犬瘟热病毒单克隆抗体夹心ELISA检测方法的建立  

Development of a sandwich ELISA based on monoclonal antibodies against canine distemper virus

作　　者：孙婧[1,2] 毕振威[2] 夏兴霞[2] 王晓丽[2] 诸玉梅[2] 董晨红[2] 贾红[3] 朱瑞良[1] 王永山[2] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室,江苏南京210014 [3]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国预防兽医学报》2013年第8期635-639,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏省农业科技自主创新资金项目[CX(12)3062];公益性行业(农业)科研专项(201303042)

摘　　要：为建立方便快捷的犬瘟热病毒(CDV)检测方法,本实验应用杂交瘤细胞融合技术,建立了4株分泌抗CDV单克隆抗体(MAb)的杂交瘤细胞株,分别命名为A2、F7、H4和G12。相加ELISA试验显示4株MAb作用于CDV不同的抗原表位。选取相加指数较高的两株MAb G12和A2分别作为捕获抗体和酶标检测抗体,建立检测CDV的夹心ELISA检测方法,并对实验条件进行了优化。结果显示,该方法与犬细小病毒、犬副流感病毒、犬腺病毒1型、犬冠状病毒等犬类病毒无交叉反应,敏感度为5μg/mL,变异系数小于6%。利用该方法与RT-PCR方法同时检测57份临床样品,两种方法的符合率为100%。本实验建立的MAb夹心ELISA方法具有特异、敏感、方便快捷等优点,适用于大批量临床样品检测。To develop a method for the detection of canine distemper virus （CDV）, four hybridoma of A2, F7, H4 and G12 secreting monoclonal antibodies （MAb） to CDV were prepared by the fusion of SP2/0 cells and splenocyte from BALB/C mice immunized with the purified CDV. Additivity ELISA revealed that the four MAbs recognized spatially independent epitopes of CDV. In addition, a sandwich ELISA was established with MAb G12 as capture antibody and HRP conjugated MAb A2 as detection antibody for the rapid detection of CDV which had no cross-reactivity with other canine virus and the detection limit was 5 ixg/mL. The coefficient of variation of reproducibility was less than 6%. A total of 57 clinical samples were detected by the ELISA and RT-PCR with agreement rate of 100%. The result indicated that the sandwich ELISA was applicable to the clinical detection of CDV.

关 键 词：犬瘟热病毒 杂交瘤细胞株 单克隆抗体 夹心ELISA 

分 类 号：S852.65[农业科学—基础兽医学]