乳汁中猪流行性腹泻IgA抗体间接ELISA方法的建立

Indirect ELISAs for Detecting IgA Antibodies Against Porcine Epidemic Diarrhea Virus in Pig's Milk

为建立检测母猪乳汁中猪流行性腹泻病毒(PEDV)IgA抗体的间接ELISA方法,研究以PEDV重组N蛋白和纯化全病毒作为包被抗原,方阵滴定法优化反应条件,建立了两种检测猪乳汁中PEDV IgA抗体的间接ELISA方法。结果显示,重组N蛋白和纯化全病毒的最佳包被抗原浓度均为2μg/mL,检测样品最佳检测稀释度分别为1∶10和1∶30,重复性试验的变异系数均小于10%,PEDV阳性乳汁的最低检测稀释倍数分别为1∶320和1∶960,对猪传染性胃肠炎病毒、猪伪狂犬病病毒等阳性乳汁均无交叉反应性,两种方法对临床样品检测符合率为97.48%。表明两种方法均具有良好的敏感性、特异性和重复性,可用于检测和评价乳汁中特异性PEDV IgA抗体的水平。

In order to establish indirect enzyme-linked immunosorbent assy(iELISA)for detecting specific IgA antibody level against porcine epidemic diarrhea virus(PEDV)in pig's milk,the two iELISAs were established with the purified PEDV and recombinant N protein as coating antigen.The results showed that the optimal coating antigen concentrations of N protein and purified PEDV were both 2μg/mL,the coefficients of variation of the intra-and inter-batch repetitive tests were all less than 10%,and the optimal dilutions of sample were 1∶10 and 1∶30,respectively.The minimum dilution ratio of PEDV positive sample were 1∶320 and 1∶960,respectively.The two iELISAs were no cross-reaction with those positive milks of transmissible gastroenteritis virus,porcine pseudorabies virus,and so on.The coincidence rate of the two methods was 97.48%.The results suggested that the two iELISAs were both specific,sensitive,and repeatable.They can be used to detect and evaluate the IgA antibody level against PEDV in pig's milk.