五年制《医学免疫学》12-immunological-techniques课件

ImmunologicaltechniquesImmunologicaltechniquesObjectivesToknowthemainlaboratorydetectionmethodusingantibody.Toknowhowtoisolatethespecificlymphocytepopulation.Beabletodescribehowtodetectthechangeofhumanimmunefunctionafterbacteriaorvirusinfection.ObjectivesToknowthemainContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantige五年制《医学免疫学》12-immunological-techniques课件ContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantige类型抗原抗体反应细胞免疫水平细胞分子基因免疫学技术类型抗原抗体反应细胞免疫水平细胞分子基因免疫学技术

1.Principles

andinfluencingfactorsofAg-AbreactionPrinciplesofAg-Abreactiona.Specificity

BindingbetweenAbandAghasveryhighspecificity.

Affinity:thestrengthofthebindingbetweenasinglebindingsiteofanAbandanAg

Avidity:theoverallstrengthofinteractionbetweenanAbandanAg.1.PrinciplesandinfluencingAffinity=attractiveandrepulsiveforcesAbAgHighAffinityAbAgLowAffinityAffinity（亲和力）StrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsiteAffinity=attractiveandrepuAvidity（亲合力）TheoverallstrengthofbindingbetweenanAgwithmanydeterminantsandmultivalentAbsYKeq=104AffinityY106AvidityYYYYY1010AvidityAvidity（亲合力）Theoverallstrengb.reversalcombinationnoncovalentforcehydrogenbondelectrostaticattractionVanderWalsforces

hydrophobicbonddegreeofdissociationAg-AbaffinityEnvironmentalfactorsb.reversalcombinationC.ConcentrationandratioofAgandAbWhentheantigensandantibodiesarepresentinanappropriateratio,theyforminsolubleimmunecomplexes(e.g.aggregationorprecipitation)largeenoughtobeseen.C.ConcentrationandratioofAsincreasingconcentrationsofAgareaddedtoaconstantamountofAb,theamountofICprecipitatedrisesandthenfalls.Theprecipitincurvegeneratedinthiswayhasthreezones.

Abexcesszone(prozone)equivalencezoneAgexcesszone(postzone)AsincreasingconcentrationImmunecomplexAntigenaddedAntibodyexcesszoneEquivalencezoneAntigenexcesszonePrecipitincurveImmunecomplexAntigenaddedAntFactorsAffectingMeasurementofAg/AbReactionsAffinityAvidityAg:AbratioPhysicalformofAgAbexcessAgexcessEquivalence–Latticeformation(Visible)FactorsAffectingMeasurementd.TwophasesSpecificcombinationVisiblephased.Twophases2)influencingfactorsofAg-AbreactionelectrolytesTemperature:37degreepH:pH6-82)influencingfactorsofAg-ContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantige

抗原：可溶性、颗粒性、细胞内膜结合、组织中抗体：体液(血清)中、培养液中、

单克隆抗体MethodsfordetectionofAgorAbMethodsfordetectionAgglutination(aggregation)Assays:ImmunodiffusionComplementFixationEIAenzymeimmunoassay(IHC/ELISA/ELISPOT)Immunofluorescence(IFAFACS)ICS（Intracellularcytokinestaining）CLIA（Chemiluminescenceimmumoassay）TraditionalImmunoassaysAntigen-AntibodyInteractionsModernImmunoassaysTraditionalImmunoassaysAntige凝集反应扩散试验补体结合反应凝集反应扩散试验补体结合反应2.MethodsfordetectionofAgorAbAgglutinationreaction凝集反应a.PrincipleWhentheparticleAgsinteractwiththeappropriateAb,theyclumptogetherandeventuallyformmassesthatbecomelargeenoughtobeseen.b.Typesdirectagglutinationreactionindirectagglutinationreaction2.MethodsfordetectionofAg五年制《医学免疫学》12-immunological-techniques课件Directcoomb’stestindirectcoomb’stestDirectcoomb’stestindirectco

B.Precipitationreaction沉淀反应a.PrincipleWhensolubleAgscomeincontactwithspecificAb,theyprecipitate.Precipitationcanbedemonstratedviaimmunodiffusioninasemisolidmedium(e.g.agar).b.Typesimmunonephelometry:theformationofICinsolutionismonitoredbyspectrometry.singleimmunodiffusiondoubleimmunodiffusionimmunoelectrophoresis

B.Precipitationreaction沉淀反应五年制《医学免疫学》12-immunological-techniques课件五年制《医学免疫学》12-immunological-techniques课件Immuno-labelingtechniques

免疫标记技术Principle

SpecificAbs(orAgs)labelledwithfluorescein,enzymes,colloidalgoldorradioisotopesareusedasprobesforthedetectionofAgs(orAbs).b.Types

Immuno-labelingtechniques

免疫标IHCWesternBlotfluorescenceFACSmicroscope

CD4CD8Immuno-labelingtechniqueshorseradishperoxidase

IHCWesternBloEIA(EnzymeImmunoassay)SubstrateColorDetectionImmuno-labelingtechniquesEIA(EnzymeImmunoassay)SubstAsecondaryantibodyisanantibodythatbindstoprimaryantibodiesorantibodyfragments.Theyaretypicallylabeledwithprobesthatmakethemusefulfordetection,purificationorcellsortingapplications.Specificsecondaryantibodiesareselectedaccordingtothesourceoftheprimaryantibody,theclassoftheprimaryantibody(e.g.,IgGorIgM),andthekindoflabelwhichispreferred.Oneconjugated2ndantibodycandetectallprimaryantibodieswithsameisotype,whichreducethelabortoconjugateallprimaryantibodiesSecondaryantibodyAsecondaryantibodyisanantSecondaryantibodySecondaryantibodyEnzymeimmunoassay(EIA)

免疫酶测定法EIAistouseenzyme-labeledAbsorAgstodetectAgandAbinteractions.Theenzymeconvertsacolorlesssubstrate(chromogen)toacoloredproduct.ELISA:AgorAbinsolutionEnzymeimmunohistochemistry:AgintissueEnzymeimmunoassay(EIA)

免疫酶测定Enzymelinkedimmunosorbentassay,ELISATheadvantagesofELISAincludespecificity,sensitivity,rapidity,inexpensiveness,andsafety.Enzyme:horseradishperoxidase,HRPSubstrates:diaminobenzidine(DAB)3,3’,5,5’-tetramethylbenzidine(TMB)EnzymelinkedimmunosorbentasELISAELISA五年制《医学免疫学》12-immunological-techniques课件todetectAbtodetectAg6.ELISAtodetectAbtodetectAg6.EELISAELISAELISABAS（Biotin-avidinsystem）-ELISA

生物素-亲和素放大系统一分子亲和素可结合四个分子生物素，敏感、快速，生物素标记抗体、亲和素标记酶。ELISABAS（Biotin-avidinsystem）Biotin-avidinsystem

-ELISABiotin-avidinsystem-ELISAImmunohistochemistrytechiniques免疫组化技术特异性标记的抗体在组织或细胞原位进行检测，对相应抗原进行定位、定性和定量免疫电镜酶免疫组化免疫金组化ImmunohistochemistrytechinIHCImmunohistochemistry免疫组织化学IHC灵敏度高，单细胞水平，可高通量筛选（Enzyme-linkedimmuno-sorbentspot，ELISPOT)灵敏度高，单细胞水平，可高通量筛选（Enzyme-linke五年制《医学免疫学》12-immunological-techniques课件TwocytokinescanbedetectedsimultaneouslyTwocytokinescanbedetectedImmunofluorescence免疫荧光Immunofluorescenceassayistouseafluorescentcompound(usuallyfluorescein)todetectthebindingofAgandAb.TheAbislabeledwiththefluorescentcompoundanditspresenceisrevealedusingafluorescencemicroscope.Direct,indirectimmunofluorescenceandindirectcomplementamplifiedimmunofluorescence(间接免疫荧光互补放大)Immunofluorescence免疫荧光ImmunoflCD4CD8Immunofluorescence

Immunofluorescence）CD4CD8ImmunofluorescenceImmu五年制《医学免疫学》12-immunological-techniques课件ImmunofluorescenceAnExampleImmunofluorescenceConfocalimagetodetectphosphorylatedAKT(green)incardiomyocytesinfectedwithadenovirusConfocalimagetodetectphosp(1)Useful

diagramforfluorochomesproperties/research/multicolor/spectrumguide/index.jspUsefultools(1)Usefuldiagramforfluoroc(2)Useful

websitetoolsBDFluorescenceSpectrumViewer:/research/multicolor/spectrum_viewer/index.jspUsefultools(2)UsefulwebsitetoolsBDFlGoldnanoparticlelabeledanti-HCG（mouseIgG）Ag（HCG，humanchorionicgonadotropin）

BGTRAmouseanti-HCG(immobilized)Anti-mouseIgG(immobilized)AbsorbentmaterialImmunologiccolloidalgoldsignature,ICSGoldnanoparticlelabeledanti

positivenegativepositivenIdentificationandPurificationofProteinsImmunoprecipitation.

Aproteinmixtureisincubatedwithspecificantibody.Anyantigen–antibodycomplexesthatformareprecipitatedfromsolutionbytheadditionofProteinA-coatedbeadsthatbindtotheantibodiesandcollectatthebottomofthetubeundertheforceofcentrifugation.Afterwashing,thedesiredantigenisreleasedfromtheantibody-boundbeadsusingalteredpHand/orhighsaltconcentrationIdentificationandPurificatioIdentificationandPurificationofProteinsAffinityChromatographyAgarosebeadsbearingimmobilizedspecificantibodyareplacedintoacolumnwithasemi-permeableplugatthebottom.Asolutioncontainingantigenispassedslowlythroughthecolumn,allowingthebindingofspecificantigentotheimmobilizedantibody.Unboundentitiespassthroughtheplugandanymoleculethatbindstothebeadsnon-specificallyisremovedbyextensivewashing.AsolutionwiththeappropriatepHandsaltconcentrationtodisruptAg–Abbindingisthenpassedthroughthecolumntoelute(washoff)theantigenofinterest.IdentificationandPurificatioWesternblottingWesternblottingWesternBlotWesternBlotWesternBlotWesternBlot五年制《医学免疫学》12-immunological-techniques课件ContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantigeIsolationofimmunecellsAIsolationofPBMC:Ficoll-Urografindensity-gradientseparation

B:Isolationoflymphocytesandsubsets.

a.immunoabsorbingassayb.immunomagneticseparationc.FACSd.peptide-MHCtetramertechniqueIsolationofimmunecellsIsolationoflymphocytesIsolationoflymphocytesMagneticcellsorting(MACS)Threebasicsteps1)Targetcellsarelabeledwithantibody-conjugatedmagneticparticles.2)Thelabeledcellsareplacedwithinamagneticfield.3)Thelabeledcellsareretainedinthemagneticfieldwhiletheunlabeledcellsarewashedaway.Magneticcellsorting(MACS)ThCellsubsetsPurificationCellsubsetsPurificationFACSseparation(流式细胞术分离)ThebasicprincipleofFACSisimmunofluorescenceandthereforeflowcytometerscanbeconsideredtobespecializedfluorescencemicroscopes.Themodernflowcytometerconsistsofalightsource,collectionoptics,electronicsandacomputertotranslatesignalstodataIsolationofdifferentcellpopulationsbyFACSreliesonthedifferentexpressionofsurfaceAgs.FACSseparation(流式细胞术分离)ThebaIsolationofdifferentcellpopulationsbyFACSlasersheathfluidsamplebeamsplitersredfluorescencegreenfluorescenceforwardlightscatter90olightscatterchargingcollardeflectionplatescollectiontubeswastevibratingflowcellIsolationofdifferentcellpoIdentificationofcellsubsetsbyFACSImmuneCelltypesandsubtypesdefinedbysurfacemarkers(CDs)BcellTcellCD4+TcellCD8+TcellsTregs(CD4+CD25+)ConventionalCD4Typeofcell

CDmarkers

stemcellsCD34+,CD31-allleukocytegroupsCD45+GranulocyteCD45+,CD15+MonocytesCD45+,CD14+TlymphocyteCD45+,CD3+ThelpercellCD45+,CD3+,CD4+CytotoxicTcellCD45+,CD3+,CD8+BlymphocyteCD45+,CD19+orCD45+,CD20+ThrombocytesCD45+,CD61+NaturalkillercellCD16+,CD56+,CD3-IdentificationofcellsubsetsFluidicsCellsinsuspensionflowinsingleOpticsscatterlightandemitfluorescencethatiscollected,filteredElectronicsconvertedtodigitalvaluesStored&analyzedonacomputer*Collection*DropchargedandcollectedBasicsofFlowCytometryFluidics*CollectionBasicsofFFlowcytometryorFluorescenceActivatedCellSorter(FACS)Quantitative,multi-parameteranalysisoflargenumbersofindividualcellsCellsurfacemarkersIntracellularproteinsCa++mobilization12colorsand15parametersSorting:70,000cell/secondFACSFlowcytometryorFluorescenceFluoresceinatedavidin荧光-亲和素Biotin生物素

AntigenicpeptideMHCIp-MHCtetramertechnique抗原肽-MHC分子四聚体技术分析CTLFluoresceinatedavidinMHCIp-MContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantigeTLymphocyteFunctionAssaysFunctionMeasurement--Proliferation--CTLkilling--DTH--HLAdetection--Apoptosis--CytokineTLymphocyteFunctionAssaysTcellproliferationMorphology3H-thymidinelabelingMTTFACS-CFSEstainingTcellproliferation五年制《医学免疫学》12-immunological-techniques课件TcellproliferationMorphologyTcellproliferationMorpholog

Lymphoblast(morphologicalfeatures):Lymphoblastsare12-20µmindiameterwitharoundtoovalnucleus.Theperipheryofboththenucleusandthecellmaybeirregular

inoutline.Thefine,highlydispersednuclearchromatinstainsalightreddish-purple,andoneortwopaleblueorcolorlesslargenucleoliarevisible.Thecytoplasmisusuallybasophilic,withmarginal(peripheral)intensityacommoncharacteristic.LymphoblastTcellproliferation3H-thymidinelabelingTcellproliferationTcellproliferation-MTTMitochondriaenzymecatalyzethereductionofMTT–TurnblueTcellproliferation-MTTMitocTcellproliferationFACS-CFSEstainingTcellproliferationCTLAssayCTLAssayCTLCytolytictestAssaysforCTLinpatientscanbeperformedasavariantofamixedcellcultureusingthetargetcellsthatlabelledby

radioisotopes.

51CrreleasingLDHcellstainingmethodTetramerstaining

CTLCytolytictestCTLAssaySupernatantCTLAssaySupernatantControlreleaseratio(》5%):averagevalue Controlcpm–Naturalrelease Maximalrelease–NaturalreleaseSpecificreleaseratio(》10%): Specificcpm–Naturalrelease Maximalrelease–NaturalreleaseNaturalreleaseratio(《15%)Controls:Naturalreleasewell–medium;Maximalreleasewell–DetergentLysedNoantigencontrol51CrReleaseAssayControlreleaseratio(》5%):a五年制《医学免疫学》12-immunological-techniques课件五年制《医学免疫学》12-immunological-techniques课件MHCtetramerscanbeusedtoquantitatenumbersofantigen-specificTcells(especiallyCD8+Tcells).BiotinylatedrecombinantclassImoleculesfoldedwiththepeptideofinterestandβ2Mandtetramerizedbyafluorescentlylabeledstreptavidin(Streptavidinbindstofourbiotinspermolecule.).ThistetramerreagentwillspecificallylabelTcellsthatexpressTcellreceptorsthatarespecificforagivenpeptide-MHCcomplex.AntigenspecificresponsescanbemeasuredasCD8+,tetramer+TcellsasafractionofallCD8+lymphocytes.TetramerMHCtetramerscanbeusedtoqTetramerscanbindtothreeTCRsatonce,allowingspecificbindinginspiteofthelow(10-6molar)affinityofthetypicalclassI-peptide-TCRinteraction.TetramerTetramerscanbindtothreeTCCD8responserelated(nowCD4too)andknownepitopeInfectiousDiseaseHIV-AIDS,EBV,CMV,HPV,HBV,HCV,Influenza,Measles,Malaria,TBandRSVBreast,Prostate,Melanoma,Colon,Lung,Cervical,OvarianandLeukemiaDiabetes,Lymedisease,Multiplesclerosis,Rheumatoidarthritis,AutoimmunevitiligoTransplantationAutoimmuneDiseaseTumorImmunologyUtilityofTetramerCD8responserelateDTHdetection:‘OT’testorPPDtest

结核杆菌素、念珠菌素、麻风菌素、链激酶-链道酶、腮腺炎病毒ImmunizationChallenge(Ear/Foogpad)Measurement(Calipers)DTHdetection:‘OT’testorPPApoptosisdetectionApoptosisdetectionFlowcytometryofapoptoticcelldeathPhospholipidredistributionFlowcytometryofapoptoticce五年制《医学免疫学》12-immunological-techniques课件IntheTUNELassay,fragmentedDNAislabeledbyterminaldeoxynucleotidyltransferase(TdT)torevealapoptoticcells.WhenApoptosisdetectionIntheTUNELassay,ApoptosisdBcellfunctionassayA.DetectionofIgB.Ab-formingcelldetection

溶血空斑试验五年制《医学免疫学》12-immunological-techniques课件Real-timePCR(mRNALevel)ELISA/ELISPOTIntracellularstaining(FACS)4.BiologicalFunctionCytokinesdetectionCytokinesdetectionIntracellularStainingIdentificationofdifferentThelpercellscharacterizedbydifferentcytokineproductionIntracellularStainingIdentifi灵敏度高，单细胞水平，可高通量筛选（Enzyme-linkedimmuno-sorbentspot，ELISPOT)灵敏度高，单细胞水平，可高通量筛选（Enzyme-linkeTwocytokinescanbedetectedsimultaneouslyTwocytokinescanbedetectedAssessmentofhumanimmunefunctionAssessmentofhumanimmunefunAssessmentofhumanimmunefunctionAssessmentofhumanimmunefunThankyou!Thankyou!ImmunologicaltechniquesImmunologicaltechniquesObjectivesToknowthemainlaboratorydetectionmethodusingantibody.Toknowhowtoisolatethespecificlymphocytepopulation.Beabletodescribehowtodetectthechangeofhumanimmunefunctionafterbacteriaorvirusinfection.ObjectivesToknowthemainContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantige五年制《医学免疫学》12-immunological-techniques课件ContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantige类型抗原抗体反应细胞免疫水平细胞分子基因免疫学技术类型抗原抗体反应细胞免疫水平细胞分子基因免疫学技术

1.Principles

andinfluencingfactorsofAg-AbreactionPrinciplesofAg-Abreactiona.Specificity

BindingbetweenAbandAghasveryhighspecificity.

Affinity:thestrengthofthebindingbetweenasinglebindingsiteofanAbandanAg

Avidity:theoverallstrengthofinteractionbetweenanAbandanAg.1.PrinciplesandinfluencingAffinity=attractiveandrepulsiveforcesAbAgHighAffinityAbAgLowAffinityAffinity（亲和力）StrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsiteAffinity=attractiveandrepuAvidity（亲合力）TheoverallstrengthofbindingbetweenanAgwithmanydeterminantsandmultivalentAbsYKeq=104AffinityY106AvidityYYYYY1010AvidityAvidity（亲合力）Theoverallstrengb.reversalcombinationnoncovalentforcehydrogenbondelectrostaticattractionVanderWalsforces

hydrophobicbonddegreeofdissociationAg-AbaffinityEnvironmentalfactorsb.reversalcombinationC.ConcentrationandratioofAgandAbWhentheantigensandantibodiesarepresentinanappropriateratio,theyforminsolubleimmunecomplexes(e.g.aggregationorprecipitation)largeenoughtobeseen.C.ConcentrationandratioofAsincreasingconcentrationsofAgareaddedtoaconstantamountofAb,theamountofICprecipitatedrisesandthenfalls.Theprecipitincurvegeneratedinthiswayhasthreezones.

Abexcesszone(prozone)equivalencezoneAgexcesszone(postzone)AsincreasingconcentrationImmunecomplexAntigenaddedAntibodyexcesszoneEquivalencezoneAntigenexcesszonePrecipitincurveImmunecomplexAntigenaddedAntFactorsAffectingMeasurementofAg/AbReactionsAffinityAvidityAg:AbratioPhysicalformofAgAbexcessAgexcessEquivalence–Latticeformation(Visible)FactorsAffectingMeasurementd.TwophasesSpecificcombinationVisiblephased.Twophases2)influencingfactorsofAg-AbreactionelectrolytesTemperature:37degreepH:pH6-82)influencingfactorsofAg-ContentTheprincipleofantigen-antibodyinteractionLaboratorymethodsdetectedAgandAbLymphocytesisolationMethodsforstudyinglymphocyteresponsesContentTheprincipleofantige

抗原：可溶性、颗粒性、细胞内膜结合、组织中抗体：体液(血清)中、培养液中、

单克隆抗体MethodsfordetectionofAgorAbMethodsfordetectionAgglutination(aggregation)Assays:ImmunodiffusionComplementFixationEIAenzymeimmunoassay(IHC/ELISA/ELISPOT)Immunofluorescence(IFAFACS)ICS（Intracellularcytokinestaining）CLIA（Chemiluminescenceimmumoassay）TraditionalImmunoassaysAntigen-AntibodyInteractionsModernImmunoassaysTraditionalImmunoassaysAntige凝集反应扩散试验补体结合反应凝集反应扩散试验补体结合反应2.MethodsfordetectionofAgorAbAgglutinationreaction凝集反应a.PrincipleWhentheparticleAgsinteractwiththeappropriateAb,theyclumptogetherandeventuallyformmassesthatbecomelargeenoughtobeseen.b.Typesdirectagglutinationreactionindirectagglutinationreaction2.MethodsfordetectionofAg五年制《医学免疫学》12-immunological-techniques课件Directcoomb’stestindirectcoomb’stestDirectcoomb’stestindirectco

B.Precipitationreaction沉淀反应a.PrincipleWhensolubleAgscomeincontactwithspecificAb,theyprecipitate.Precipitationcanbedemonstratedviaimmunodiffusioninasemisolidmedium(e.g.agar).b.Typesimmunonephelometry:theformationofICinsolutionismonitoredbyspectrometry.singleimmunodiffusiondoubleimmunodiffusionimmunoelectrophoresis

B.Precipitationreaction沉淀反应五年制《医学免疫学》12-immunological-techniques课件五年制《医学免疫学》12-immunological-techniques课件Immuno-labelingtechniques

免疫标记技术Principle

SpecificAbs(orAgs)labelledwithfluorescein,enzymes,colloidalgoldorradioisotopesareusedasprobesforthedetectionofAgs(orAbs).b.Types

Immuno-labelingtechniques

免疫标IHCWesternBlotfluorescenceFACSmicroscope

CD4CD8Immuno-labelingtechniqueshorseradishperoxidase

IHCWesternBloEIA(EnzymeImmunoassay)SubstrateColorDetectionImmuno-labelingtechniquesEIA(EnzymeImmunoassay)SubstAsecondaryantibodyisanantibodythatbindstoprimaryantibodiesorantibodyfragments.Theyaretypicallylabeledwithprobesthatmakethemusefulfordetection,purificationorcellsortingapplications.Specificsecondaryantibodiesareselectedaccordingtothesourceoftheprimaryantibody,theclassoftheprimaryantibody(e.g.,IgGorIgM),andthekindoflabelwhichispreferred.Oneconjugated2ndantibodycandetectallprimaryantibodieswithsameisotype,whichreducethelabortoconjugateallprimaryantibodiesSecondaryantibodyAsecondaryantibodyisanantSecondaryantibodySecondaryantibodyEnzymeimmunoassay(EIA)

免疫酶测定法EIAistouseenzyme-labeledAbsorAgstodetectAgandAbinteractions.Theenzymeconvertsacolorlesssubstrate(chromogen)toacoloredproduct.ELISA:AgorAbinsolutionEnzymeimmunohistochemistry:AgintissueEnzymeimmunoassay(EIA)

免疫酶测定Enzymelinkedimmunosorbentassay,ELISATheadvantagesofELISAincludespecificity,sensitivity,rapidity,inexpensiveness,andsafety.Enzyme:horseradishperoxidase,HRPSubstrates:diaminobenzidine(DAB)3,3’,5,5’-tetramethylbenzidine(TMB)EnzymelinkedimmunosorbentasELISAELISA五年制《医学免疫学》12-immunological-techniques课件todetectAbtodetectAg6.ELISAtodetectAbtodetectAg6.EELISAELISAELISABAS（Biotin-avidinsystem）-ELISA

生物素-亲和素放大系统一分子亲和素可结合四个分子生物素，敏感、快速，生物素标记抗体、亲和素标记酶。ELISABAS（Biotin-avidinsystem）Biotin-avidinsystem

-ELISABiotin-avidinsystem-ELISAImmunohistochemistrytechiniques免疫组化技术特异性标记的抗体在组织或细胞原位进行检测，对相应抗原进行定位、定性和定量免疫电镜酶免疫组化免疫金组化ImmunohistochemistrytechinIHCImmunohistochemistry免疫组织化学IHC灵敏度高，单细胞水平，可高通量筛选（Enzyme-linkedimmuno-sorbentspot，ELISPOT)灵敏度高，单细胞水平，可高通量筛选（Enzyme-linke五年制《医学免疫学》12-immunological-techniques课件TwocytokinescanbedetectedsimultaneouslyTwocytokinescanbedetectedImmunofluorescence免疫荧光Immunofluorescenceassayistouseafluorescentcompound(usuallyfluorescein)todetectthebindingofAgandAb.TheAbislabeledwiththefluorescentcompoundanditspresenceisrevealedusingafluorescencemicroscope.Direct,indirectimmunofluorescenceandindirectcomplementamplifiedimmunofluorescence(间接免疫荧光互补放大)Immunofluorescence免疫荧光ImmunoflCD4CD8Immunofluorescence

Immunofluorescence）CD4CD8ImmunofluorescenceImmu五年制《医学免疫学》12-immunological-techniques课件ImmunofluorescenceAnExampleImmunofluorescenceConfocalimagetodetectphosphorylatedAKT(green)incardiomyocytesinfectedwithadenovirusConfocalimagetodetectphosp(1)Useful

diagramforfluorochomesproperties/research/multicolor/spectrumguide/index.jspUsefultools(1)Usefuldiagramforfluoroc(2)Useful

websitetoolsBDFluorescenceSpectrumViewer:/research/multicolor/spectrum_viewer/index.jspUsefultools(2)UsefulwebsitetoolsBDFlGoldnanoparticlelabeledanti-HCG（mouseIgG）Ag（HCG，humanchorionicgonadotropin）

BGTRAmouseanti-HCG(immobilized)Anti-mouseIgG(immobilized)AbsorbentmaterialImmunologiccolloidalgoldsignature,ICSGoldnanoparticlelabeledanti

positivenegativepositivenIdentificationandPurificationofProteinsImmunoprecipitation.

Aproteinmixtureisincubatedwithspecificantibody.Anyantigen–antibodycomplexesthatformareprecipitatedfromsolutionbytheadditionofProteinA-coatedbeadsthatbindtotheantibodiesandcollectatthebottomofthetubeundertheforceofcentrifugation.Afterwashing,thedesiredantigenisreleasedfromtheantibody-boundbeadsusingalteredpHand/orhighsaltconcentrationIdentificationandPurificatioIdentificationandPurificationofProteinsAffinityChromatographyAgarosebeadsbearingimmobilizedspecificantibodyareplacedintoacolumnwithasemi-permeableplugatthebottom.Asolutioncontainingantigenispassedslowlythroughthecolumn,allowingthebindingofspecificantigentotheimmobilizedantibody.Unboundentitiespassthroughtheplugandanymoleculethatbindstothebeadsnon-specificallyisremovedbyextensivewashing.AsolutionwiththeappropriatepHandsaltconcentrationtodisruptAg–Abbindingisthenpassedthroughthecolumntoelute(washoff)theantigenofinterest.IdentificationandPurificatioWesternblottingWesternblottingWesternBlotWesternBlotWesternBlotWesternBlot五年制《医学免疫学》12-immunological-techniques课件Co