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猪流行性腹泻病毒双抗夹心ELISA的建立 被引量:3

Development of a Double Antiby Sandwich ELISA for Detection of Porcine Epidemic Diarrhea Virus
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摘要 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.
出处 《中国动物检疫》 CAS 2014年第12期65-69,共5页 China Animal Health Inspection
基金 深圳市科创委技术创新计划专项基金资助项目 No.CXZZ20130320154210829
关键词 猪流行性腹泻病毒 卵黄抗体 双抗夹心ELISA porcine epidemic diarrhea virus (PEDV) IgY double antibody sandwich ELlS
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