免疫学检测与免疫学技术.ppt

Chapter10 免疫学检测方法与免疫技术一 概述免疫学检验方法和免疫化学技术主要包括 抗原抗体的制备 纯化和鉴定 免疫扩散 免疫电泳 免疫凝集试验 补体结合试验 免疫细胞分离 纯化和鉴定 免疫功能检测 细胞因子检测 放射免疫检测 免疫酶标检测 荧光和发光免疫技术 免疫组化实验方法 原位杂交免疫组化 免疫PCR技术 免疫微球的应用 免疫电镜技术 细胞凋亡的检测方法 膜受体分析 胞内钙镁浓度的测定和细胞间通讯 流氏细胞仪技术及应用等 从上述内容可以看出 免疫技术是免疫学和物理 化学及电子信息和分子生物学理论和技术的结合产物 其应用涉及生命科学的各个领域 已成为现代医学和生物学研究工作不可缺少的有效工具 免疫技术的原理和特点 基于免疫应答的理论 即抗原抗体的特异性反应 基于免疫细胞的结构 应答特性和分子基础 具有特异性高度灵敏性 可重复性 广泛适用性 快速反应性 可观察性 可定性 定量 即可控性 组化定位特性等特点 概括起来可分为 沉淀反应 即可溶性抗原和抗体间的反应 凝集反应 即颗粒性抗原和抗体间的反应 免疫标记 即用酶 同位素 荧光素或电子致密物质标记 免疫印渍 即用标记抗体与待测蛋白质印迹结合 单抗及工程抗体技术 即分子生物技术 流氏细胞术 即荧光标记 流体喷射 激光和能谱检测 电脑分析 Ag AbreactionsTestsforAg Abreactions NatureofAg AbReactions LockandKeyConcept Non covalentBonds HydrogenbondsElectrostaticbondsVanderWaalforcesHydrophobicbonds Reversible MultipleBonds Affinity attractiveandrepulsiveforces Affinity StrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsite CalculationofAffinity Ag Ab Ag Ab ApplyingtheLawofMassAction Avidity TheoverallstrengthofbindingbetweenanAgwithmanydeterminantsandmultivalentAbs Specificity Theabilityofanindividualantibodycombiningsitetoreactwithonlyoneantigenicdeterminant Theabilityofapopulationofantibodymoleculestoreactwithonlyoneantigen CrossReactivity TheabilityofanindividualAbcombiningsitetoreactwithmorethanoneantigenicdeterminant TheabilityofapopulationofAbmoleculestoreactwithmorethanoneAg FactorsAffectingMeasurementofAg AbReactions Affinity Avidity Ag Abratio PhysicalformofAg TestsBasedonAg AbReactions AlltestsbasedonAg AbreactionswillhavetodependonlatticeformationortheywillhavetoutilizewaystodetectsmallimmunecomplexesAlltestsbasedonAg AbreactionscanbeusedtodetecteitherAgorAb AgglutinationTests LatticeFormation Agglutination Hemagglutination Definition teststhathaveastheirendpointtheagglutinationofaparticulateantigenAgglutinin hemagglutinin Agglutination Hemagglutination QuantitativeagglutinationtestTiterProzone Agglutination Hemagglutination DefinitionQualitativetestQuantitativetest ApplicationsBloodtypingBacterialinfectionsFourfoldriseintiter PracticalconsiderationsEasySemi quantitative PassiveAgglutination Hemagglutination Definition agglutinationtestdonewithasolubleantigencoatedontoaparticle ApplicationsMeasurementofantibodiestosolubleantigens Coombs Antiglobulin Tests IncompleteAbDirectCoombsTestDetectsantibodiesonerythrocytes Coombs Antiglobulin Tests IndirectCoombsTestDetectsanti erythrocyteantibodiesinserum Coombs Antiglobulin Tests ApplicationsDetectionofanti RhAbAutoimmunehemolyticanemia Agglutination HemagglutinationInhibition Definition testbasedontheinhibitionofagglutinationduetocompetitionwithasolubleAg Agglutination HemagglutinationInhibition ApplicationsMeasurementofsolubleAgPracticalconsiderationsSameasagglutinationtest Definition PrecipitationTests LatticeFormation RadialImmunodiffusion Mancini InterpretationDiameterofringisproportionaltotheconcentrationQuantitativeIglevels MethodAbingelAginawell Immunoelectrophoresis MethodAgsareseparatedbyelectrophoresis InterpretationPrecipitinarcrepresentindividualantigens Abisplacedintroughcutintheagar Immunoelectrophoresis MethodInterpretationQualitativeRelativeconcentration Countercurrentelectrophoresis MethodAgandAbmigratetowardeachotherbyelectrophoresisUsedonlywhenAgandAbhaveoppositecharges QualitativeRapid Radioimmuoassays RIA Enzyme LinkedImmunosorbentAssays ELISA Latticeformationnotrequired CompetitiveRIA ELISAforAg MethodDetermineamountofAbneededtobindtoaknownamountoflabeledAg UsepredeterminedamountsoflabeledAgandAbandaddasamplecontainingunlabeledAgasacompetitor CompetitiveRIA ELISAforAg Methodcont DetermineamountoflabeledAgboundtoAb NH4SO4 anti IgImmobilizetheAb QuantitativeMostsensitivetest ConcentrationdeterminedfromastandardcurveusingknownamountsofunlabeledAg SolidPhaseNon CompetitiveRIA ELISA AbdetectionImmobilizeAgIncubatewithsampleAddlabeledanti IgAmountoflabeledAbboundisproportionaltoamountofAbinthesample Quantitative SolidPhaseNon CompetitiveRIA ELISA AgdetectionImmobilizeAbIncubatewithsampleAddlabeledantibodyAmountoflabeledAbboundisproportionaltotheamountofAginthesample Quantitative TestsforCellAssociatedAntigens Latticeformationnotrequired Immunofluorescence DirectAbtotissueAgislabeledwithfluorochrome Immunofluorescence IndirectAbtotissueAgisunlabeledFluorochrome labeledanti IgisusedtodetectbindingofthefirstAb QualitativetoSemi Quantitative Immunofluorescence FlowCytometryCellsinsuspensionarelabeldwithfluorescenttagDirectorIndirectFluorescenceCellsanalyzedonaflowcytometer Immunofluorescence FlowCytometrycont Datadisplayed GreenFluorescenceIntensity NumberofCells Unstainedcells FITC labeledcells OneParamet