牛病毒性腹泻病毒双单克隆抗体夹心ELISA检测方法的建立及初步应用  

Development and Evaluation of Double Monoclonal Antibody Sandwich ELISA for Bovine Viral Diarrhea Virus Detection

作　　者：杨俊兴[1,2] 曹琛福[1] 曾少灵[1] 卢体康[1] 孙洁[1,2] 张彩虹[1,2] 唐金明[1,2] 陶虹[1,2] 吕建强[1] 花群义[1,2] 

机构地区：[1]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518045 [2]深圳市外来有害生物检测技术研发重点实验室,广东深圳518045

出　　处：《动物医学进展》2013年第5期11-16,共6页Progress In Veterinary Medicine

基　　金：国家质量监督检验检疫总局科技项目(2011IK015);国家科技支撑计划项目(2011BAK10B01)

摘　　要：为了建立牛病毒性腹泻病毒(BVDV)抗原快速检测ELISA方法,用于BVDV抗原的快速检测。用纯化的BVDV作为抗原,经动物免疫、细胞融合、间接ELISA筛选和亚克隆,得到了3株抗BVDV的单克隆抗体(2A6、2F4和5C9)。选用单克隆抗体2A6和5C9包被ELISA板,用HRP标记的单克隆抗体2F4作为夹心抗体,建立了检测BVDV的双单克隆抗体夹心ELISA方法。通过对BVDV阳性样品、其他病毒阳性样品和阴性对照样品及系列稀释的阳性样品检测,表明该方法具有良好的特异性、敏感性和重复性。用ELISA检测326份临床样品,结果表明该方法与RT-PCR和商品化ELISA试剂盒检测结果符合率分别为98.8%和99.0%。该方法可以用于BVDV抗原快速检测和对大量样本的筛选。In order to establish a rapid test for detecteion of bovine viral diarrhea virus （BVDV）, a sanwich ELISA was developed based on monclonal antibodies against BVDV, which were obtained by screening the fused SP2/0 cells and spleen cells isolated from Balb/c mouse immunized with purified BVDV. The mono- clonal antibodies 2A6 and 5C9 were selected as the first antibody for coating ELISA plate, and the HRP labelled monclonal antibody 2F4 was used as the second antibody. For detecting the BVDV antigen, other reference virus antigens, negetive control samples, and series dilluted BVDV antigen, the results indicated the ELISA had good specificity and sensitivity. This ELISA was also used to test 326 clinical samples, RT-PCR and a commercial BVDV ELISA kit were used as reference methods, the agreement ratio between ELISA and reference methods were 98.8% and 99.0% respectively.

关 键 词：牛病毒性腹泻病毒 酶联免疫吸附试验 单克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]