ETEC K88菌毛蛋白抗体PPA-ELISA检测方法的建立  

Establishiment of PPA-ELISA Assay to Detect ETEC K88 Fimbria Antibody Levels

作　　者：李书光[1,2] 李峰[1,2] 苗立中[1,2] 张娜[1,2] 肖跃强[1,2] 陈金龙 孙翠平 沈志强[1,2] 

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600

出　　处：《动物医学进展》2012年第6期95-98,共4页Progress In Veterinary Medicine

摘　　要：以纯化的大肠埃希菌K88菌毛蛋白为包被抗原,建立了检测大肠埃希菌K88IgG抗体的间接ELISA方法。确定了间接ELISA的最适反应条件,即最佳抗原包被浓度为1μg/mL,兔抗体稀释倍数为1∶10,猪抗体稀释倍数为1∶100,确定最佳封闭液为50g/L脱脂奶粉,最佳封闭时间为0.5h,血清反应时间0.5h,二抗反应时间1h,底物室温反应时间为15min,阴阳性临界值兔血清为0.33、猪血清为0.45。证实该间接PPA-ELISA方法特异性强、重复性好、敏感度高,可以作为疫苗效力检验和流行病学调查的参考方法。A PPA-ELISA to detect the anti-K88 IgG levels were developed by using the porcine enterotoxigenic Escherichia coli （ETEC） purified K88 fimbrial as coating antigen. The PPA-ELISA was checked with serum samples obtained from rabbits and pigs, and the optimal parameters affecting the method were also determined as follows. The optimal coating concentration of K88,1 μg/mL; the optimal dilution of corresponding serum samples, rabbits 1 ： 10, pigs 1 ： 100; the optimal blocking solution,50 g/L skimmed milk powder;the optimal blocking time,0.5 h;the optimal serum sample combining time,0.5 h;the optimal PPA combining time, 1 hl the optimal substrate reaction time at room temperature, 15 min; the positive- negative cut-off point, in rabbit sera, OD450 nm 0.33, in pig sera, OD450 nm 0.45. The results proved that the PPA-ELISA method was sensitive, specific and repeatable.

关 键 词：产肠毒素大肠埃希菌 K88 辣根过氧化物酶标记的葡萄球菌A蛋白-酶联免疫吸附试验 

分 类 号：S852.612[农业科学—基础兽医学]