副猪嗜血杆菌抗体间接ELISA检测方法的建立与应用

本试验采用121 ℃高压处理副猪嗜血杆菌4型和5型耐热蛋白,混合作为包被抗原,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:抗原包被浓度为10 μg/mL,37 ℃包被2 h;封闭液选择含20 g/L脱脂奶粉的PBST,封闭30 min;血清的稀释度为1∶80;抗原抗体反应时间为45 min;酶标二抗稀释度为1∶12000,作用时间为30 min;底物显色时间为15 min。特异性、重复性和敏感性试验及对200份送检血清的检测结果表明,建立的间接ELISA方法特异性和重复性良好,敏感性比间接血凝试验高,对已知阴阳性血清的临床样本检测结果与国外ELISA试剂盒一致,可用于副猪嗜血杆菌的血清抗体检测和血清流行病学调查。

Establishment and Application of an Indirect ELISA for Detection of Antibody Against Haemophilus parasuis

An indirect ELISA coated with 121 ℃ high pressure Haemophilus parasuis serovar 4 and 5 hot-torlerance protein was established. The results of chessboard titration tests showed that coating antigen at a concentration of 10 μg/mL incubated optimally at 37 ℃ for 2 h and then coated for 30 min with 20 g/mL skim milk powder. The optimal dilution of serum was 1∶80. The reaction time of coating antigen with serum 30 min. The optimal dilution of ELISA secondary antibody was 1∶12000. The reaction time was 30 min. The time of color development reaction was 15 min. 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 and the clinical test result was consistent to overseas ELISA reagent kits. So this assay can be used as a tool for diagnosis and quarantine of H.parasuis.