中国兽医科学2025，Vol.55Issue(8)：1026-1032,7.DOI:10.16656/j.issn.1673-4696.2025.0152
产气荚膜梭菌β2毒素抗体竞争ELISA检测方法的建立
Establishment of competitive ELISA for detection of Clostridium perfringens β2 toxin antibody
摘要
Abstract
In order to detect the antibody to Clostridium perfringens(Cp)β2 toxin,the Cpβ2 toxin gene was synthesized and cloned into pET-32a vector,and the recombinant Cp β2 toxin protein was obtained after induced expression.The purified Cp β2 toxin recombinant protein was used as the coating antigen,Cp β2 toxin monoclonal antibody 2A4 is the primary competitive antibody with sheep serum in competitive reaction,HRP-Sheep anti-mouse IgG is the secondary antibody.A competitive ELISA procedure was developed and refined for the identification of antibodies targeting the Cpβ2 toxin.The results of the study revealed that the best reaction conditions were achieved at a protein coating dosage of 1.5 μg/mL,the serum to be tested was not diluted,1∶20 000 dilution of secondary antibody and 15 min of color development away from light.The negative critical value(PI)is 31.44％,and the positive critical value(PI)is 37.65％,the sensitivity of positive serum detection was 1∶64,and the coefficient of variation of inter-batch and intra-batch tests was less than 10％.The positive sera against α,β1,epsilon of Cp,Clostridium septicum and Escherichia coli were all nonspecific.The positive coincidence rate between the cELISA and iELISA was 85.7％,the negative coincidence rate was 95.9％and the total coincidence rate was 92％.The coin-cidence rate of 100 samples of bovine serum was 100％.The results showed that the proposed method has good repeatability,specificity and sensitivity.It can be used to detect the presence of antibodies against Cp β2 toxin in animals,providing an effective means to assess whether animals are infected with Cp,and has a wide application prospect in the epidemiological investigation of Cp and the evaluation of vaccine immune effect.关键词
产气荚膜梭菌/β2重组蛋白/单克隆抗体/竞争ELISA/抗体检测Key words
Clostridium perfringens/β2 recombinant protein/monoclonal antibody/competitive ELISA/antibody detection分类
农业科技引用本文复制引用
赵佳慧,马祎芳,李学瑞,高鹏程,狄娜,郑福英,储岳峰..产气荚膜梭菌β2毒素抗体竞争ELISA检测方法的建立[J].中国兽医科学,2025,55(8):1026-1032,7.基金项目
国家重点研发子课题:羊梭菌三联四防基因工程亚单位疫苗研发(2023YFD1802504-04) （2023YFD1802504-04）
兰州市科技计划项目(2023-1-14) （2023-1-14）
中国农业科学院创新工程项目(CAAS-ASTIP-2021-LVRI) （CAAS-ASTIP-2021-LVRI）
