水泡性口炎病毒双抗体夹心ELISA检测方法的建立

为建立方便快捷的水泡性口炎病毒(VSV)检测方法,本研究以抗VSV单克隆抗体(MAb)为捕获抗体,兔抗VSV多克隆抗体为检测抗体,建立VSV双抗体夹心ELISA检测方法。结果显示,该方法的最佳工作条件为:抗VSV MAb 1A2的包被浓度为3.09μg/mL,兔抗VSV多克隆抗体和酶标抗体的工作浓度分别为5.16μg/mL和1∶5 000,以OD450nm≥0.231作为阳性判定标准。该ELISA方法对猪水泡病病毒、猪水疱疹病毒及羊传染性脓疱病毒等均无交叉反应;敏感度可达3.125μg/mL(101TCID50);其重复性变异系数小于10%。采用建立的ELISA方法与RT-PCR方法同时检测187份临床样品,符合率达到97.9%,具有良好的相关性。本实验建立的VSV双抗体夹心ELISA检测方法具有特异性好、敏感性高、成本低及方便快捷等优点,可以用于VSV的快速检测。

Development of a double antibody sandwich ELISA for detection of vesicular stomatitis virus

To develop a method for vesicular stomatitis virus(VSV) detection,a double-antibody sandwich ELISA(DAS-ELISA) was developed using VSV monoclonal antibody(MAb) as capture antibody and rabbit polyclonal antibodies against VSV as detecting antibody.The optimized reaction conditions showed that the optimal coating concentration of VSV MAb was 3.09 g/mL,the optimal working concentration of rabbit polyclonal antibodies against VSV was 5.16 μg/mL,and the optimal working dilution of HRP-labelled goat-anti-rabbit IgG was 1∶5 000 with a cutoff value of 0.231(OD450nm).The ELISA had no cross-reaction with swine vesicular disease virus,vesicular exanthema of swine virus and orf virus,and at least 3.125 μg/mL antigen(101 TCID50) of VSV was able to be detected.The coefficient of variation of reproducibility was less than 10%.A total of 187 clinical samples were detected by the ELISA and RT-PCR with agreement rate of 97.9%.The results revealed that the ELISA method was quick,sensitive and repeatable,which was applicable to the detection of VSV.