免疫学检测与免疫学技术

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-Abreactions

TestsforAg-AbreactionsNatureofAg/AbReactionsLockandKeyConceptNon-covalentBondsHydrogenbondsElectrostaticbondsVanderWaalforcesHydrophobicbondsReversibleMultipleBonds

Source:Li,Y.,Li,H.,Smith-Gill,S.J.,Mariuzza,R.A.,Biochemistry39,6296,2000

:85/chime2/lyso-abfr.htmAffinity=

attractiveandrepulsiveforcesAbAgHighAffinityAbAgLowAffinityAffinityStrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsiteCalculationofAffinityAg+Ab

Ag-AbKeq=

[Ag-Ab][Ag]x[Ab]ApplyingtheLawofMassAction:AvidityTheoverallstrengthofbindingbetweenanAgwithmanydeterminantsandmultivalentAbsYKeq=104AffinityY106AvidityYYYYY1010AviditySpecificityTheabilityofanindividualantibodycombiningsitetoreactwithonlyoneantigenicdeterminant.Theabilityofapopulationofantibodymoleculestoreactwithonlyoneantigen.CrossReactivityTheabilityofanindividualAbcombiningsitetoreactwithmorethanoneantigenicdeterminant.TheabilityofapopulationofAbmoleculestoreactwithmorethanoneAgAnti-AAbAgAAnti-AAbAgBSharedepitopeAnti-AAbAgCSimilarepitopeCrossreactionsFactorsAffectingMeasurementofAg/AbReactions

Affinity

Avidity

Ag:Abratio

PhysicalformofAgAbexcessAgexcessEquivalence–LatticeformationTestsBasedonAg/AbReactionsAlltestsbasedonAg/AbreactionswillhavetodependonlatticeformationortheywillhavetoutilizewaystodetectsmallimmunecomplexesAlltestsbasedonAg/AbreactionscanbeusedtodetecteitherAgorAbAgglutinationTestsLatticeFormationAgglutination/HemagglutinationDefinition-teststhathaveastheirendpointtheagglutinationofaparticulateantigenAgglutinin/hemagglutininYYY+

QualitativeagglutinationtestAgorAbAgglutination/HemagglutinationQuantitativeagglutinationtestTiterProzone1/21/41/81/161/321/641/1281/2561/5121/1024Pos.Neg.Titer648512<232128324Patient12345678Agglutination/HemagglutinationDefinitionQualitativetestQuantitativetest

Applications

BloodtypingBacterialinfectionsFourfoldriseintiter

Practicalconsiderations

EasySemi-quantitative1/21/41/81/161/321/641/1281/2561/512PassiveAgglutination/HemagglutinationDefinition-agglutinationtestdonewithasolubleantigencoatedontoaparticleYYY+

ApplicationsMeasurementofantibodiestosolubleantigensCoombs(Antiglobulin)Tests

IncompleteAbDirectCoombsTest

Detectsantibodiesonerythrocytes+

YYYYYYYYYYYYYYYYYYYPatient’sRBCsCoombsReagent(Antiglobulin)Coombs(Antiglobulin)Tests

IndirectCoombsTestDetectsanti-erythrocyteantibodiesinserumYYYYYPatient’sSerumTargetRBCs+

Step1+

YYYYYYYYYYYYYYYYYYYCoombsReagent(Antiglobulin)Step2Coombs(Antiglobulin)Tests

ApplicationsDetectionofanti-RhAbAutoimmunehemolyticanemiaAgglutination/HemagglutinationInhibitionDefinition-testbasedontheinhibitionofagglutinationduetocompetitionwithasolubleAgYYY+

PriortoTestY+

YY+TestPatient’ssampleAgglutination/HemagglutinationInhibitionApplicationsMeasurementofsolubleAgPracticalconsiderationsSameasagglutinationtestDefinitionPrecipitationTestsLatticeFormationRadialImmunodiffusion(Mancini)InterpretationDiameterofringisproportionaltotheconcentrationQuantitativeIglevels

MethodAbingelAginawellAgConcentrationDiameter2AgAgAgAgAbingelImmunoelectrophoresisMethodAgsareseparatedbyelectrophoresis

InterpretationPrecipitinarcrepresentindividualantigensAg-+AgAbAgAbAbisplacedintroughcutintheagarImmunoelectrophoresisMethodInterpretationQualitativeRelativeconcentrationCountercurrentelectrophoresisMethodAgandAbmigratetowardeachotherbyelectrophoresisUsedonlywhenAgandAbhaveoppositecharges

QualitativeRapidAgAb-+Radioimmuoassays(RIA)

Enzyme-LinkedImmunosorbentAssays(ELISA)LatticeformationnotrequiredCompetitiveRIA/ELISAforAg

MethodDetermineamountofAbneededtobindtoaknownamountoflabeledAgYY+

PriortoTestLabeledAgYY+

Test+Patient’ssampleLabeledAg+UsepredeterminedamountsoflabeledAgandAbandaddasamplecontainingunlabeledAgasacompetitorCompetitiveRIA/ELISAforAg

Methodcont.DetermineamountoflabeledAgboundtoAb

NH4SO4

anti-IgImmobilizetheAb

Quantitative

Mostsensitivetest

YY+

Test+Patient’ssampleLabeledAg+ConcentrationdeterminedfromastandardcurveusingknownamountsofunlabeledAgSolidPhaseSolidPhaseSolidPhaseNon-CompetitiveRIA/ELISAAbdetectionImmobilizeAgIncubatewithsampleAddlabeledanti-IgAmountoflabeledAbboundisproportionaltoamountofAbinthesample

QuantitativeSolidPhaseYAgImmobilizedYAbinPatient’ssampleLabeledAnti-IgSolidPhaseNon-CompetitiveRIA/ELISAAgdetectionImmobilizeAbIncubatewithsampleAddlabeledantibodyAmountoflabeledAbboundisproportionaltotheamountofAginthesample

QuantitativeSolidPhaseYAgImmobilizedYAginPatient’ssampleLabeledAbTestsforCellAssociatedAntigensLatticeformationnotrequiredImmunofluorescence

DirectAbtotissueAgislabeledwithfluorochromeAgYFluorochromeLabeledAbTissueSectionImmunofluorescenceIndirectAbtotissueAgisunlabeledFluorochrome-labeledanti-IgisusedtodetectbindingofthefirstAb.AgYYFluorochromeLabeledAnti-IgTissueSectionUnlabeledAbQualitativetoSemi-QuantitativeImmunofluorescence

FlowCytometryCell