猪流行性腹泻病毒双抗夹心ELISA的建立

Development of a Double Antiby Sandwich ELISA for Detection of Porcine Epidemic Diarrhea Virus

用猪流行性腹泻病毒(PEDV)免疫蛋鸡后提取抗猪流行性腹泻病毒的特异性卵黄抗体为捕获抗体,鼠抗猪流行性腹泻病毒多克隆抗体为检测抗体,建立了猪流行性腹泻病毒病原检测的双抗夹心ELISA,其最适包被抗体浓度为1:4000(12.35μg/m L),鼠抗猪流行性腹泻病毒的多克隆抗体最佳浓度为1:6000(10.25μg/m L),样品反应时间为30min,酶标抗体工作浓度为1:6000,并以OD450≥0.130作为阳性判断标准。该方法与猪传染性胃肠炎、猪轮状病毒、猪流感病毒、大肠杆菌K88、K99、987P、F41等病原无交叉反应。对经猪流行性腹泻病毒PCR检测的100份粪便样本进行检测表明,8份PCR检测阳性样本中6份为本法阳性;PCR阴性者本法全部阴性。实验结果表明该方法具有良好的特异性和敏感性,可用于猪流行性腹泻病毒的快速检测。

Porcine epidemic diarrhea virus（PEDV）was used to immunize laying hens for preparation of speciifc yolk antibody（IgY）as capturing antibody. A double antibody sandwich ELISA（DAS-ELISA）was developed for the detection of PEDV in pigs. The optimal coating concentration of anti-PEDV IgY was 1：4000（12.35μg/mL）;the optimal concentration of mouse-anti-PEDV polyclonal antibody was 1：6000（10.25μg/mL）;optimal reaction time was 30 minutes;optimal working concentration of HRP-labelled goat-anti-mouse IgG was 1：6000;and the positive standard value was OD450≥0.130. The DAS-ELISA showed no cross-reaction with porcine transmissible gastroenteritis virus, porcine rotavirus,Escherichia coli k88/k99/987P/F41. One hundred clinical fecal samples which had been detected by real-time PCR were analyzed by this method. 6 out of 8 real-time PCR positive samples were positive in the DASELISA,and all the negative samples in real-time PCR were negative in the DAS-ELISA. The results indicated that the DAS-ELISA was highly speciifc,reproducible and sensitive,and suitable for rapid detection of PEDV.