抗原抗体反应及应用

第一节抗体的制备•抗血清：指抗原人工免疫实验动物，获得含特异性抗体的血清。•多克隆抗体(PAb)：多个抗原决定簇免疫机体所产生的多种抗体的混合物。•单克隆抗体（monoclonalantibody，MAb）：•只针对某一特定的抗原决定簇，纯度高的抗体。第四章抗原抗体反应及应用Back一、抗血清的制备1、免疫动物•抗原•佐剂•免疫动物2、抗血清的纯化与保存•采血•纯化与保存3、抗血清的鉴定•效价•亲和力BACKAffinity=attractiveandrepulsiveforcesAbAgHighAffinityAbAgLowAffinityAffinity•StrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsiteCalculationofAffinityAg+AbAg-AbKeq=[Ag-Ab][Ag]x[Ab]ApplyingtheLawofMassAction:二、单抗的制备1、单抗的制备原理2、单抗的制备方法•抗原提纯和动物免疫•骨髓瘤细胞及饲养细胞的制备•细胞融合•筛选阳性株•单克隆抗体的制备和冻存•单克隆抗体的纯化GO骨髓瘤细胞脾脏淋巴细胞（HGPRT-TK-)（HGPRT+TK+）在HAT中不能生长体外培养过程中死亡↓←PEG杂交瘤细胞(HGPRT+、TK+)↓在HAT中大量长期繁殖↓克隆化↓产生单抗BACKBACKBACKBACK三、单抗的应用临床检测、抑制器官移植排斥、治疗自身免疫疾病、生物导弹等。第二节抗原抗体反应的原理一、抗原抗体反应的原理1.亲水胶体转化疏水胶体2.抗原抗体结合力•电荷引力（静电引力）•范登华引力•氢键结合力•疏水作用力二、抗原抗体反应的特性NatureofAg/AbReactionsLockandKeyConcept•antibodycombiningsitetoreactwithonlyoneantigenicdeterminant.•Theabilityofapopulationofantibodymoleculestoreactwithonlyoneantigen.SpecificityCrossReactivity•TheabilityofanindividualAbcombiningsitetoreactwithmorethanoneantigenicdeterminant.•TheabilityofapopulationofAbmoleculestoreactwithmorethanoneAgAnti-AAbAgAAnti-AAbAgBSharedepitopeAnti-AAbAgCSimilarepitopeCrossreactionsNon-covalentBonds–Hydrogenbonds–Electrostaticbonds–VanderWaalforces–HydrophobicbondsReversible•MultipleBondsSource:Li,Y.,Li,H.,Smith-Gill,S.J.,Mariuzza,R.A.,Biochemistry39,6296,2000三、影响抗原抗体反应的因素FactorsAffectingMeasurementofAg/AbReactions•Affinity•Ag:Abratio•PhysicalformofAgAbexcessAgexcessEquivalence–Latticeformation•PH•Temperament•Electrolyte第三节常见的免疫分析方法一、凝聚反应(Agglutination)二、沉淀反应1、溶液中的沉淀反应2、凝胶扩散沉淀3、免疫电泳血清免疫电泳火箭电泳对流免疫电泳三、补体参与的反应四、免疫标记的抗原抗体技术酶联免疫吸附（ELISA）Agglutination/Hemagglutination•Definition-teststhathaveastheirendpointtheagglutinationofaparticulateantigen+•Qualitativeagglutinationtest–AgorAbAgglutination/Hemagglutination•Quantitativeagglutinationtest–Titer–Prozone1/21/41/81/161/321/641/1281/2561/5121/1024Pos.Neg.Titer648512232128324Patient12345678Agglutination/Hemagglutination•Definition•Qualitativetest•Quantitativetest•Applications–Bloodtyping–Bacterialinfections–Fourfoldriseintiter•Practicalconsiderations–Easy–Semi-quantitative1/21/41/81/161/321/641/1281/2561/512BACKImmunoelectrophoresis•Method–Agsareseparatedbyelectrophoresis•Interpretation•PrecipitinarcrepresentindividualantigensAgAgAbAgAb–AbisplacedintroughcutintheagarBACK+-Countercurrentelectrophoresis•Method–AgandAbmigratetowardeachotherbyelectrophoresis–UsedonlywhenAgandAbhaveoppositecharges•Qualitative–RapidAgAb-+BACKBACKComplementFixation–AgmixedwithtestserumtobeassayedforAb–Standardamountofcomplementisadded–ErythrocytescoatedwithAbsisadded–AmountoferythrocytelysisisdeterminedAgPatient’sserumAgNoAgAg•MethodologyBACKBACK酶标记抗体技术免疫酶技术的基本程序：将酶分子与抗体或抗抗体分子共价结合，酶标记抗体可与存在于组织细胞或吸附于固相载体上的抗原或抗体发生特异性结合。滴加底物溶液后，底物可在酶作用下催化底物水解、氧化或还原等反应，产生有颜色的物质。酶降解底物的量与呈现的颜色成正比。用于标记的酶：1.辣根过氧化物酶(HorseradishPeroxidase,HRP);2.碱性磷酸酶(Alkalinephosphatase,AP)。直接法－抗原包被AgLabeledAbTissueSection间接法AgLabeledAnti-IgTissueSectionUnlabeledAb夹心法－直接夹心SolidPhaseAgImmobilizedAginPatient’ssampleLabeledAb直接夹心法直接夹心法直接法