognize,entire immunogen molecules;instead,they recognize discre
18、te sites on the macromolecule called epitopes,or antigenic determinants.-epitopes:immunologically active regions of an immunogen,that bind to Ag-specific membrane receptors on lymphocytes or to secreted Abs.T-cell and B-cell epitopes-The recognition of antigens by T cells and B cells is fundamentall
19、y different.-Because B cells bind antigen that is free in solution,the epitopes they recognize tend to be highly accessible sites on the exposed surface of the immunogen.-T-cell epitopes are peptides combined with MHC molecules.Thus,there is no requirement for solution accessibility such as B-cell e
20、pitope.Properties of B-cell epitopes 1.B-cell epitopes on native proteins generally are composed of hydrophilic a.a.on the protein surface that are topographically accessible to membrane-bound or free Ab.2.B-cell epitopes may be composed of sequential or nonsequential amino acids.Sperm whale myoglob
21、ulin contains 5 sequential B-cell epitopesHen egg-white lysozyme(HEL)composesone nonsequential(conformational)epitopeContact with Ab light chainContact with Ab heavy and light chainsContact with Ab heavy chainAb to native HEL does not bind to reduced HEL3.B-cell epitopes tend to be located in flexib
22、le regions of an immunogen and display site mobility.-site mobility of epitopes maximizes complementarity with the Abs binding site4.Complex proteins contain multiple overlapping B-cell epitopes,some of which are immunodominant.-Most of the surface of a globular protein is potentially immunogenic.-S
23、ome epitopes,called immunodominant,induce a more pronounced immune response in a particular animal than other epitopes.Basic structure of AbsElectrophoresis of immune serum(Tiselius&Kabat,1939)Immune seraafter reactionwith Agg-g-globulin(g gG)Immunoglobulin(Ig):IgG,IgM,IgA,IgE,IgDAntibody(Ab)Ab mole
24、cules contain 4 peptide chains -A dimer of heterodimersV:variableC:constantm m:IgMg g:IgGa a:IgAd d:IgDe e:IgE(H)(L)50-55 kDa22 kDa100 kDa numerous small peptides150 kDaFab fragment:antigen binding45 kDaFc fragment:crystallizable50 kDaAntibody to the Fab fragment could react with both the H and L ch
25、ains,whereas antibody to the Fc fragment reacted only with the H chain.Fab consists of portions of an H and a L chain.Fc contains only H chain components.-A heterogeneous spectrum of antibodies in the serum g-globulin fraction-Multiple myeloma:a cancer of Ab-producing plasma cells-Myeloma protein:95
26、%of the serum Ig-Bence-Jones proteins:the excess light chains in the urine.-MOPC:mineral-oil induced plasmacytoma in mice Heavy and light chains are folded into“domains”(IgG,IgD,IgA)(IgM,IgE)Associations between domains of an Ab molecule2 Ag-binding sites:Immunoglobulin Domains -each contains about
27、110 a.a.residues and an intrachain disulfide bond that forms a loop of 60 a.a.Variable-Region Domains -hypervariable regions:(15%-20%of the variable domain)=complementarity-determining regions(CDR)CDR1,CDR2,CDR3 -framework regions(FR)-diversity in the VH domain is concentrated in CDRsVariability of
28、a.a.residues in the VH and VL domainsCDRs bind Ags number of different amino acids at a given positionVariability =_ frequency of the most common amino acid at a given positionConformational changes may be induced by antigen binding:after binding to the Ag :before binding to the AgCDRs of L chain:L1
29、,L2,L3CDRs of H chain:H1,H2,H3FabAb-mediated effector functions-Antibodies generally do not kill or remove pathogens solely by binding to them.-While V regions bind to Ag,the is responsible for a variety of collaborative interactions with other proteins,cells,and tissues that result in the effector
30、functions of the Ab responses.Ab-Mediated Effector Functions-Opsonization(調理作用)is promoted by Ab-Abs activate complement(補體)(chapter 7)-Antibody-dependent cell-mediated cytotoxicity (ADCC)kills cells (p.366 chapter 14)-Some Abs can cross epithelial layers by transcytosisAb promotes opsonization thro
31、ugh abinding to Fc receptors(FcR)on phagocytes Ab activates complement-mediated cytolysis or promotes opsonization through a binding to C receptors on phagocytesAntibody-dependent cell-mediated cytotoxicity(ADCC)(Figure 14-15)Transcytosis -movement of Ab across epithelial layer -delivery of IgA to t
32、he mucosal surfaces of the respiratory,gastrointestinal,and urogenital tracts,as well as its export to breast milk -transplacental transport of IgG from mother to fetus Secretory IgA in Breast Milk Bind to microbes in babys digestive tract and thereby prevent their attachment to the walls of the gut
33、 and their subsequent passage into the bodys tissues.Ab classes and biological activities5 major classes of secreted antibodyIgG-most abundant in serum-80%of total serum Ig-4 IgG subclasses4 subclasses of human IgG-size of the hinge region-no.&position of the interchain-S-S-bond-IgG1IgG2IgG3IgG4 in
34、serum conc.-90%-95%homologous in DNA sequences-varied effectiveness in placenta transfer and C activation IgM Pentamer-monomer on the membrane&pentamer in secretion -5%-10%serum Ig -1st Ab in neonates -1st Ab in primary response-more efficient in agglutination&C fixation-J(joining)chain is required
35、for polymerization of the monomers to form pentameric IgM-also present in mucosal surfaces IgA Dimer-10%-15%of total serum Ig-monomers,dimers,trimers and tetramers in serum-predominant in external secretions,e.g.,breast milk,saliva,tears,and mucus of the bronchial,genitourinary,and digestive tracts
36、Secretory IgADimers and tetramers in secretion with a secretory componentFormation of Secretory IgATranscytosisIgE-potent biological activity-extremely low conc.in serum-mediates the immediate hyper-sensitivity reactions-responsible for the symptoms of hay fever,asthma,hives,and anaphylactic shockAl
37、lergen cross-linkage of receptor-bound IgE on mast cells-induces degranulation,causing release of substances that mediate allergic manifestations IgD-0.2%of total serum Ig-together with IgM,is the major membrane-bound Ig on mature B cells-thought to function in the activation of B cells-no biologica
38、l effector function has been identifiedAntigenic determinants on Igs3 Antigenic Determinantsof ImmunoglobulinsIsotypesAllotypesIdiotypes Isotypic Determinants-constant-region determinants that collectively define each H-chain class and subclass,and each L-chain type and subtype within a species Allo
39、typic Determinants -differences in amino acids in C regions,which occur in some,but not all,members of a species Idiotypic Determinants-The unique amino acid sequence of the V regions of a given Ab.In some cases,an idiotype is the actual antigen-binding siteB-cell Receptor Fc receptors bound to Fc r
40、egions of Absneonatal Fc receptorFc receptors-essential for many of the biological functions of Abs-movement of Abs across cell membranes,e.g.,the transfer of IgG from mother to fetus across the plancenta-passive acquisition of Ab by many cell types,including B and T lymphocytes,neutrophils,mast cel
41、ls,eosinophils,macrophages,and natural killer cells(a linker between Ab molecules and various types of cells)The Ig superfamilyThe Ig superfamily-1The Ig superfamily-2 Monoclonal AbsClonal Selection of B LymphocytesProduction of monoclonal antibodies(mAb)QuestionsHow to predict whether a molecule is a“good”immunogen?What are the differences between B-cell epitopes and T-cell epitopes?Draw a schematic diagram of a typical IgG molecule and label each of the chains,bonds,regions,sites,fragments and domains.How does Ab kill or remove pathogens?What is the principal of making monoclonal antibody?
