中国兽医杂志2024，Vol.60Issue(7)：65-70,6.DOI:10.20157/j.cnki.zgsyzz.2024.07.009
牛病毒性腹泻病毒Erns-ELISA抗体检测试剂盒的制备
Preparation of Erns-ELISA Antibody Test Kit for Bovine Viral Diarrhea Virus
摘要
Abstract
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 Erns as the coating antigen.The reaction conditions were optimized to assemble an Erns-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 4 μg/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∶20 000 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∶6 400,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 Erns protein-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/Erns蛋白/抗体/间接ELISAKey words
bovine viral diarrhea viru/Erns protein/antibody/indirect ELISA分类
农业科技引用本文复制引用
王静,陈柯源,王胜华,丁成志,杨光辉,刘一,尹金花,王九峰..牛病毒性腹泻病毒Erns-ELISA抗体检测试剂盒的制备[J].中国兽医杂志,2024,60(7):65-70,6.基金项目
国家重点研发计划(2023YFD1801100) （2023YFD1801100）
国家自然科学基金(31873034,31960721) （31873034,31960721）
