抗体专题知识讲座

AntibodiesSecretedbyBlymphocytesGreatdiversityandspecificity:>109differentantibodies;candistinguishbetweenverysimilarmoleculesTagparticlesforclearance/destructionProtectagainstre-infection(vaccines)AntibodyStructureIgdomain:110aminoacids;globulardomainusedinmanyproteins;Variabledomains,Constantdomains,HingeFab:fragmentantigenbindingFc:fragmentcrystallizable(effectorfunctions)©NewSciencePressLtd.2023TheImmunoglobulinSuperfamily

(afewexamples)VariabilityinantibodiesisclusteredintheloopsintheVariabledomainsoftheheavyandlightchains(green);theseregionsareresponsibleforbindingtoantigenAntibodyClasses:Structure©NewSciencePressLtd.2023AffinityandAvidityAffinity:thestrengthofbindingbetweenasinglebindingsiteandasingleligand

[A][B][AB]Avidity:thestrengthofbindingbetweenamoleculeandacomplexligand,e.g.iftherearemultiplebindingsitesthentheaviditymaybeincreasedbyincreasingthenumberofbindingsitesorbyincreasingtheaffinityofthosebindingsitesKD=AffinityandAvidityIIIgMisproducedearlyinanimmuneresponsewhentheaffinityforantigenoftenislow;asanimmuneresponsecontinues,antibodyaffinityisimproved,thisiscombinedby“classswitching”totheuseofsmallermolecules(IgG,IgEandIgA).Theincreasedaffinitycompensatesforthedecreaseinnumberofbindingsitesinmaintainingtheoverallavidityforantigen©NewSciencePressLtd.2023MonoclonalAntibodiesSingleantibody(allsameHandLchains)MadebyfusionofBcellstoatransformedcelllineoftheplasmacelltypeandselectionfor“hybridomas”thatproduceantibodywiththedesiredpropertiesStandardized,unlimitedreagentfordiagnosisortherapy(humanantibodiesor“humanized”antibodiescanbemade)Polyclonalvs.Monoclonal

AntibodiesImmunizeIndividualSerumPolyclonalantibodies7+daysPurifyantibodiesBlymphocytes3daysImmortalize“hybridoma”cDNAcloningMonoclonalAntibodiesGenerationofMonoclonalAntibodies©NewSciencePressLtd.2023MonoclonalantibodiesusedinmedicineStandardized,unlimitedreagentsfordiagnosisortherapy(humanantibodiesor“humanized”antibodiescanbemade)©NewSciencePressLtd.2023GenerationofantibodydiversityHowdowemake>109differentantibodies?GenesforantibodiesarepresentinpiecesthatcanbecombinedinmanydifferentcombinationsindifferentlymphocytesVregionofIgLandHchainsareconstructedfrom2and3differentpieceseachhavingmultiplecopiesV(D)J

recombination©NewSciencePressLtd.2023GenerationofAntibodyDiversityklightchains:40Vkx5Jk=200llightchains:30Vlx4Jl=120Hchains:40VHx27DHx6JH=6,480320Lchainsx6,480Hchains=2.1x106

Junctionaldiversity(additionordeletionofnucleotidesatrecombinationsites,especiallyofHchain),estimatedtoadd3x107foldtooveralldiversityDiscoveryofRag1,2genes“RecombinationActivatingGene”MechanismofV(D)JrecombinationCACCGTGRecombinationsignalsRag-1/Rag-2/ArtemisNon-homologousendjoiningproteins(defects:SevereCombinedImmunodeficiency(SCID))©NewSciencePressLtd.2023CreationofJunctionalDiversity

byP-regionsandTdTDefectsinLymphocytedevelopmentleadingtoseverecombinedimmunodeficiency(SCID)Note:SCIDcanalsoresultfromdefectsthatinterferewithlymphocyteactivation(adenosinedeaminasedeficiency,purinenucleotidephosphorylasedeficiency,MHCdefects,etc.)©NewSciencePressLtd.2023LymphoidmalignanciesresultingfromerrorsinV(D)Jrecombination©NewSciencePressLtd.2023VDJRecombinationreactionscontributestotranslocationleadingtoover-expressionofacellulargrowthorsurvivalpromotinggene44CHCHIgHeavychainclass(isotype)switchingAffinitymaturationandantibodyresponsesfromLongacreandStorbCell102:541,2023.ProposedmechanismoftargetingofsomatichypermutationfromLongacreandStorbCell102:541,2023.ComparisonofVDJrecombination,classswitchrecombinationandsomatichypermutationssDNAnicks?ComparisonofVDJrecombination,classswitchrecombinationandsomatichypermutationssDNAnicks?Activation-inducedcytidinedeaminase(AID)Discoveredasaninducedgeneinacelllinewithinducibleclass-switchrecombination(subtractivehybridization)TransfectionintoBcelllinesinducesclassswitchrecombinationAIDKOmicehavestrongdefectinclassswitchrecombinationANDinsomatichypermutationHyper-IgMsyndrometype2(autosomal)isduetomutationinAID;verysimilarphenotypetomice(noIgG,IgA,IgE;verymuchreducedsomaticmutation)AID:Howdoesitwork?AIDishighlyrelatedtoABOBEC-1,acytidinedeaminasethateditsmRNAforApolipoproteinB(viaatargetingsubunit)indirectactionordirectactioninclassswitchandhypermutation? AIDcouldeditmRNAsforfactorsthatactinclassswitchandfactorsthatactinclassswitch OR itcouldactdirectlyinbothprocessesAIDasamutatorofDNAAIDismutagenicinbacteriaandmutationsareincreasedbydeficiencyinUracil-DNAglycosylase(enzymethatremovesUfromDNAandtriggersDNArepair)ClassswitchisinhibitedandhypermutationperturbedinUNG-deficientmiceTheseresultsfavorthehypothesisthatAIDdirectlyactsonCresiduesin