猪流行性腹泻病毒间接ELISA抗体检测方法建立与应用

Development and application of an indirect ELISA method for detection of antibody to porcine epidemic diarrhea virus

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是目前引起我国猪急性肠炎并水样腹泻的重要病原之一,但尚无商品化病毒血清抗体检测试剂盒。本研究以纯化的PEDV为包被抗原,通过优化ELISA反应条件,建立了间接ELISA抗体检测方法,其反应条件为:抗原最佳包被浓度为3μg/mL,血清样品最佳稀释度为1∶100,包被时间为37℃作用2 h,1.5%BSA 37℃封闭3 h,二抗1∶10 000稀释,37℃作用30 min,抗体临界值为OD450nm≥0.306判为阳性,OD450nm≤0.268判为阴性,介于二者之间为可疑。该方法检测6份已知PEDV阳性血清效价为1∶3 200,检测猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、伪狂犬病毒和口蹄疫病毒血清抗体均为阴性,批间和批内重复试验变异系数2.5%～8.3%,对江苏、上海、浙江、安徽地区587份猪血清样品进行检测,抗体阳性率达56.76%,证明该方法具有较好敏感性、特异性和重复性,可用于PEDV抗体检测和流行病学调查。

Porcine epidemic diarrhea virus （PEDV） is one of the most important pathogens causing severe enteritis and watery diarrhea. There is no effective antibody test kit in China. In this paper, an indirect ELISA was successfully developed to detect anti-PEDV using the purified PEDV as coating antigen. The optimized reaction conditions were as follows： antigen working concentration was 3 μg/mL. Serum sample dilution was 1 ： 100. It was coated at 37 ℃ for 2h. The plates were blocked by 1.5% BSA incubated at 37 ℃ for 3h. The secondary antibody was diluted at 1 ： 10000, incubated at 37 ℃ for 0. 5h. It was judged as positive when the cutoff value OD450nm ≥0. 306, as negative when OD450nm ≤0. 268, and as suspicious between 0. 268 and 0. 306. The difference value among wells in a plate and among plates for ELISA was 5.65 % and 5.81%. It could not react with the positive sear of other five viruses, such as porcine respiratory and reproductive syndrome virus, porcine circovirus 2, classical swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. And it had good inter-and intra-bateh reproducibility. A total of 587 clinical serum samples obtained from pig farms in Jiangsu Shanghai Zhejiang Anhui province were detected. The positive rate was 56. 76%. It indicated that this method could be used for PEDV epidemiological surveys and diagnosis in the future.