牛病毒性腹泻/黏膜病毒抗体间接ELISA检测方法的建立  

Establishment of the Indirect ELISA Detection Method of Bovine Viral Diarrhea Virus

作　　者：常敬伟 邢思毅 常春龙[1] 毕莹[1] 王士霞[1] 李田田[1] 周玉龙[1] 倪宏波[1] Chang Jingwei Xing Siyi Chang Chunlong Bi Ying Wang Shixia Li Tiantian Zhou Yulong Ni Hongbo(College of Animal Science and Technology, Heilongjiang Bayi Agriculture University, Daqing 163319)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《黑龙江八一农垦大学学报》2016年第6期74-78,共5页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省农垦总局"十二五"重点科技攻关项目(HNK125B-11-10A;HNK125B-11-02)

摘　　要：为建立一种检测牛病毒性腹泻、黏膜病病毒抗体的间接ELISA方法,利用昆虫细胞真核表达系统成功表达E2蛋白并纯化,使用纯化后的蛋白作为包被抗原。确定最佳抗原包被浓度为6μg·m L-1,最佳血清稀释度为1∶80,最佳包被液为碳酸缓冲液,最佳盐封闭液为5%脱脂乳,最佳血清作用时间为60 min,酶标抗体作用最佳浓度为1∶10 000;最佳底物作用时间为5 min,阳性临界值为D450 nm≥0.282。与IDEXX的BVDV间接ELISA试剂盒比较,总符合率为91.5%,板内和板间重复性试验变异系数均小于10%。该方法与牛副流感病毒Ⅲ型、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒和牛冠状病毒阳性血清无交叉反应。因此,该ELISA诊断方法可用于大批量样本抗体水平监测和流行病学调查。To establish a ELISA method to detect the antibody of bovine viral diarrhea virus （BVDV）,the envelope protein E2 was successfully expressed by insect cells eukaryotic expression systems and purified,using the recombinant and purified E2 protein as antigen. The optimal working parameters were determined as follows,the antigen concentration was 6 μg·mL^-1,the serum dilution was 1：80,the optimal coating buffer was carbonate buffer solution,the block liquid was 5% skimmed milk,the block time was 60 min at 37 ℃ ,the antibody concentration was 1：10 000,the optimum substrate reaction time was 5 min,The cut-off value was 0.282. Compared with foreign commercial kits,the total coincidence rate was 91.5%. There was no cross-reaction with the positive sera of BPIV-3, IBRV, BRSV or BCV in the method. The ELISA method was suitable to a bulk sample BVDV antibody level monitoring and epidemiological investigation.

关 键 词：牛病毒性腹泻病毒 E2蛋白 间接ELISA 

分 类 号：S858.23[农业科学—临床兽医学]