猪流行性腹泻病毒IgA抗体ELISA检测方法的建立和初步应用  

Establishment and preliminary application of an ELISA method for detection of porcine epidemic diarrhea virus IgA antibody

作　　者：王婉莹 石正旺 罗俊聪 廖焕程 冯露 郑海学[2] 田宏[2] 蔺国珍[1] WANG Wanying;SHI Zhengwang;LUO Juncong;LIAO Huancheng;FENG Lu;ZHENG Haixue;TIAN Hong;LIN Guozhen(College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China;State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]中国农业科学院兰州兽医研究所,兰州大学动物医学与生物安全学院,动物疫病防控全国重点实验室,甘肃兰州730000

出　　处：《中国兽医科学》2024年第10期1302-1308,共7页Chinese Veterinary Science

基　　金：甘肃省科技重大专项(22ZD6NA012,22ZD6NA001);甘肃省自然科学基金项目(21JR1RA203);中央高校基本科研业务费专项(lzujbky-2022-ct02);国家生猪技术创新中心先导科技项目(NCTIP-XD/C03)。

摘　　要：为检测猪流行性腹泻病毒(PEDV)Ig A抗体,以PEDV重组S蛋白作为包被抗原,辣根过氧化物酶标记的小鼠抗猪Ig A单克隆抗体为检测抗体,建立并优化了PEDV Ig A抗体的ELISA检测方法,并评估了其特异性、敏感性、重复性及临床样品检测性能。结果显示:该方法抗原最佳包被质量浓度为2μg/m L,酶标抗体的最佳工作浓度为1:25000,血清最佳稀释度为1:50;与猪伪狂犬病病毒、非洲猪瘟病毒、猪瘟病毒、塞内卡病毒、口蹄疫病毒阳性血清均无交叉反应;灵敏度为1:800;批内、批间变异系数均小于10%;与IDEXX商品化试剂盒的检测结果具有高度一致性。结果证明,本研究中建立的PEDV Ig A抗体检测方法特异性强、敏感性高、重复性好,适用于猪流行性腹泻的临床诊断与防控、流行病学调查以及免疫效果评估。To identify the IgA antibody against porcine epidemic diarrhea virus(PEDV),an ELISA technique was developed and refined using PEDV recombinant S protein as the coating antigen and horseradish peroxidase-conjugated murine anti-porcine IgA monoclonal antibody as the identifying antibody,and its specificity,sensitivity,reproducibility,and performance of clinical samples were evaluated.The findings indicated that the optimal antigen encapsulation mass concentration was 2μg/mL,the best enzyme-labeled antibody working concentration was 1:25000,and the most effective serum dilution was 1:50.There was no cross-reactivity with positive serums of porcine pseudorabies virus,African swine fever virus,classical swine fever virus,Senecavirus A and foot-and-mouth disease virus.The sensitivity was determined to be 1:800.The coefficients of variation were less than 10%within and among different batches,and the consistency rate with IDEXX commercial kit was very high in testing result.These results prove that the detection method for PEDV IgA antibody developed in this research has good specificity,sensitivity and reproducibility,making it suitable for clinical diagnosis,disease prevention,and control of porcine epidemic diarrhea,as well as for epidemiological studies and evaluationof vaccinationefficacy.

关 键 词：猪流行性腹泻病毒 S蛋白 IGA抗体 ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]