包被LPS抗原检测鸡源鲍氏志贺氏菌抗体的间接ELISA方法的建立

The establishment of the indirect ELISA method for the detecting of Shigella boydii antibody from chicken by using LPS as a coating antigen

以鸡源鲍氏志贺氏菌菌体提取的脂多糖(LPS)成分作为检测抗原,以辣根过氧化物酶标记的兔抗鸡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菌抗体的间接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:鸡源鲍氏志贺氏菌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,horse radish peroxidase labeled rabbitanti-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 ℃ over-night;the best dilution of serum to be found is 1：160,with the coating antigen at 37 ℃ for 1h;Rabbitanti-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 substrateis 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.