牛病毒性腹泻病毒(BVDV)抗原捕获ELISA检测方法的建立

用牛病毒性腹泻病毒(BVDV)单克隆抗体包被酶标板,以兔多克隆抗体作为夹心抗体,建立BVDV抗原捕获ELISA(AC-ELISA)检测方法,优化反应条件并对该方法的稳定性等指标进行了测试和评价。结果表明,单抗最佳包被质量浓度为5μg/mL,兔多抗血清最佳质量浓度为10μg/mL;单抗在4℃包被12~24 h,多抗在37℃作用1 h为双抗体的最佳反应条件;酶标抗体最适稀释度1∶10000,最适作用时间为1 h;采用1%BSA和1%明胶分别在抗体包被后和加入待检抗原反应后进行两次封闭效果好。用AC-ELISA方法检测临床采集的11份牛腹泻病料和12份健康牛组织样品,同时以病毒分离和RT-PCR检测方法做对比,3种方法符合率很高。研究表明AC-ELISA方法稳定性好,可用于BVDV的临床快速检测。

Development of an antigen-capture ELISA for detecting bovine viral diarrhea virus

An antigen-capture enzyme-linked immunosorbent assay(AC-ELISA) for detecting bovine viral diarrhea virus(BVDV) was developed with a monoclonal antibody(McAb) capturing virus,while the rabbit anti-BVDV serum was used as the second antibody to identify virus.Working conditions of the AC-ELISA were optimized and then its capabilities were evaluated.The results showed that the optimum working concentration of McAb was 5 μg/mL and that of rabbit anti-BVDV serum was 10 μg/mL.The dilution of conjugate was 1∶10 000,and the reaction time was one hour.Twice blockings,firstly 1% BSA after the 96 plate was coated with McAb and then 1% gelatin after the detected antigen reacted with the coated McAb,achieved the best effect.Eleven diarrhea samples and 12 healthy tissue samples from cattle were detected paralelly by the AC-ELISA,virus isolation and PCR,respectively.The accordant rate was higher.These findings suggest that the AC-ELISA has good repeatability and can be applicable to the rapid detection of BVDV.