赤羽病病毒N蛋白阻断ELISA抗体检测方法的建立及应用  

Development of a Blocking ELISA for Detecting the Antibodies Against Akabane Virus N Protein

作　　者：陈冬杰 魏方 李昊轩 王晶晶 许晓琳 吴绍强[1,2] CHEN Dongjie;WEI Fang;LI Haoxuan;WANG Jingjing;XU Xiaolin;WU Shaoqiang(Institute of Animal Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Technology Innovation Center of animal and plant product quality,safety and control,State Administration for Market Regulation,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院动物检验与检疫研究所,北京100176 [2]国家市场监督管理总局技术创新中心(动植物产品质量与安全),北京100176

出　　处：《中国动物传染病学报》2024年第6期57-62,共6页Chinese Journal of Animal Infectious Diseases

基　　金：基本科研业务费(2024JK003);国家重点研发计划项目(2022YFD1802000)。

摘　　要：本研究旨在建立一种赤羽病病毒(AKAV)N蛋白阻断ELISA抗体检测方法。以纯化的AKAV N重组蛋白作为包被抗原,HRP标记的单克隆抗体(mAb)1C5作为阻断抗体,通过对反应条件进行优化,建立了基于AKAV N蛋白的AKAV阻断ELISA抗体检测方法。用建立的阻断ELISA方法检测67份临床阴性血清,计算血清阻断率PI并确定了临界值及判定标准:当PI>53.1%时判定为AKAV抗体阳性;PI<46.8%时判定为AKAV抗体阴性;当PI介于两者之间时,判定为可疑。该方法与牛病毒性腹泻病毒、牛结节性皮肤病病毒和蓝舌病毒的抗体阳性血清无交叉反应。用该方法与商品化ID screen AKAV抗体检测试剂盒同时检测79份牛临床血清样品,两种方法的符合率为91.1%。本实验室建立的AKAV阻断ELISA抗体检测方法可用于国内临床中AKAV血清抗体的检测,对AKAV的临床诊断具有非常重要的参考意义。To develop a blocking ELISA method for detecting antibodies of Akabane virus(AKAV)N protein,the purified recombinant N protein was used as the coating antigen and HRP-labeled monoclonal antibody(mAb)1C5 was used as the blocking antibody.After optimization,a blocking ELISA assay based on AKAV N protein was developed for detecting AKAV antibodies.The optimal blocking ELISA was used to test 67 negative serum samples and the blocking rate(PI)was calculated to determine the cut-off value and criterion.A serum sample tested was defined as AKAV antibody positive at PI>53.1%,negative at PI<46.8%and suspicious at PI 46.8%-53.1%.The blocking ELISA had no cross reaction with Bovine viral diarrhea virus(BVDV),Lumpy skin disease virus(LSDV)and Bluetongue virus(BTV),indicating its specificity.In addition,the 79 clinical serum samples were tested using this blocking ELISA and a commercial ID screen AKAV antibody detection kit and their coincidence was 91.1%.The results showed that the blocking ELISA developed here could be used for the detection of AKAV serum antibodies in domestic clinical practice and had very important reference significance for diagnosis of AKAV.

关 键 词：赤羽病病毒 单克隆抗体 阻断ELISA 蛋白表达 

分 类 号：S852.65[农业科学—基础兽医学]