副猪嗜血杆菌抗体间接ELISA检测方法的建立与应用

Establishment and application of an indirect ELISA for detection of antibody against Haemophilus parasuis

采用超声裂解的副猪嗜血杆菌菌体上清作为包被抗原,建立了检测副猪嗜血杆菌抗体的间接ELISA方法。通过对试验条件进行筛选优化,确定了最佳反应条件:抗原包被浓度80μg/mL,37℃作用2 h后4℃过夜包被,待检血清的稀释度为1∶320,抗原抗体反应时间为1 h,酶标二抗稀释度为1∶15000,作用时间为1 h,底物显色时间为15 min。待检血清S/N值≥2.5时判为阳性,S/N值<2.5时判为阴性。特异性、重复性和敏感性试验以及对200份送检血清的检测结果表明,建立的间接ELISA方法的特异性和重复性良好,敏感性比间接血凝试验高,可用于副猪嗜血杆菌的检疫和流行病学调查。

An indirect ELISA coated with supernatant of sonicated Haemophilus parasuis serovar 5 cell culture was established. The results of chessboard titration tests showed that coating antigen at a concentration of 80μg/mL incubated optimally at 37 ℃ for 2 h and then coated overnight at 4 ℃. The optimal dilution of serum and HRP-IgG was 1 ：320 and 1 ： 15 000,respectively. The reaction time of coating antigen with serum and serum with HRP-IgG was 1 h. The time of color development reaction was 15 min. The criteria evaluation was as follows：the serum was positive if S/N≥ 2.5,and the reverse is negative. Reproducibility,specificity,sensitivity tests and detection for 200 sera by the established ELISA confirmed that the developed ELISA had good stability and specificity. Compared with indirect haemagglutination test,the indirect ELISA was more sensitive. So this assay can be used as a tool for diagnosis and quarantine of H. parasuis.