牛病毒性腹泻病毒E^(rns)-ELISA抗体检测试剂盒的制备  

Preparation of E~(rns)-ELISA Antibody Test Kit for Bovine Viral Diarrhea Virus

作　　者：王静[1] 陈柯源 王胜华 丁成志 杨光辉 刘一 尹金花 王九峰[1] WANG Jing;CHEN Keyuan;WANG Shenghua;DING Chengzhi;YANG Guanghui;LIU Yi;YIN Jinhua;WANG Jiufeng(College of Veterinary Medicine,China Agricultural University,Beijing 100193,China;China Animal Disease Control Center,Beijing 102618;College of Animal Science and Technology,Tarim University,Alar 843300,China)

机构地区：[1]中国农业大学动物医学院,北京海淀100193 [2]中国动物疾病预防控制中心,北京大兴102618 [3]塔里木大学动物科学与技术学院,新疆阿拉尔843300

出　　处：《中国兽医杂志》2024年第7期65-70,共6页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划(2023YFD1801100);国家自然科学基金(31873034,31960721)。

摘　　要：为建立快速检测牛病毒性腹泻病毒(BVDV)的血清学方法,本试验以BVDV NADL毒株结构蛋白E^(rns)作为包被抗原,优化反应条件并组装BVDV酶联免疫吸附试验(ELISA)抗体检测试剂盒,通过重复性试验、灵敏性试验、符合率试验和保存期试验验证该试剂盒的准确性、灵敏性和稳定性。结果显示,抗原包被浓度为4μg/mL,1%酪蛋白溶液37℃封闭45 min,被检血清以1∶400稀释孵育30 min,兔抗牛HRP标记二抗以1∶20000稀释孵育30 min,TMB底物反应5 min时,ELISA的反应背景下降,区分度最好。优化后方法的批内重复性试验和批间重复性试验的变异系数均在10%以内,表明该试剂盒具有较好的稳定性;灵敏性试验结果显示,当阳性血清稀释度达到1∶6400时仍可以检测为阳性,说明该试剂盒灵敏度较高;该试剂盒与美国爱德士生物科技公司(IDEXX)BVDV总抗体检测试剂盒共同检测466份临床血清样品,两者总符合率为89.1%;保存期试验结果显示,第4个月时该试剂盒仍能检测到阳性血清,说明稳定性较好。综上表明,本试验利用E^(rns)蛋白建立的BVDV间接ELISA抗体检测试剂盒灵敏度高且重复性好,可为BVDV抗体检测和流行性调查提供新的方法。In order to establish a serological method for rapid detection of bovine viral diarrhea virus(BVDV),this study utilized the BVDV NADL strain structural protein E^(rns)as the coating antigen.The reaction conditions were optimized to assemble an E^(rns)-based enzyme-linked immunosorbent assay(ELISA)antibody test kit for BVDV.The accuracy,sensitivity,and stability of the test kit were validated through repeatability tests,sensitivity tests,conformity tests,and storage tests.The results showed that the optimal antigen coating concentration was 4μg/mL,and sealing with 1%casein solution at 37℃for 45 minutes yielded the best results.Serum samples diluted at 1∶400 and incubated for 30 minutes,followed by incubation with rabbit anti-bovine HRP-conjugated secondary antibody at 1∶20000 for 30 minutes,showed reduced background and optimal differentiation during the TMB substrate reaction for 5 minutes.Intra-assay and inter-assay coefficients of variation were both within 10%,indicating good stability of the test kit after optimization.Sensitivity tests demonstrated detection of positive results even at a serum dilution of 1∶6400,indicating high sensitivity.Comparative testing with the IDEXX BVDN total antibody test kit in 466 clinical serum samples showed an overall agreement rate of 89.1%.Storage tests revealed detectable positive serum samples even after four months,indicating good stability.In conclusion,the Ernsprotein-based indirect ELISA antibody test kit developed in this study exhibits high sensitivity and good repeatability,providing a new method for BVDV antibody detection and epidemiological surveys.

关 键 词：牛病毒性腹泻病毒 E^(rns)蛋白 抗体 间接ELISA 

分 类 号：S855.3[农业科学—临床兽医学]