A型口蹄疫抗体固相竞争ELISA检测方法的建立  

A study of solid phase competition ELISA for detection of A-type foot-and-mouth disease virus antibody

作　　者：李敏杰 许智强 刘彦玲 周国辉[1] 于力[1] LI Min-jie;XU Zhi-qiang;LIU Yan-ling;ZHOU Guo-hui;YU Li(Harbin Veterinary Research Institute,Livestock Infectious Disease Division,Chinese Academy of Agricultural Science,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所牛羊病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第9期899-904,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0501505)。

摘　　要：为评价A型口蹄疫疫苗免疫水平,本研究以本实验室前期制备的口蹄疫病毒(FMDV)的单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 9A9作为检测抗体,建立了基于MAb的检测A型FMDV抗体的固相竞争ELISA(SPCE)方法,并对其条件进行优化。结果显示,MAb 3D9的包被浓度为1.16μg/mL,A型FMDV抗原的最佳稀释度为1:5,HRP标记的MAb 9A9的最佳稀释度为1:5000,当血清1:32稀释时,检测的临界值确定为35%。利用该方法分别检测A型、O型口蹄疫抗体阳性标准血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清。结果显示,除A型口蹄疫阳性标准血清为阳性结果外其余均为阴性结果,未出现交叉反应,表明本研究建立的方法特异性强。该方法对经病毒中和试验(VNT)检测为阳性的3份血清进行敏感性试验,敏感性分别为1:1024、1:256、1:128,均高于VNT,表明其敏感性较高;重复性试验结果显示,该方法批内和批间重复试验的变异系数均小于10%,表明其重复性较好。利用该方法与液相阻断ELISA方法(LPBE)和VNT对112份血清样品同时检测,分析三者相关性。结果显示,本实验建立的SPCE方法与二者的相关系数分别为0.901和0.916,表明该方法可靠性较好且与VNT的相关性更高。进一步利用本研究建立的SPCE方法和韩国Jeno A型FMDV ELISA抗体检测试剂盒同时检测470份临床血清样品(90份羊血清、170份牛血清和210份猪血清),结果显示该方法检测羊血清、牛血清和猪血清与Jeno试剂盒总体符合率分别为90.0%、91.8%和89.0%。表明该方法的检测结果较为准确。本研究为建立A型口蹄疫检测方法奠定了基础。In order to evaluate the immune levels of type A FMD vaccine,the monoclonal antibody(MAb)3D9 of FMDV prepared in our lab was used as the capture antibody,and the HRP-labeled MAb 9A9 was used as the detection antibody.A solidphase competition ELISA method(SPCE)for the detection of antibody against FMDV serotype A based on monoclonal antibodies was developed and its conditions were optimized.The results showed that the optimal concentration of MAb 3D9 was 1.16μg/mL,the optimal dilution of A-type FMDV antigen was 1:5,and the optimal dilution of HRP-labeled MAb 9A9 was 1:5000.When the serum diluted at 1:32,the critical value of the assay was determined to be 35%.The method was used to detect positive serum of type A FMDV,type O FMDV,BCV,BRV,PRRSV,PCV and CSFV,respectively.The results showed that the solid phase competition ELISA method could specifically detect type A FMDV,but had no cross-reaction with other viruses.It showed that the method established in this study has strong specificity.The sensitivity test was performed on 3 positive sera samples by VNT(virus neutralization test).The sensitivity is 1:1024,1:256,1:128,respectively,indicating the method has high sensitivity.Repeatability results showed the coefficient of variation of the intra-assay and inter-assay repeated tests were less than 10%,indicating that the repeatability is good.This method was used to detect 112 serum samples along with the liquid-phase blocking ELISA method(LPBE)and VNT and analyze the correlation.The results showed the correlation coefficient between the method established in this study and LPBE and VNT was 0.901 and 0.916,respectively.It indicated that this method had a good reliability and has a higher correlation with VNT.Using the SPCE and Korean Jeno A FMDV ELISA antibody detection diagnosis kitto detect 470 clinical samples(90 sheep serum samples,170 cow serum samples and 210 pig serum samples).The results showed the overall coincidence rate between this method and Jeno kit is 90.3%,91.8%and 89.0%,respectively,indicating that

关 键 词：口蹄疫病毒 单克隆抗体 固相竞争ELISA 

分 类 号：S852.65[农业科学—基础兽医学]