猪流行性腹泻病毒N蛋白阻断ELISA抗体检测方法的建立及初步应用  

Development and Application of N-protein Blocking ELISA for Detecting Porcine Epidemic Diarrhea Virus Antibodies

作　　者：万颖 周改静 麻园 石正旺 肖书奇 罗俊聪 宋锐 曹丽艳[1] 杨波[1] 王丽娟 田宏[1] 郑海学[1] WAN Ying;ZHOU Gaijing;MA Yuan;SHI Zhengwang;XIAO Shuqi;LUO Juncong;SONG Rui;CAO Liyan;YANG bo;WANG Lijuan;TIAN Hong;ZHENG Haixue(National Foot-and-Mouth Disease Reference Laboratory, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute of Chinese Academy of Agriculture Sciences, Lanzhou 730046, China;College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China)

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室,兰州730046 [2]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2022年第4期1173-1181,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：中国农业科学院科技创新工程(CAAS-ASTIP-2020-LYRI)。

摘　　要：旨在建立一种猪流行性腹泻病毒(PEDV)N蛋白阻断ELISA抗体检测方法。本研究将纯化的N蛋白作为包被抗原,通过棋盘滴定法优化ELISA反应条件,建立了检测PEDV抗体的阻断ELISA方法,并对其进行特异性、敏感性和重复性试验。对140份临床血清样品进行检测,并将检测结果与市售IDvet PEDV间接ELISA抗体检测试剂盒试剂盒进行比较。结果显示,建立的阻断ELISA方法的抗原最佳包被浓度为625 ng·mL^(-1),血清最佳稀释比例为1∶1;酶标抗体的最佳工作浓度为1∶5000;检测健康猪血清以及猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)和传染性胃肠炎病毒(TGEV)等常见猪病病原的阳性血清,均无交叉反应;PEDV阳性血清的灵敏度为1∶16,与市售IDvet PEDV间接ELISA抗体检测试剂盒(效价1∶32)的敏感性相当;批内、批间重复性试验的变异系数均小于10%,说明具有良好的重复性和稳定性;对140份临床血清样品的检测并与商品化试剂盒检测结果进行比较,两者的kappa值为0.87,为高度一致。综上表明,本研究建立的阻断ELISA抗体检测方法可以应用于PEDV的防控、流行病学调查以及疫苗免疫后抗体水平的监测。The purpose of this study is to establish a blocking ELISA antibodies detection method for porcine epidemic diarrhea virus(PEDV).The purified N protein was used as the coating antigen,and the ELISA reaction conditions were optimized by the chess rboard titration.A blocking ELISA method for detecting PEDV antibodies was established,and its specificity,sensitivity and repeatability tests were carried out.One hundred and forty clinical serum samples were tested,and the results were compared with commercially IDvet PEDV indirect ELISA antibodies detection kit.The results showed that the best antigen coating concentration was 625 ng·mL^(-1),and the best dilution ratio of serum was 1:1;The best dilution of the HRP-conjugated antibody working solution was 1:5000;There was no cross-reaction with healthy pig serum and the positive sera of common pig disease pathogens,such as classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV2),and transmissible gastroenteritis virus(TGEV).The sensitivity of PEDV positive serum was 1:16,which was equivalent to that of IDvet ELISA kit(titer 1∶32).The coefficient of variation of within-run and between-run repeatability test is less than 10%,so it showed that the blocking ELISA established in this study had good repeatability and stability;the kappa value of detected 140 clinical porcine serum using this method was 0.87 when compared with IDvet ELISA.The above results indicated that the established blocking ELISA method for detecting PEDV antibodies in this study could be applied to the prevention and control of PEDV,epidemiological investigation and the monitoring of antibody levels after vaccine immunization.

关 键 词：猪流行性腹泻病毒 N蛋白 抗体检测 阻断ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]