抗原抗體反應(yīng)及應(yīng)用.ppt

1、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（monoclonal antibody，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,一、抗血清的制備,1、免疫動(dòng)物 抗原 佐劑 免疫動(dòng)物 2、抗血清的純化與保存 采血 純化與保存 3、抗血清的鑒定 效價(jià) 親和力,BACK,Affinity = attractive and repulsive forces,Affinity,Strength of the reaction b

2、etween a single antigenic determinant and a single Ab combining site,Calculation of Affinity,Ag + Ab Ag-Ab,Applying the Law of Mass Action:,二、單抗的制備,1、單抗的制備原理 2、單抗的制備方法 抗原提純和動(dòng)物免疫 骨髓瘤細(xì)胞及飼養(yǎng)細(xì)胞的制備 細(xì)胞融合 篩選陽(yáng)性株 單克隆抗體的制備和凍存 單克隆抗體的純化 GO,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 (HGP

3、RT+、TK+) 在HAT中大量長(zhǎng)期繁殖 克隆化 產(chǎn)生單抗,BACK,BACK,BACK,BACK,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 Nature of Ag/Ab Reactions,Lock and Key Concept antibody combining site to react with only one antigenic determinant. The

4、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,Non-covalent Bonds,H

5、ydrogen bonds Electrostatic bonds Van der Waal forces Hydrophobic bonds,Reversible,Multiple Bonds,三、影響抗原抗體反應(yīng)的因素 Factors Affecting Measurement of Ag/Ab Reactions,Affinity,Ag:Ab ratio,Physical form of Ag,PH,Temperament,Electrolyte,第三節(jié) 常見(jiàn)的免疫分析方法,一、凝聚反應(yīng)(Agglutination) 二、沉淀反應(yīng) 1、溶液中的沉淀反應(yīng) 2、凝膠擴(kuò)散沉淀 3、免疫電泳 血

6、清免疫電泳 火箭電泳 對(duì)流免疫電泳 三、補(bǔ)體參與的反應(yīng) 四、免疫標(biāo)記的抗原抗體技術(shù) 酶聯(lián)免疫吸附（ELISA）,Agglutination/Hemagglutination,Definition - tests that have as their endpoint the agglutination of a particulate antigen,Agglutination/Hemagglutination,Quantitative agglutination test Titer Prozone,Agglutination/Hemagglutination,Definition Qual

7、itative test Quantitative test,Applications Blood typing Bacterial infections Fourfold rise in titer,Practical considerations Easy Semi-quantitative,BACK,Immunoelectrophoresis,Method Ags are separated by electrophoresis,Interpretation Precipitin arc represent individual antigens,Ab is placed in trou

8、gh 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,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,Ag,Methodology,BACK,BACK,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