文档介绍：摘要本研究通过weStem blot方法对大肠杆菌乳房炎分离株进行菌体蛋白分析,找出了主要免疫原性蛋白为约33kDa的膜蛋白,应用超声破碎、超速离心、电洗脱等方法对其进行纯化,以其作为包被抗原,建立了间接ELIsA方法。以膜蛋白作为抗原包被酶标板,通过对ELISA各反应条件的优化,确定了最佳反应条件为: ;∥mL;最佳封闭液为 5%脱脂奶粉,封闭条件为37℃lh:最佳血清稀释液为5%脱脂奶粉,血清稀释倍数为loo倍; 000。采用已确立的ELISA反应条件,对210份阴性血清的检测结果进行统计学分析,确定了间接ELISA方法阳性血清判定标准,即OD4。,。应用建立的间接ELISA方法对72份乳房炎疫苗免疫牛血清和224份未免疫牛血清的检测结果表明,ELISA方法的特异性为93%,%。采用建立的间接ELISA方法对奶牛乳房炎多联灭活疫苗免疫牛血清中大肠杆菌特异性抗体水平进行了检测,揭示了疫苗免疫牛大肠杆菌抗体水平的动态变化,为该疫苗I临床应用效果评价提供依据。关键词:奶牛乳房炎,大肠杆菌,间接ELISA,抗体 Abstract from coliform mastiffs WaS analyzed by Western blottofind out the main protein∞ indirectELISA was developed withthepurified protein∞the coating antigen. The optimal reactionconditions of ELISA optimal coating buffer WaS PBS (),and theoptimal coating antigen formicroplate percent skiIIlmilk Was added as theoptimal blocking agent and incubatedfor 2hours at37"( dilution bufferWas 5%skim milk,and thedilution of sera samples was 1: working concentratinn of HRP-labeled rabbitanti-bovine IgG was l: cut off value fortheindirect ELISA Was determinated with 210 negative bovine serum OD valueis more than or equal to the testsample will two positive serum samples fromthe COWS immunized withbovine mastitis ine and 224 negative serum samples fromthe COWS ascontrol were detected fortheantibodiesagainst thedeveloped Was found thatthe specificity andsensitivity ofthedeveloped ELISA WaS93%%.respectively. The developed ELISA Was employed todetectthe level ofthe COWS immunized with polyvalent inactivated ine in order to reveal the dynamic change of serum antibodiesagainst inimmunized some referencefortheevaluationofclinical application ofthe ine. Keywords:bovinemastitis,Escherichiacoli,indirectELISA,antibo