检测猪流行性腹泻病毒S1蛋白抗体的间接ELISA方法  

Indirect ELISA for Detecting Antibody to Porcine Epidemic Diarrhea Virus S1 Protein

作　　者：姚作俊 郝达仁 白云[1] 颜国华[2] 王海敏[1] 宋勤叶[1] 李潭清[1] 

机构地区：[1]河北农业大学动物医学院河北省兽用生物制品工程技术研究中心,保定071000 [2]河北省畜牧兽医研究所,保定071000

出　　处：《畜牧兽医学报》2017年第6期1085-1091,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划(2016YFD0500703);国家自然科学基金(31372441)

摘　　要：为了建立检测猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)抗体的间接ELISA方法,初步分析PED灭活疫苗免疫母猪所产仔猪的血清母源特异抗体动态,笔者应用重组PEDV部分纤突(spike,S)蛋白作为检测抗原,建立了基于PEDV S1蛋白的间接ELISA抗体检测方法,并用该方法检测了产前4周用PED灭活疫苗免疫母猪的初乳、分娩当天血清及其仔猪1、7、14、21、28和35日龄(d)时血清中的PEDV抗体。结果显示:建立的间接ELISA方法能够特异地检测PEDV抗体,与猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪传染性胃肠炎病毒及猪轮状病毒的抗血清无交叉反应;敏感性高于免疫过氧化物酶单层试验(immunoperoxidase monolayer assay,IPMA),与IPMA检测结果的符合率为97.14%;批内和批间重复性试验的变异系数小于10%。免疫母猪初乳、分娩当天血清中的PEDV抗体阳性率为100%,初乳比血清中的平均抗体水平低;1d仔猪血清PEDV抗体阳性率为50%,平均抗体水平低,与初乳中的抗体水平接近,7d以后所有仔猪血清全部转为PEDV抗体阴性。建立了检测PEDV抗体的间接ELISA方法;产前用PEDV灭活疫苗给怀孕母猪免疫1次时,其所产仔猪血清中的母源特异抗体阳性率和抗体水平低,且维持时间短暂。The present study aimed to develop an indirect ELISA for detecting antibody to porcine epidemic diarrhea virus （PEDV） spike （S） protein and analyze the dynamic of maternal special antibodies in the serum from neonatal piglets born by sows immunized with PEDV inactivated vaccine. An indirect ELISA was established using recombinant partial PEDV spike （$1） protein as detecting antigen. And anti-PEDV antibodies in the immunized sows＇ colostrum, sera from sows at the delivery day and from the neonatal piglets at 1, 7, 14, 21, 28 and 35 clays were detec- ted with the established ELISA. Results suggested that the ELISA method could be used to de- tect the special antibodies against PEDV without cross reactions with those antibodies to classical swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus,pseudorabies virus, porcine transmissible gastroenteritis virus, and porcine rotavirus. The sensi- tivity of ELISA was higher than that of the immunoperoxidase monolayer assay （IPMA） and the coineidence rate was 97.14% when the same serum samples were detected using the ELISA and IPMA. The coefficients of variation （CV） of repeated in-batch and batch-to-batch tests of ELISA were less than 10%. Positive rate of anti-PEDV antibody in the colostrum and serum of all immu- nized sows was 100% with a higher average antibody level at the delivery day in the serum than that in the colostrum, and the specific antibody positive rate of one-day old neonatal piglets was 50% with a lower average antibody level in serum similar to that in the eolostrum. And anti- PEDV antibodies in all of the piglet serum turned negative after day 7. The present study shows that PEDV antibodies could be detected using the established ELISA. The low positive rate and levels of maternal special antibodies are present in the serum of neonatal piglets born by the sows immunized once with PEDV inactivated vaccine before farrowing.

关 键 词：猪流行性腹泻病毒 重组PEDV S1蛋白 间接ELISA 初生仔猪 母源抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]