ETEC菌毛蛋白K99抗体PPA-ELISA检测方法的建立及初步应用  

Development and preliminary application of PPA-ELISA in detection of antibodies against K99 fimbrial protein

作　　者：谢金文[1,2] 白耀国 李书光[1,2] 王金良[1,2] 曲光刚[1,2] 苗立中[1,2] 沈志强[1,2] 

机构地区：[1]山东省滨州畜牧兽医研究院山东省畜禽用蜂胶疫苗研究开发推广中心,滨州256600 [2]山东绿都生物科技有限公司,滨州256600

出　　处：《安徽农业大学学报》2015年第2期237-242,共6页Journal of Anhui Agricultural University

基　　金：山东省自然科学科学基金青年基金项目(ZR2014CQ009);滨州市2013年科技发展计划项目(2013GG0304);山东省现代农业产业技术体系生猪产业创新团队(SDAIT-06-011-14)共同资助

摘　　要：以纯化的大肠埃希菌K99菌毛蛋白为包被抗原,建立检测大肠埃希氏菌K99 Ig G抗体的间接ELISA方法。确定间接ELISA的最适反应条件,即抗原包被的ELISA板4℃过夜,最适抗原包被浓度为4.94μg·m L-1,兔抗体稀释倍数为1:50,猪抗体稀释倍数为1:200,确定最佳封闭液为100 g·L-1脱脂奶粉,最佳封闭时间为0.5 h,血清反应时间为1 h,二抗最适浓度梯度为1:2000,反应时间为37℃0.5 h,底物室温反应时间为37℃10 min,阴阳性临界值兔血清为0.25、猪血清为0.35。试验证实该间接PPA-ELISA方法的特异性强、敏感性高、重复性好,可以为疫苗效力检验和流行病学调查提供参考方法。A PPA-ELISA method for detecting anti-K99 IgG levels was developed by using the porcine en- terotoxigenic Escherichia coli （ETEC）-purified K99 fimbrial protein as the coating antigen. The PPA-ELISA was tested with serum samples obtained from rabbits and pigs. The optimal parameters affecting the method were also determined. They were： antigen coated ELISA plates at 4℃ overnight; the optimal coating concentration of K99 was 4.94 μg/mL; the optimal dilution of corresponding serum samples for rabbits was 1：50 and for pigs was 1：200; the optimal blocking solution was 100 g/L skimmed milk powder. The best blocking time, reaction time for serum samples, PPA concentration gradient, PPA reaction time and substrate reaction time at room temperature were 0.5 h, 1 h, 1：2000, 0.5 h, and 10 min, respectively. The positive-negative threshold values of rabbit and pig sera were 0.25 and 0.35, respectively. The results proved that the PPA-ELISA method was sensitive, specific and repeatable and can be applied to the vaccine potency test and epidemiological investigation.

关 键 词：ETEC K99 PPA-ELISA 抗体 检测 建立 

分 类 号：S852.61[农业科学—基础兽医学]