猪初乳中PEDV IgA抗体捕获ELISA检测方法的建立及其初步应用  

Establishment and application of capture ELISA method for PEDV IgA detection in porcine colostrum

作　　者：杨利[1,2] 张浩明 于晓明[1,2] 侯立婷 郑其升[1,2] YANG Li;ZHANG Hao-ming;YU Xiao-ming;HOU Li-ting;ZHENG Qi-sheng(Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences/National Research Center of Engineering and Technology for Veterinary Biologicals/Jiangsu Key Laboratory for Food Quality and Safety,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所/国家兽用生物制品工程技术研究中心/江苏省食品质量与安全重点实验室,江苏南京210014 [2]兽用生物制品(泰州)国泰技术创新中心,江苏泰州225300

出　　处：《中国预防兽医学报》2023年第8期822-828,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：十四五重点研发专项(2022YFD1800800);江苏省农业自主创新资金[CX(21)3135];江苏现代农业产业化单项技术研发[CX(20)3096]。

摘　　要：为建立评价猪流行性腹泻病毒(PEDV)疫苗免疫效力的方法,本研究利用纯化的PEDV免疫BALB/c小鼠,按照常规方法制备PEDV全病毒单克隆抗体(MAb)作为捕获抗体,PEDV细胞培养上清为夹心抗原,HRP标记的羊抗猪IgA为二抗,采用棋盘滴定法对各反应条件优化后建立了用于猪初乳中PEDV IgA检测的捕获ELISA方法,并确定其临界值为0.25。采用该捕获ELISA对PEDV、PCV2b、PRRSV、PRV、JEV、PPV、FMDV与CSFV IgA抗体阳性猪初乳进行检测,结果显示,除PEDV IgA抗体阳性猪初乳检测结果为阳性外,其他病毒抗体阳性猪初乳检测结果均为阴性,表明该方法特异性强。采用该方法和BIONOTE PEDV IgA试剂盒对从1∶10开始2倍倍比稀释的16个稀释度的阳性猪初乳样品检测,结果显示该方法对阳性样品检测的最大稀释度为1∶40960,是BIONOTE PEDV IgA试剂盒敏感性的16倍(1∶2560),表明本研究建立方法的敏感性高。分别采用同一批次和不同批次制备的MAb包被的ELISA板分别检测5份PEDV IgA抗体阳性和1份PEDV IgA抗体阴性猪初乳,评估该方法的重复性。结果显示,批内和批间重复性试验的变异系数分别为0.34%~4.49%和1.90%~8.71%,均小于10%,重复性好。利用该捕获ELSIA和IDEXX PEDV IgA抗体试剂盒分别对50份猪初乳检测,结果显示两种方法对样品检测的OD450nm值趋势高度一致,表明该方法检测结果与IDEXX试剂盒相关性强。利用该捕获ELISA和BIONOTE PEDV IgA试剂盒对100份临床采集的猪初乳样品检测,结果显示二者总体符合率为97.0%。利用该方法对90份免疫背景不同的猪初乳检测,结果显示,未感染PEDV且未免疫PED疫苗的母猪初乳检测结果均为阴性结果,感染了PEDV或接种过PED疫苗的母猪初乳均为阳性结果,检测结果与样品的免疫背景相符。本研究建立的捕获ELISA方法特异性强、敏感性高、重复性好,与同类进口产品检测结果符合率高,对临床样品的检测结果与其�To evaluate the efficacy of PEDV vaccine,a capture ELISA for PEDV IgA detection in colostrum was established by using anti-PEDV monoclonal antibody(MAb)as coating antibody,cell cultures of PEDV as a capture antigen and a goat anti-porcine IgA as secondary antibody.Subsequently,the sensitivity,specificity,repeatability and coincidence rate of the capture ELISA were determined.The porcine colostrum with IgA anti-PEDV,PCV2b,PRRSV,PRV,JEV,PPV,FMDV and CSFV was detected by the capture ELISA,and the results showed that only the colostrum with IgA anti-PEDV was positive,while the colostrum with IgA anti-other viruses were negative,indicating good specificity of this method.Besides,capture ELISA and BIONOTE PEDV IgA kit were used to detect positive samples with a 2-fold ratio dilution,respectively.The results showed that the maximum dilution of samples detected positive by this method was 1∶40960,which was 16 times that of BIONOTE PEDV IgA kit(1∶2560),showing that the method established in this study was highly sensitive.The intra-batch and inter-batch coefficients of variation of this method were 0.34%-4.49%and 1.90%-8.71%,respectively,illustrating good repeatability of this method.Capture ELISA and BIONOTE PEDV IgA kit were used to detect 100 samples of porcine colostrum,and the compliance rate of the two methods was 97.0%.The detection results of 50 porcine colostrum samples by capture ELSIA and IDEXX PEDV IgA kit showed that the OD450nm value trend for the same group of samples detected by the two methods were highly consistent,indicating that the results detected by this method correlated well with the IDEXX.The method was used to detect the 90 porcine colostrum samples with different immune background,and the results showed that the colostrum of sows not infected with PEDV and not vaccinated against PEDV was negative,while that of sows infected with PEDV or vaccinated against PEDV was positive,demonstrating that the test results were consistent with the immunological background of samples.In summary,the capture

关 键 词：猪流行性腹泻病毒 IgA检测 捕获ELISA 猪初乳 

分 类 号：S852.65[农业科学—基础兽医学]