抗原抗體反應(yīng)及應(yīng)用課件

第一節(jié)抗體的制備抗血清：指抗原人工免疫實(shí)驗(yàn)動(dòng)物，獲得含特異性抗體的血清。多克隆抗體(PAb)：多個(gè)抗原決定簇免疫機(jī)體所產(chǎn)生的多種抗體的混合物。單克隆抗體（monoclonalantibody，MAb）：只針對(duì)某一特定的抗原決定簇，純度高的抗體。

第四章抗原抗體反應(yīng)及應(yīng)用Back

BACKAffinity=attractiveandrepulsiveforcesAbAgHighAffinityAbAgLowAffinityAffinityStrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsiteCalculationofAffinityAg+AbAg-AbKeq=

[Ag-Ab][Ag]x[Ab]ApplyingtheLawofMassAction:

骨髓瘤細(xì)胞脾臟淋巴細(xì)胞（HGPRT-TK-)（HGPRT+TK+）在HAT中不能生長(zhǎng)體外培養(yǎng)過(guò)程中死亡↓←PEG雜交瘤細(xì)胞(HGPRT+、TK+)↓在HAT中大量長(zhǎng)期繁殖↓克隆化↓產(chǎn)生單抗BACKBACKBACK

三、單抗的應(yīng)用

臨床檢測(cè)、抑制器官移植排斥、治療自身免疫疾病、生物導(dǎo)彈等。

第二節(jié)抗原抗體反應(yīng)的原理一、抗原抗體反應(yīng)的原理1.親水膠體轉(zhuǎn)化疏水膠體2.抗原抗體結(jié)合力電荷引力（靜電引力）范登華引力氫鍵結(jié)合力疏水作用力

二、抗原抗體反應(yīng)的特性

NatureofAg/AbReactions

LockandKeyConcept

antibodycombiningsitetoreactwithonlyoneantigenicdeterminant.Theabilityofapopulationofantibodymoleculestoreactwithonlyoneantigen.SpecificityNon-covalentBonds

HydrogenbondsElectrostaticbondsVander

WaalforcesHydrophobicbondsReversibleMultipleBonds

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

三、影響抗原抗體反應(yīng)的因素FactorsAffectingMeasurementofAg/AbReactionsAffinity

Ag:AbratioPhysicalformofAgAbexcessAgexcessEquivalence–LatticeformationPHTemperamentElectrolyte

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

BloodtypingBacterialinfectionsFourfoldriseintiterPracticalconsiderations

EasySemi-quantitative1/21/41/81/161/321/641/1281/2561/512BACKImmunoelectrophoresisMethodAgsareseparatedbyelectrophoresis

InterpretationPrecipitinarcrepresentindividualantigensAgAgAbAgAbAbisplacedintroughcutintheagarBACK+-CountercurrentelectrophoresisMethodAgandAbmigratetowardeachotherbyelectrophoresisUsedonlywhenAgandAbhaveoppositecharges

QualitativeRapidAgAb-+BACKBACKComplementFixationAgmixedwithtestserumtobeassayedforAbStandardamountofcomplementisaddedErythrocytescoatedwithAbsisaddedAmountoferythrocytelysisisdeterminedAgYYPatient’sserum

YYYYYYYYAgNoAgAgMethodologyBACKBACK

酶標(biāo)記抗體技術(shù)

免疫酶技術(shù)的基本程序：將酶分子與抗體或抗抗體分子共價(jià)結(jié)合，酶標(biāo)記抗體可與存在于組織細(xì)胞或吸附于固相載體上的抗原或抗體發(fā)生特異性結(jié)合。滴加底物溶液后，底物可在酶作用下催化底物水解、氧化或還原等反應(yīng)，產(chǎn)生有顏色的物質(zhì)。酶降解底物的量與呈現(xiàn)的顏色成正比。用于標(biāo)記的酶：1.辣根過(guò)氧化物酶(HorseradishPeroxidase,HRP);2.堿性磷酸酶(Alkalinephosphatase,AP)。直接法－抗原包被AgYLabeledAbTissueSection間接法Ag