摘要
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12～24h，多抗在37℃作用1h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1h；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。
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.
出处
《畜牧与兽医》
北大核心
2009年第8期13-19,共7页
Animal Husbandry & Veterinary Medicine
基金
吉林大学青年基金资助项目(430505010219)
