以鸡源鲍氏志贺氏茵菌体提取的脂多糖(LPS)成分作为检测抗原,以辣根过氧化物酶标记的免抗鸡IgG抗体为第二抗体,以TMB为显色剂,成功建立了检测鸡源鲍氏志贺氏茵抗体的间接ELISA方法.通过对间接ELISA反应条件的一系列研究,确定了各种最适工作条件:鸡源鲍氏志贺氏茵LPS抗原最适包被质量浓度为5mg·L-1,最适包被条件为4 ℃过夜;待检血清的最佳稀释度为1∶160,与包被抗原在37 ℃作用1 h;酶标二抗的最适工作浓度为1∶2 000,血清与酶标二抗的反应条件为37 ℃ 1 h;最适封闭条件为37 ℃作用1 h;底物最适反应条件为室温避光20 min;阴阳性临界值为0.385.经特异性试验、敏感性试验和重复性试验,表明建立的间接ELISA方法具有特异性强、敏感性高、重复性好等特点.%This paper aims to establish an indirect ELISA method of detecting chicken shigellosis by using extracted LPS from S. boydii in chicken as a coating antigen, horseradish peroxidase labeled rabbit anti-chicken IgG-HRP as the second antibody, TMB as color reagent. By a series of trial on indirect ELISA reaction conditions, the optimal working conditions are defined: the optimum coating antigen concentration of LPS from chicken S. boydii is 5 mg · L-１; the optimum coating condition is 4 ℃ overnight; the best dilution of serum to be found is 1: 160, with the coating antigen at 37 ℃ for Ih; Rabbit anti-chicken IgG-HRP concentration for optimal work is 1: 2 000, and the reaction condition is 37 ℃ for 1 h; the optimum condition for the closure is 37 ℃ for 1 h; the best reaction condition for substrate is at room temperature in dark for 20 min; the critical value of negative and positive was 0. 385. The specificity test,sensitivity test and repeatability test show that the establishment of an indirect ELISA method had high specificity, sensitivity, repeatability.
展开▼
