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.
出处
《动物医学进展》
CSCD
北大核心
2012年第6期95-98,共4页
Progress In Veterinary Medicine
