犬瘟热病毒单克隆抗体夹心ELISA检测方法的建立

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5 μg/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 μg/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.