抗体专题知识讲座

抗体专题知识讲座抗体专题知识讲座第1页AntibodyStructureIgdomain:110aminoacids;globulardomainusedinmanyproteins;Variabledomains,Constantdomains,HingeFab:fragmentantigenbindingFc:fragmentcrystallizable(effectorfunctions)©NewSciencePressLtd.抗体专题知识讲座第2页抗体专题知识讲座第3页TheImmunoglobulinSuperfamily

(afewexamples)抗体专题知识讲座第4页VariabilityinantibodiesisclusteredintheloopsintheVariabledomainsoftheheavyandlightchains(green);theseregionsareresponsibleforbindingtoantigen抗体专题知识讲座第5页AntibodyClasses:Structure©NewSciencePressLtd.抗体专题知识讲座第6页AffinityandAvidityAffinity:thestrengthofbindingbetweenasinglebindingsiteandasingleligand

[A][B][AB]Avidity:thestrengthofbindingbetweenamoleculeandacomplexligand,e.g.iftherearemultiplebindingsitesthentheaviditymaybeincreasedbyincreasingthenumberofbindingsitesorbyincreasingtheaffinityofthosebindingsitesKD=抗体专题知识讲座第7页AffinityandAvidityIIIgMisproducedearlyinanimmuneresponsewhentheaffinityforantigenoftenislow;asanimmuneresponsecontinues,antibodyaffinityisimproved,thisiscombinedby“classswitching”totheuseofsmallermolecules(IgG,IgEandIgA).Theincreasedaffinitycompensatesforthedecreaseinnumberofbindingsitesinmaintainingtheoverallavidityforantigen抗体专题知识讲座第8页©NewSciencePressLtd.抗体专题知识讲座第9页MonoclonalAntibodiesSingleantibody(allsameHandLchains)MadebyfusionofBcellstoatransformedcelllineoftheplasmacelltypeandselectionfor“hybridomas”thatproduceantibodywiththedesiredpropertiesStandardized,unlimitedreagentfordiagnosisortherapy(humanantibodiesor“humanized”antibodiescanbemade)抗体专题知识讲座第10页Polyclonalvs.Monoclonal

AntibodiesImmunizeIndividualSerumPolyclonalantibodies7+daysPurifyantibodiesBlymphocytes3daysImmortalize“hybridoma”cDNAcloningMonoclonalAntibodies抗体专题知识讲座第11页GenerationofMonoclonalAntibodies©NewSciencePressLtd.抗体专题知识讲座第12页MonoclonalantibodiesusedinmedicineStandardized,unlimitedreagentsfordiagnosisortherapy(humanantibodiesor“humanized”antibodiescanbemade)©NewSciencePressLtd.抗体专题知识讲座第13页GenerationofantibodydiversityHowdowemake>109differentantibodies?GenesforantibodiesarepresentinpiecesthatcanbecombinedinmanydifferentcombinationsindifferentlymphocytesVregionofIgLandHchainsareconstructedfrom2and3differentpieceseachhavingmultiplecopies抗体专题知识讲座第14页V(D)J

recombination©NewSciencePressLtd.抗体专题知识讲座第15页GenerationofAntibodyDiversityklightchains:40Vkx5Jk=200llightchains:30Vlx4Jl=120Hchains:40VHx27DHx6JH=6,480320Lchainsx6,480Hchains=2.1x106

Junctionaldiversity(additionordeletionofnucleotidesatrecombinationsites,especiallyofHchain),estimatedtoadd3x107foldtooveralldiversity抗体专题知识讲座第16页DiscoveryofRag1,2genes“RecombinationActivatingGene”抗体专题知识讲座第17页MechanismofV(D)JrecombinationCACCGTGRecombinationsignalsRag-1/Rag-2/ArtemisNon-homologousendjoiningproteins(defects:SevereCombinedImmunodeficiency(SCID))©NewSciencePressLtd.抗体专题知识讲座第18页CreationofJunctionalDiversity

byP-regionsandTdT抗体专题知识讲座第19页DefectsinLymphocytedevelopmentleadingtoseverecombinedimmunodeficiency(SCID)Note:SCIDcanalsoresultfromdefectsthatinterferewithlymphocyteactivation(adenosinedeaminasedeficiency,purinenucleotidephosphorylasedeficiency,MHCdefects,etc.)©NewSciencePressLtd.抗体专题知识讲座第20页LymphoidmalignanciesresultingfromerrorsinV(D)Jrecombination©NewSciencePressLtd.VDJRecombinationreactionscontributestotranslocationleadingtoover-expressionofacellulargrowthorsurvivalpromotinggene44CHCH抗体专题知识讲座第21页IgHeavychainclass(isotype)switching抗体专题知识讲座第22页Affinitymaturationandantibodyresponses抗体专题知识讲座第23页fromLongacreandStorbCell102:541,.抗体专题知识讲座第24页ProposedmechanismoftargetingofsomatichypermutationfromLongacreandStorbCell102:541,.抗体专题知识讲座第25页ComparisonofVDJrecombination,classswitchrecombinationandsomatichypermutationssDNAnicks?抗体专题知识讲座第26页ComparisonofVDJrecombination,classswitchrecombinationandsomatichypermutationssDNAnicks?抗体专题知识讲座第27页Activation-inducedcytidinedeaminase(AID)Discoveredasaninducedgeneinacelllinewithinducibleclass-switchrecombination(subtractivehybridization)TransfectionintoBcelllinesinducesclassswitchrecombinationAIDKOmicehavestrongdefectinclassswitchrecombinationANDinsomatichypermutationHyper-IgMsyndrometype2(autosomal)isduetomutationinAID;verysimilarphenotypetomice(noIgG,IgA,IgE;verymuchreducedsomaticmutation)抗体专题知识讲座第28页AID:Howdoesitwork?AIDishighlyrelatedtoABOBEC-1,acytidinedeaminasethateditsmRNAforApolipoproteinB(viaatargetingsubunit)indirectactionordirectactioninclassswitchandhypermutation? AIDcouldeditmRNAsforfactorsthatactinclassswitchandfactorsthatactinclassswitch OR itcouldactdirectlyinbothprocesses抗体专题知识讲座第29页AIDasamutatorofDNAAIDismutagenicinbacteriaandmutationsareincreasedbydeficiencyinUracil-DNAglycosylase(enzymethatremovesUfromDNAandtriggersDNArepair)ClassswitchisinhibitedandhypermutationperturbedinUNG-deficientmiceTheseresultsfavorthehypothesisthatAIDdirectlyac