基于FIPV S蛋白单抗的竞争ELISA方法建立及应用  

Establishment and Application of Competitive ELISA Method based on the Anti-FIPV S Protein Monoclonal Antibody

作　　者：刘妍 杜吉革[1] 朱真 王诗研[1] 张石豪 王团结 焦文杰[1] 印春生 孙卫东[2] LIU Yan;DU Ji-ge;ZHU Zhen;WANG Shi-yan;ZHANG Shi-hao;WANG Tuan-jie;JIAO Wen-jie;YIN Chun-sheng;SUN Wei-dong(China Institute of Veterinary Drug Control,Beijing 102629,China;Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]中国兽医药品监察所(农业农村部兽药评审中心),北京100081 [2]南京农业大学,南京210095

出　　处：《中国兽药杂志》2024年第9期7-14,共8页Chinese Journal of Veterinary Drug

基　　金：“十四五”国家重点研发计划项目(2022YFD1800603)。

摘　　要：为给猫传染性腹膜炎病毒(FIPV)的流行和地理分布提供有价值的检测手段,以FIPV S蛋白为包被抗原,利用FIPV的特异性单克隆抗体(FIPV MAb 3D8)为竞争抗体,经条件优化,建立一种快速检测FIPV抗体的竞争ELISA法。优化结果表明最佳条件为:包被抗原质量浓度10μg/mL,血清稀释度1∶4;FIPV MAb 3D8稀释倍数1∶100,酶标二抗稀释倍数1∶5000;CBS在37℃包被1 h,5%BSA在37℃封闭2 h,FIPV MAb 3D8在37℃孵育1 h,TMD底物显色液37℃避光5 min。临界值PI≥25.76%时为阳性,PI<20.22%为阴性。该方法特异性较强,重复性较好。利用该方法和已发表的其他方法同时对来自宠物医院的62份临床血清样本进行检测,总体符合率为85.5%,表明该方法准确性较高。本研究建立了快速准确测定FIPV抗体的竞争ELISA方法,适用于临床样本检测,为猫传染性腹膜炎的鉴定、防控和流行病学调查提供支持。In order to provide a valuable detection method for the prevalence and geographical distribution of feline infectious peritonitis virus(FIPV),a competitive ELISA for rapid detection of FIPV antibodies was established using the S protein of FIPV as the coating antigen and the specific monoclonal antibody of FIPV(FIPV MAb 3D8)as the competitive antibody after conditional optimization.The results showed that the optimum conditions were as follows:coating antigen concentration 10μg/mL,serum dilution 1∶4;FIPV MAb 3D8 dilution 1∶100,enzyme-labeled secondary antibody dilution 1∶5000;CBS coating at 37℃for 1 h,5%BSA blocking at 37℃for 2 h,FIPV MAb 3D8 incubation at 37℃for 1 h,TMD substrate chromogen at 37℃for 5 min in the dark.A cut-off value of PI≥25.76%was considered positive and PI<20.22%was considered negative.This method is specific and reproducible.Sixty-two clinical serum samples from pet hospitals were tested simultaneously using it and other published methods,and the overall coincidence rate was 85.5%,indicating high accuracy.A competitive ELISA method for rapid and accurate determination of FIPV antibodies was established,which is suitable for the detection of clinical samples and provides support for its identification,prevention and control and epidemiological investigation.

关 键 词：猫传染性腹膜炎病毒 竞争ELISA 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]