免疫學(xué)檢測與免疫學(xué)技術(shù)

Chapter10.免疫學(xué)檢測方法與免疫技術(shù)一、概述

免疫學(xué)檢驗方法和免疫化學(xué)技術(shù)主要包括：抗原抗體的制備、純化和鑒定，免疫擴散、免疫電泳、免疫凝集試驗、補體結(jié)合試驗，免疫細胞分離、純化和鑒定，免疫功能檢測，細胞因子檢測，放射免疫檢測，免疫酶標(biāo)檢測，熒光和發(fā)光免疫技術(shù)，免疫組化實驗方法，原位雜交免疫組化，免疫PCR技術(shù)，免疫微球的應(yīng)用，免疫電鏡技術(shù)，細胞凋亡的檢測方法，膜受體分析，胞內(nèi)鈣鎂濃度的測定和細胞間通訊，流氏細胞儀技術(shù)及應(yīng)用等。從上述內(nèi)容可以看出，免疫技術(shù)是免疫學(xué)和物理、化學(xué)及電子信息和分子生物學(xué)理論和技術(shù)的結(jié)合產(chǎn)物。其應(yīng)用涉及生命科學(xué)的各個領(lǐng)域，已成為現(xiàn)代醫(yī)學(xué)和生物學(xué)研究工作不可缺少的有效工具。免疫技術(shù)的原理和特點:基于免疫應(yīng)答的理論，即抗原抗體的特異性反應(yīng)?；诿庖呒毎慕Y(jié)構(gòu)、應(yīng)答特性和分子基礎(chǔ)。具有特異性高度靈敏性；可重復(fù)性；廣泛適用性；快速反應(yīng)性；可觀察性；可定性、定量，即可控性；組化定位特性等特點。概括起來可分為:沉淀反應(yīng)，即可溶性抗原和抗體間的反應(yīng)。凝集反應(yīng)，即顆粒性抗原和抗體間的反應(yīng)。免疫標(biāo)記，即用酶、同位素、熒光素或電子致密物質(zhì)標(biāo)記。免疫印漬，即用標(biāo)記抗體與待測蛋白質(zhì)印跡結(jié)合。單抗及工程抗體技術(shù)，即分子生物技術(shù)。流氏細胞術(shù)，即熒光標(biāo)記，流體噴射，激光和能譜檢測，電腦分析。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