A型塞内卡病毒VP2蛋白间接ELISA检测方法的建立

Development of an indirect ELISA for detection of VP2 protein against Senecavirus A

为建立一种检测塞内卡病毒VP2蛋白的间接ELISA方法,本试验将A型塞内卡病毒(SVA)VP2蛋白作为抗原包被酶标板,采用棋盘式方法优化反应的最佳血清稀释度和抗原包被量,同时对间接ELISA方法的其他反应条件进行了筛选。结果显示,VP2蛋白包被量为2μg/m L(1∶1000稀释);用2%BSA封闭液稀释标准阳性血清为1∶20;二抗做1∶5000稀释;TMB底物显色液室温避光显色反应10 min。特异性试验结果显示,同FMDV、PRRSV、CSFV、PRV和APP阳性血清均无交叉反应。此方法对塞内卡病毒阳性血清的敏感性为1∶160。批间、批内重复性试验结果的变异系数均小于10%。对200份猪血清样本分别进行间接荧光抗体免疫方法和间接ELISA方法的检测,结果符合率为95.5%。上述结果表明,该方法具有良好的特异性、敏感性和重复性,可用于塞内卡病毒血清抗体检测和流行病学调查。

In this experiment,an indirect(ELISA)method for detecting antibodies against Seneca virus using the recombinant protein VP2 was developed.The structural protein VP2 of SVA was used as the coating antigen.The chessboard method was used to optimize the optimal serum dilution and antigen enveloping conditions for ELISA,and other reaction conditions were screened by indirect ELISA.Moreover,the results showed that the VP2 protein coating amount was 2μg/mL(1∶1000 dilution);the standard positive serum was diluted 1∶20 with 2%BSA blocking solution;the second antibody was diluted1∶5000;TMB substrate color reaction at room temperature and light for 10 min.Specific test results showed no cross-reactivity with other FMDV,PRRSV,CSFV,PRV and APP positive sera.This method had a sensitivity of 1∶160 to seneca-positive serum.The intre-assay repeatability and the inter-assay repeatability coefficient of variation were both<10%.In comparison with results obtained from indirect immunofluorescence assay(IFA)by testing of 200 serum samples to evaluate the sensitivity and specificity of the ELISA,agreement 95.5%was reached.The method has good specificity,sensitivity and reproducibility and can be used for seneca virus serum antibody detection and epidemiological investigation.