抗原抗体反应和应用.ppt

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,资料仅供参考,不当之处，请联系改正。,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,资料仅供参考,不当之处，请联系改正。,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,资料仅供参考,不当之处，请联系改正。,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,BACK,Affinity=,a,ttractive and repulsive forces,Ab,Ag,High Affinity,Ab,Ag,Low Affinity,Affinity,Strength of the reaction between a single antigenic determinant and a single Ab combining site,Calculation of Affinity,Ag+Ab,Ag-Ab,K,eq,=,Ag-Ab,Ag x Ab,Applying the Law of Mass Action:,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,BACK,BACK,BACK,BACK,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,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.,Specificity,Cross Reactivity,The 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 Ag,Anti-A Ab,Ag A,Anti-A Ab,Ag B,Shared epitope,Anti-A Ab,Ag C,Similar epitope,Cross reactions,Non-covalent Bonds,Hydrogen bonds,Electrostatic bonds,Van der Waal forces,Hydrophobic bonds,Reversible,Multiple Bonds,Source:Li,Y.,Li,H.,Smith-Gill,S.J.,Mariuzza,R.A.,Biochemistry 39,6296,2000,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,Ag,Ab excess,Ag excess,Equivalence 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/Hemagglutination,Definition-tests that have as their endpoint the agglutination of a particulate antigen,Y,Y,Y,+,Qualitative agglutination test,Ag or Ab,Agglutination/Hemagglutination,Quantitative agglutination test,Titer,Prozone,1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256,1/512,1/1024,Pos.,Neg.,Titer,64,8,512,2,32,128,32,4,Patient,1,2,3,4,5,6,7,8,Agglutination/Hemagglutination,Definition,Qualitative test,Quantitative test,Applications,Blood typing,Bacterial infections,Fourfold rise in titer,Practical considerations,Easy,Semi-quantitative,1/2,1/4,1/8,1/16,1/32,1/64,1/128,1/256,1/512,BACK,Immunoelectrophoresis,Method,Ags are separated by electrophoresis,Interpretation,Precipitin arc represent,individual antigens,Ag,Ag,Ab,Ag,Ab,Ab is placed in trough cut in the agar,BACK,+,-,Countercurrent electrophoresis,Method,Ag and Ab migrate toward each other by electrophoresis,Used only when Ag and Ab have opposite charges,Qualitative,Rapid,Ag,Ab,-,+,BACK,BACK,Complement Fixation,Ag mixed with test serum to be assayed for Ab,Standard amount of complement is added,Erythrocytes coated with Abs is added,Amount of erythrocyte lysis is determined,Ag,Y,Y,Patients,serum,Y,Y,Y,Y,Y,Y,Y,Y,Ag,No Ag,Ag,Methodology,BACK,BACK,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,Ag,Y,Labeled Ab,Tissue Section,间 接 法,Ag,Y,Y,Labeled Anti-Ig,Tissue Section,Unlabeled,Ab,夹 心 法直 接 夹 心,Solid,Phase,Y,Ag,Immobilized,Y,Ag in,Patients,sample,Labeled,Ab,直接夹心,法,直接,夹心,法,直接,法,