抗原抗体反应及应用.ppt
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（monoclonal antibody，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=attractive and repulsive forcesAbAgHigh AffinityAbAgLow AffinityAffinityStrength of the reaction between a single antigenic determinant and a single Ab combining siteCalculation of AffinityAg+Ab Ag-AbKeq=Ag-AbAg x AbApplying the Law of Mass Action: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。第二节第二节 抗原抗体反应的原理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二、抗原抗体反应的特性 Nature of Ag/Ab Reactions Lock and Key Concept antibody combining site to react with only one antigenic determinant.The ability of a population of antibody molecules to react with only one antigen.SpecificityCross ReactivityThe ability of an individual Ab combining site to react with more than one antigenic determinant.The ability of a population of Ab molecules to react with more than one AgAnti-A AbAg AAnti-A AbAg BShared epitopeAnti-A AbAg CSimilar epitopeCross reactionsNon-covalent Bonds Hydrogen bonds Electrostatic bonds Van der Waal forces Hydrophobic bondsReversible Multiple Bonds Source:Li,Y.,Li,H.,Smith-Gill,S.J.,Mariuzza,R.A.,Biochemistry 39,6296,2000 http:/www.med.sc.edu:85/chime2/lyso-三、影响抗原抗体反应的因素Factors Affecting Measurement of Ag/Ab Reactions Affinity Ag:Ab ratio Physical form of AgAb excessAg excessEquivalence Lattice formation 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/HemagglutinationDefinition-tests that have as their endpoint the agglutination of a particulate antigen+Qualitative agglutination testAg or AbAgglutination/HemagglutinationQuantitative agglutination testTiterProzone1/21/41/81/161/321/641/1281/2561/5121/1024Pos.Neg.Titer648512232128324Patient12345678Agglutination/HemagglutinationDefinition Qualitative testQuantitative test Applications Blood typing Bacterial infectionsFourfold rise in titer Practical considerations Easy Semi-quantitative1/21/41/81/161/321/641/1281/2561/512BACKImmunoelectrophoresisMethodAgs are separated by electrophoresis Interpretation Precipitin arc represent individual antigensAgAgAbAgAbAb is placed in trough cut in the agar BACK+-Countercurrent electrophoresisMethodAg and Ab migrate toward each other by electrophoresisUsed only when Ag and Ab have opposite charges QualitativeRapidAgAb-+BACKBACKComplement FixationAg mixed with test serum to be assayed for AbStandard amount of complement is addedErythrocytes coated with Abs is addedAmount of erythrocyte lysis is determinedAgPatientsserum AgNo AgAgMethodologyBACKBACK 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(Horseradish Peroxidase,HRP);2.碱 性 磷 酸 酶(Alkaline phosphatase,AP)。直接法抗原包被AgLabeled AbTissue Section间 接 法AgLabeled Anti-IgTissue SectionUnlabeledAb 夹 心 法直 接 夹 心 SolidPhaseAgImmobilizedAg in PatientssampleLabeledAb直接夹心直接夹心法法直接直接夹心夹心 法法直接直接 法法
