基于IPV S蛋白单抗的竞争ELISA方法建立及应用

Establishment and application of competitive ELISA method for anti-FIPV S protein monoclonal antibody

关键词:  猫传染性腹膜炎病毒  竞争ELISA  单克隆抗体

Key words:  Feline Infectious Peritonitis Virus  Competitive ELISA  Monoclonal Antibody

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10 μg/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1h，5% BSA在37℃封闭2h，FIPV MAb 3D8在37℃孵育1h，TMD底物显色液37℃避光5min。临界值为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。利用其和已发表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%，表明准确性较高。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，为其鉴定防控和流行病学调查提供支持。

Abstract:

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 10 μg/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.