免疫学实验技术

Chapter25免疫学技术（TechnologiesinImmunology）AntigenandAntibody

IgsecretedbyB-lymphocytes

Eachantigenmaygenerateseveralantibodiesfordifferentsites(epitopes)onantigenAg-AbinteractionisspecificbindingAgglutinationReactionsGranularantigens(particulate)suchascellsorbacteria,orsolubleantigensthatconjugatewithparticles(RBC,latexparticles)canreactwithdi-ormultivalentantibodiesresultedinvisibleclumpingofthecomplexesformed.Theclumpingreactioniscalledagglutination,andantibodiesthatproducesuchreactionsarecalledagglutinins.AgglutinationDirectIndirect:RFIndirectinhibition:influenzavirusDirectCoombs:autoimmunehemolyticanemiaIndirectCoombs:bloodtransfusion

Hemagglutination:ABObloodtypeBacterialagglutination:SalmonellatyphiHemagglutinationusingantibodiesagainstSRBCABOBloodTypesTest++-BacterialagglutinationSalmonellatyphiQuantitativetestagglutinintiterofanantiserumcanbeusedtodiagnoseabacterialinfectionWidal

reactionIndirectagglutinationSolubleantigensCarrier:bloodcells,latexparticle

Fast,specificity,highsensitivityRheumatoidfactorsHemagglutinationassaynodetectablevirusSampleDcauseshemagglutinationuptothe1:512dilutionInfluenzavirusparticleshaveanenvelopeproteincalledthehemagglutinin,orHA,whichbindstosialicacidreceptorsoncells.Theviruswillalsobindtoerythrocytes,),causingtheformationofalattice.determinethelevelofantibodiestoinfluenzaviruspresentinserumsamples

afixedamountofvirus+

testedserum+redbloodcells

hemagglutinationisinhibitedwhenantibodiesarepresent

hemagglutination(-):Abagainstinfluenzavirus(+)

HItiteroftheserum=ThehighestdilutionofserumHIassayisapowerfulepidemiologicaltool

Hemagglutinationinhibition(HI)assayDirectCoombstest1)detectAbsorcomplementproteinsthatareboundtothesurfaceofRBCsinvivo.2)ApositiveCoombstestindicatesanimmunemechanismisattackingthepatient'sownRBCs3)autoimmunehemolyticanemiaindirectCoombstest1)isusedtodetectin-vitroantibody-antigenreactions.

2)bloodtransfusionAgglutinationDirectIndirect:RFIndirectinhibition:influenzavirusDirectCoombs:autoimmunehemolyticanemiaindirectCoombs:bloodtransfusion

Hemagglutination:ABObloodtypeBacterialagglutination:SalmonellatyphiImmunoprecipitationReactionsPolyvalentsolubleantigenbindmultipledifferentantibodiesresultingcross-linkedcomplexbecomessolargethatitfallsoutofsolutionasaprecipitate.Immunoprecipitationcanbeperformedinsolution,alsoingelmatrices(Agar).precipitation:AbandAgCon.areequivalentnoprecipitation:monovalentbindingAg,orAbexcessSingleradialimmunodiffusionPrecipitinline:visiblelineofprecipitationAgandAbdiffusetowardoneanotherinagelmatrix

Immunodiffusioninagargelcanbeusedtoassayforthepresenceofantibodiesanddeterminecross-reactivitypatternsbetweencomplexantigensandantibodysamples.DoubleimmunodiffusionCounterimmunoelectrophoresis,CIEPRocketimmunoelectrophoresis,RIEP血清蛋白电泳图谱蛋白电泳各组分含量表达（1）百分比（％）表示：白蛋白57-68α1球蛋白1.0-5.7α2球蛋白4.9-11.2β球蛋白7-13γ球蛋白9.8-18.2（2）绝对浓度（g/L）表示：绝对浓度＝百分浓度×总蛋白浓度immunofixationelectrophoresis免疫固定电泳检测速度较快，整个过程约1.5至2h敏感性高，能检测到0.5～1.5g/L的单克隆抗体分辨率高，很短的电泳距离分离出单克隆蛋白组分能确定蛋白质的单克隆属性，从而诊断浆细胞病等。在1张琼脂糖凝胶上对1个样品重复做6份电泳，1份为普通电泳，其他5份经过普通电泳分离蛋白后分别加入羊抗人IgG、IgA、IgM、κ链和λ链单克隆抗血清，被分离出的免疫球蛋白或轻链与其相应的抗体发生反应而沉淀，未反应的蛋白被清洗掉，抗原抗体反应物经染色后形成可见条带，比较普通电泳条带与免疫固定后的沉淀带，可对结果做出定性解释WesternBlottingsamplesweretreatedbySDSSDS-polyacrylamidegelelectrotransferPVDFmembraneenzyme-linkedantibodieschemiluminescent+enhancingagent(greatersensitivity)generatelightbyexposureoftheblottoapieceofphotographicfilmEdwinMellorSouthernSouthernblotDNA(1975)AgarNorthernblotRNA(1977)AgarGeorgeStarkWesternBlotProtein(1979-81)SDS聚丙烯酰胺凝胶电泳，被检测物是蛋白质探针是抗体显色用标记的二抗经过PAGE（聚丙烯酰胺凝胶电泳）分离的蛋白质样品转移到固相载体（PVDF膜）上固相载体以非共价键形式吸附蛋白质，且能保持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋白质或多肽作为抗原，与对应的抗体起免疫反应再与酶或同位素标记的第二抗体起反应经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。免疫比浊测定免疫比浊测定方式：监测散射光或透射光的变化DnDt光源样品杯Dn=散射检测器Dt=透射检测器WesternBlotfluorescenceFACSmicroscope

CD4CD8AntibodyconjugationTheprimaryantibody(inpurple)bindstoanantigen(inred).Alabeledsecondaryantibody(ingreen),thenbindstotheprimaryantibody.Thelabelisthenusedtoindirectlydetecttheantigen.Second

Antibody

enzyme-basedimmunoassay

radio-basedimmunoassay

gold-basedimmunoassay

fluorescence-basedimmunoassay

ImmunolabellingtechniqueEnzyme-basedimmunoassayEnzymeSubstrateAbsorbslightAlkalinephosphatase(ALP)p-nitrophenylphosphate(pNPP)磷酸对硝基苯460nmyellowHorseradishperoxidase(HRP)Orthophenylenediamine(OPD)邻苯二胺492nmorange3,3,5,5-tetramethylbenzidine(TMB)四甲基联苯胺450nmblueLuminol鲁米诺H2O2/EnhanceSystem

enzyme-basedimmunoassay

ImmunohistochemistryELISAELISPOTWesternBlotting

ImmunolabellingtechniqueImmunohistochemistryUseenzyme-conjugatedantibodiestocreateimagesoffixedtissuesenzymes+differentsubstratedifferentcoloredprecipitatevisibleinlightmicroscope

reagents:enzyme-conjugatedAb(2ndAb)antigen-specificAb(1stAb)

Samples:tissuefixed--paraffinimbeddingorfrozensectioning(cutting)Immunohistochemicalstainingoflymphnode.Humanlymphnodematerialwasembeddedinparaffin,fixed,andstainedwithantibodytoCD4(redstainedcells).Thelymphnodewasthencounter-stainedwithhematoxylin(苏木精复染)(blue).TypicalELISAProcedureApplicationQualitativeorquantitativemeasurementtodetectthepresenceofantibodyorantigenIndirectELISA——forAbSandwichELISA——forAgCompetitiveELISA——forAgAgorAbconcentrationisdirectlyproportionaltothecolorproducedinindirectorSandwichELISA.CompetitveELISAisaninhibition-typeassay,theconcentrationofantigenisinverselyproportionaltothecolorproduced.ELISAPrincipleELISAisbasedonhighspecificityofAg-Abreactions.adsorbedtothesurfaceofasolid-phasecarriersuchaspolystyrene.Enzyme-linkedAbamplifiesAg-Abreaction.Substrate(absorbancemaybereadatwavelengthsfrom450nmto650nmdependinguponavailablefilters)QuantitativedeterminationofAgorAbconcentrationwithamicroplatereader(spectrophotometer)toreadOD(opticaldensityvalue)dependoncoloredproduct.ng、pg/mlBiotin-streptavidinbondinginteractionsBiotinisawater-solubleBcomplexvitaminBiotinbindstobacterialproteinstreptavidinwithhighaffinityinteractionisstableunderawidevarietyofconditionsbiotin-conjugatdantibodiesstreptavidin-enzymeconjugate

Enzyme-linkedimmuno-sorbentspot(ELISPOT)ItwasdevelopedbyCecilCzerkinskyin1983amodificationofELISA

visualizationofthesecretaryproductofindividualcellsEachspotrepresentsasinglereactivecell

qualitative(typeofimmuneprotein)

quantitative(numberofrespondingcells)ELISPOT

1.CoatplatewithCapturingAntibody(Y)2.Washcoatedplate3.AddAntigenandCellsYYYYYYYYYYYY2hr@37ºCoro/n@4ºCYYYYYYYYYYYBlock2hr@37ºCYYYYYYYYYYYAPCTcellsWash4.O/NstimulationforcytokineproductionYYYYYYYYYYYAPCTcellsYYYYYYYYYYYYYYY2hr@37ºC6.AddSubstrateYYYYYYYYYYYYYYYWash7.SpotsDevelopmentNoresponsePositiveresponseYY5.AddEnzyme-conjugatedDetectingAntibodiesSpotsrepresentthesiteofacellthatsecretedCKThemainadvantage:sensitivityELISPOT>ELISAELISpotallowsfordetectionofasinglecell

thatsecretsaproteinofinterestTwocytokinescanbedetectedsimultaneouslyThespotscanbecountedRadioimmunoassay,RIA

RosalindSussmanYalow.CreatoroftheradioimmunoassaytechniqueandNobelLaureate,1977.HormonesandproteinsinbodyfluidsconcentrationsaretoolowtobedetectedRIAmadeitpossibletomeasuresubstancesradioactivelabel125IAcompetitive,solid-phaseradioimmunoassay(RIA)tomeasurecytokineconcentrationsinserum.HCGCollodalgold:instriperedlinesappear

HCG:Stripe:3kindsofAbAb1:+againstHCG(couldmoveinurine)Ab2:againstHCGinstripeAb3:againstAb1-FcfragementGoldimmunoassayimmunofluorescence-basedassayImmunofluorescencemicroscopyFlowCytometryFlowcytometryorFluorescenceActivatedCellSorter(FACS)Quantitative,multi-parameteranalysisoflargenumbersofindividualcellsCellsurfacemarkersIntracellularproteinsCa++mobilization12colorsand15parametersSorting:70,000cell/secondFACSBasicsofFlowCytometryFluidicsCellsinsuspensionflowinsingleOpticsscatterlightandemitfluorescencethatiscollected,filteredElectronicsconvertedtodigitalvaluesStored&analyzedonacomputer*Collection*DropchargedandcollectedCell

NumberUnderstandaFACS

DataHistogramCell

NumberCell

NumberCD3

FITCCD19

PEPopulation

identified

2，4…….2n

CellstructureSize细胞粒度细胞表面面积核浆比例DNA含量与细胞周期RNA含量蛋白质含量染色体分析Cellfunction细胞表面/胞浆/核的特异性抗原细胞活性细胞内细胞因子酶活性激素结合位点细胞受体细胞凋亡TraditionalAnalysis--PercentpositivePositivecellNegativeCellCD4PECD4PEAbsoluteCounts--CellspermLPositiveCellAbsoluteCountBeadsNegativeCellCD4PECD4PEIntracellular

StainingIdentificationofdifferentThelpercellscharacterizedbydifferentcytokineproductionDetectionofmulti-targetcells100101102103104100101102103104PBMC050100150200250FSC-H050100150200250SSC-HCD20PerCP-Cy5.5CD3PEZap70(Y319)/Syk(Y352)Alexa647

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