猪流行性腹泻病毒血清IgA抗体间接ELISA方法的建立  

Indirect ELISA for detecting specific IgA antibody in serum to porcine epidemic diarrhea virus

作　　者：孙立旦 陈立功[1] 周双海[2] 李艳琴[1] 郭海勇[3] 李潭清[1] 宋勤叶[1] SUN Li-dan;CHEN Li-gong;ZHOU Shuang-hai;LI Yan-qin;GUO Hai-yong;LI Tan-qing;SONG Qin-ye(Hebei Agricultural University , Baoding , Hebei 071001, China;College of Animal Science and Technology ^Beijing University of Agriculture ,Beijing 102206,China;College of Life Sciences,Jilin Normal University Siping, Jilin 136000 , China)

机构地区：[1]河北农业大学,河北保定071000 [2]北京农学院动物科学技术学院,北京102206 [3]吉林师范大学生命科学学院,吉林四平136000

出　　处：《中国兽医学报》2019年第3期381-386,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31372441);国家重点研发计划资助项目(2016YFD0500703);河北省科技计划资助项目(17226613D-2)

摘　　要：建立了检测猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)IgA抗体的间接ELISA方法,旨在为PEDV感染检测与免疫效果评价提供技术手段。以重组PEDV结构蛋白(S1蛋白)作为包被抗原,采用方阵滴定法确定抗原包被浓度、封闭液、稀释液以及待测血清和酶标抗体的最佳工作浓度,建立了PEDV IgA抗体检测间接ELISA方法。进而检测方法的特异性、批内与批间重复性,评价建立方法与国外试剂盒的符合率,分析ELISA检测血清特异性IgA抗体水平与中和抗体的相关性。结果显示,ELISA方法的最佳抗原包被浓度为1 mg/L,封闭液和抗体稀释液为含5%犊牛血清的PBS,待检血清和酶标抗体的工作浓度分别为1∶40与1∶1 500倍稀释。结果表明,该ELISA能够特异地检测PEDV抗体,与猪繁殖与呼吸综合征病毒、猪瘟病毒、伪狂犬病毒、猪圆环病毒2型等病毒的抗血清无交叉反应,批内和批间重复性试验的变异系数低于10%,与现有试剂盒的总符合率为94.8%,血清特异性IgA抗体水平与中和抗体呈正相关(r=0.69,P<0.001)。The object of this study is to establish an indirect ELISA method for detecting IgA antibody against porcine epidemic diarrhea virus(PEDV) and provide a technique for PEDV infection detection and immune effect evaluation.Antigen coating concentration,blocking and dilution buffer,optimal dilutions of test serum and enzyme-labeled antibody were determined and the indirect ELISA method for detecting PEDV-IgA antibody was developed.Furthermore,the specificity,intra-and inter-batch repeat ability tests of the ELISA were examined,the coincidence rate was measured between the ELISA and the existing ELISA kit,and the correlation between the specific IgA and the neutralizing antibodies in serum was analyzed.The results showed that the optimal antigen coating concentration of the ELISA was 1 mg/L,the blocking and dilution buffers were PBS containing 5% calf serum and 0.05% Tween-20,and the working concentrations of tested serum and enzyme-labeled antibody were 1∶40 and 1∶1500,respectively.The method could be used to detect the PEDV specific antibodies without cross reactions with those antibodies against porcine reproductive and respiratory syndrome virus,classical swine fever virus,pseudorabies virus,and porcine circovirus type 2 using the ELISA.The coefficients of variation(CV) of intra-and interbatch repetitive tests of the ELISA were less than 10%.The coincidence rate was 94.8% when the same serum samples were detected using the ELISA and existing ELISA kit for detecting PEDV antibody.The level of specific IgA antibody was positively correlated with that of the neutralizing antibody in serum(r=0.69,P<0.001).

关 键 词：猪流行性腹泻病毒 S1蛋白 间接ELISA IGA 中和抗体 相关性 

分 类 号：S852.65[农业科学—基础兽医学]