基于绵羊肺炎支原体Enolase蛋白建立间接ELISA抗体检测方法

Establishment of an indirect ELISA method for antibody detection based on Mycoplasma ovipneumoniae Enolase protein

旨在建立一种检测绵羊肺炎支原体(Mo)血清抗体的间接ELISA方法,笔者构建了Mo Enolase基因原核表达载体,诱导表达后,纯化的重组蛋白用Western-blot分析其反应原性。以重组蛋白为包被抗原,建立了Mo间接ELISA抗体检测方法。结果显示,重组蛋白得到可溶性表达,Western-blot证实具有良好的反应原性。间接ELISA反应条件优化结果显示,包被抗原浓度为2 mg/L,37℃2 h,封闭条件为含10 g/L BSA的PBS,4℃过夜,待检血清稀释度为1∶50,37℃1 h,酶标二抗最佳稀释度为1∶4000,37℃1 h,底物最佳显色条件为37℃避光10 min。分别利用38份阴性血清、38份阳性血清确定临界值为S/P=0.365。用该方法与间接血凝法对180份血清进行检测,两者符合率为81.11%。结果表明,本研究建立的间接ELISA方法敏感、特异,具有良好的临床应用前景。

To develop an indirect ELISA for detecting antibodies against Mycoplasma ovipneumoniae(Mo),the Mo Enolase protein was expressed prokaryotic and analyzed by Western-blot after purification.A recombinant Enolase protein-based indirect ELISA was established after optimization of a series of reaction conditions,and its performance was evaluated.The results showed that the Mo Enolase protein was successfully expressed,purified and had good immunoreogenicity.Based on the purified Mo Enolase protein,an indirect ELISA method was successfully established.The protein coating concentration was 2 mg/L.10 g/L BSA PBS solution and overnight at 4℃showed the best blocking effect.The optimal dilution of the serum was 1∶50 and the incubation condition was 37℃1 h.The optimal dilution ratio of HRP conjugated rabbit anti-goat IgG was 1∶4000 and the incubation condition was 37℃1 h.The chromogenic substrate was incubated for 10 min at 37℃in the dark.The critical value of S/P=0.365 was determined by using 38 negative and 38 positive sera respectively.180 sera samples were detected by the IHA method and the indirect method,the coincidence rate between the two methods was 81.11%.These results indicated that the indirect ELISA method established had good sensitivity and specificity,and could have a good clinical application prospect.