浙大 高级免疫学技术 英文课件

免疫学相关技术（TechnologiesinImmunology）王迪浙江大学免疫学研究所[e-mail],immunologiststypicallyspentthebulkoftheirtimeatthelaboratorybench.Theirresearchinvolvedpeeringintoamicroscopeandprobablycharacterizingthedifferentcellsfromabloodsample.Andtheirunderstandingoftheimmuneresponsewaslimitedtowhattheycouldseeand,basedonthat,whattheycouldhypothesize.BeforeNowWhatisemergingisamorecomplexunderstandingofthesciencethatinvolvesmanyfieldsofscience.MolecularBiologyBiochemistryStructuralBiologyGenomicsProteomicsTransgenicandknockoutanimalmodelsTechnologybasedonImmunologyTechnologywhichboostimmunologyAntibodiesFACS(Analysisandsorting)ELISAELISPOTTetramercellculture(invitroculturesystem)GeneProfiling（parallelsequencing）ProteomicsTransgenicandknockoutanimalsTechnologybasedonImmunologyTechnologywhichboostimmunology1.AntibodiesRecogniseandbindtomolecules(antigens)onforeignparticles,markingthemfordestruction.Eachantigenmaygenerateseveralantibodiesfordifferentsites(epitopes)onantigen.ProteinssecretedbyB-lymphocytes(typeofwhitebloodcell),invertebrates.170suppliersinUSPolyclonalantibodyAntigenImmunizationBleedingMonoclonalantibodyFreezeAbpurification*AffinityPurification:ProteinA/G,Antigenpeptideconjugatedonsepharoseoragarosebeads.ProteinAisfromthecellwallofS.aureus，MW42kDaProteinGisfromthecellwallof-hemolyticstreptococci,Gstrain，MW30~35kDa.ProteinAorProteinGcanbindtotheFcdomainofAb.AbpurificationAntibodyPurificationAffinitychromatographyusesantigen-antibodybindingtopurifyantigensorantibodiesTopurifyaspecificantigenfromacomplexmixtureofmolecules,amonoclonalantibodyisattachedtoaninsolublematrix,suchaschromatographybeads,andthemixtureofmoleculesispassedoverthematrix.Thespecificantibodybindstheantigenofinterest;othermoleculesarewashedaway.SpecificantigenisthenelutedbyalteringthepH,whichcanusuallydisruptantibody-antigenbonds.Antibodiescanbepurifiedinthesamewayonbeadscoupledtoantigen(notshown).AffinitychromatographyAntibodyconjugation•Beads,sepharose–Targetproteinpurification•Fluorochrome–immunohistology,FACS•Enzyme–Westernblot,ELISA…•Toxin–cancerimmunotherapy•Radioactiveresidues•GoldImmunoassayWesternBlotfluorescenceFACSmicroscopeCD4CD8AntibodyconjugationSecondAntibodyAsecondaryantibodyisanantibodythatbindstoprimaryantibodiesorantibodyfragments.Theyaretypicallylabeledwithprobesthatmakethemusefulfordetection,purificationorcellsortingapplications.SecondAntibodyTheprimaryantibody(inpurple)bindstoanantigen(inred).Alabeledsecondaryantibody(ingreen),thenbindstotheprimaryantibody.Thelabelisthenusedtoindirectlydetecttheantigen.Specificsecondaryantibodiesareselectedaccordingtothesourceoftheprimaryantibody,theclassoftheprimaryantibody(e.g.,IgGorIgM),andthekindoflabelwhichispreferred.TumorCellSurfaceAntigenAntibodyconjugationtoxinAntibodyforresearch&AntibodyforclinicAgglutinationDirect:Human“ABObloodgroup”determinationIndirect:SerumrheumatoidfactorsdetectionItcanbeusedtodetectspecificantibodieswithknownantigensorconverselyspecificantigenswithknownantibodies.Indirectagglutination:Rheumatoidfactorsareagroupofauto-antibodiesproducedbymanyindividualswithrheumatoidarthritis.TheycanreactwithepitopesintheFcregionofIgG.Intheindirectagglutination,humanglobulinboundredbloodcellscancross-linkedbyrheumatoidfactortoformthelatticeworkimmunecomplexes.Goldnanoparticlelabeledmouseanti-HCGantibodyHGCBCTRAImmobilizedmouseanti-HCGantibodyImmobilizedanti-mouseIgGantibodyGoldimmunoassayAntibodiesELISAELISPOTFACS(Analysisandsorting)Tetramercellculture(cellsandinvitroculturesystem)GeneProfilingProteomicsTransgenicandknockoutanimalsTechnologybasedonImmunologyTechnologywhichboostimmunologyTypicalELISAProcedureTheEnzyme-linkedimmuno-sorbentspot(ELISPOT)assayisacommonmethodformonitoringimmuneresponsesinhumansandanimals.ItwasdevelopedbyCecilCzerkinskyin1983.TheELISPOTassayisbasedon,andwasdevelopedfromamodifiedversionoftheELISAimmunoassay.Simplyput,atappropriateconditionstheELISPOTassayallowsvisualizationofthesecretaryproductofindividualactivatedorrespondingcells.Eachspotthatdevelopsintheassayrepresentsasinglereactivecell.Thus,theELISPOTassayprovidesbothqualitative(typeofimmuneprotein)andquantitative(numberofrespondingcells)information.ELISPOT1.CoatplatewithCapturingAntibody(Y)2.Washcoatedplate3.AddAntigenandCellsYYYYYY2hr@37ºCoro/n@4ºCYYYYYYYYYYYBlock2hr@37ºCYYYYYYYYYYYAPCTcellsWash4.O/NstimulationforcytokineproductionYYYYYYYYYYYAPCTcellsYYYYYYYYYYYYYYY2hr@37ºC6.AddSubstrateYYYYYYYYYYYYYYYWash7.SpotsDevelopmentNoresponsePositiveresponseYY5.AddEnzyme-conjugatedDetectingAntibodiesYELISPOTcomparedtoELISAThemainadvantageofELISpotissensitivityofthemethod.ELISpotallowsfordetectionofasinglecellthatsecretsaproteinofinterest(cytokine,effectorprotein,receptor,surfacemarker,antibody)among1,000,000cellsintheculture.InELISA,detectionofproteinssecretedbyasinglecellisimpossible.Sometimes,cytokineproductionbyhundredsofstimulatedcellsremainsundetectedbecauseoftheinsufficientsensitivityofELISAand/ortheongoingconsumptionofthecytokinebyculturedcells.Inmanysituations,detectionofinterleuk