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.
出处
《安徽农业大学学报》
CAS
CSCD
北大核心
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
