口蹄疫病毒抗体SA-ELISA快速检测方法的建立  

Establishment of a Rapid Detection SA-ELISA Method for Anti Foot-and-Mouth Disease Virus

作　　者：周广青 刘晓庆 史喜绢 杨大鹏 袁莉刚[1] 常惠芸[2] ZHOU Guangqing;LIU Xiaoqing;SHI Xijuan;YANG Dapeng;YUAN Ligang;CHANG Huiyun(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology,OIE/National Foot-and-Mouth Disease Reference Laboratory,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]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,兰州730046

出　　处：《畜牧兽医学报》2023年第5期2020-2029,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：甘肃省动物生殖生理及繁殖调控重点实验室建设项目(20JR10RA563)。

摘　　要：旨在解决如何对现有方法进行优化创新以建立口蹄疫抗体检测新方法的问题,本研究在基于原有液相阻断ELISA的基础上进行方法优化,实现口蹄疫病毒抗体的快速、灵敏检测。本研究通过对抗口蹄疫IgG抗体标记生物素技术与HRP标记链霉亲和素(HRP-SA)相结合建立新的液相阻断ELISA检测方法(SA-LPBE),在猪,牛、羊血清样品中具有较高的符合率。结合实际情况,判定优化后牛、羊血清抗体效价≥128,猪血清效价≥64时,判定为阳性,与商用检测试剂盒结果一致。并且经符合率分析,与原试剂盒具有92.3%的符合率,其批内变异系数<5%,批间变异系数<10%。该方法改变了原有的需要制备兔抗豚鼠抗血清再标记HRP技术路线,根本上提高了酶促反应的效率,保证了方法原有敏感性的同时提高了方法的特异性;利用生物素标记抗体与HRP-SA反应迅速、标记原料稳定、检测背景值等优点实现了将液相阻断ELISA试剂盒操作时间由2 d缩短为3 h,解决了原有方法操作繁琐、用时过长、操作疲劳、不稳定、易出错等问题。建立的SA-LPBE可应用于FMDV抗体检测,为FMD流行和临床检测提供了技术方法。In order to solve the problem of how to optimize and innovate the existing methods to establish a new method for FMD antibody detection,this study aimed to optimize the method based on liquid phase blocking ELISA to achieve rapid and sensitive detection of FMD virus antibody.In this study,a novel liquid phase blocking ELISA method(SA-LPBE)was established by combining the purification of IgG labeled biotin(dolphin anti-foot-and-mouth disease)from guinea pig serum with HRP labeled streptavidin(HRP-SA).SA-LPBE has a high consistency rate in bovine and sheep serum samples.Combined with the actual situation,the optimized serum antibody titer of cattle and sheep was determined to be≥128,and that of pigs serum sample was determined to be≥64.The results were determined to be positive,which was consistent with the results of commercial detection kits.And after compliance analysis,it has a compliance rate of 92.3%with the original kit,and the intra-batch variation coefficient is less than 5%,and the inter-batch variation coefficient is less than 10%.This method changed the original route of preparing rabbit anti-guinea pig antiserum and labeling HRP,improved the efficiency of enzyma-tic reaction,guaranteed the original sensitivity of the method and improved the specificity of the method;Using the advantages of quick reaction between biotin-labeled antibody and HRP-SA,stable labeling raw material and detection background value,the operation time of liquid phase blocking ELISA kit was shortened from 2 days to 3 hours,which solved the problems of tedious,long time,operation fatigue,instability and error-prone of the original operation.It can be applied to FMDV antibody detection,providing a technical method for FMD epidemic and clinical detection.

关 键 词：口蹄疫病毒 液相竞争ELISA ELISA 抗体检测 链霉亲和素 

分 类 号：S855.3[农业科学—临床兽医学]