中国兽医科学2026，Vol.56Issue(4)：496-503,8.DOI:10.16656/j.issn.1673-4696.2026.0043
产气荚膜梭菌ε毒素单克隆抗体的制备及其竞争ELISA抗体检测方法的建立
Preparation of monoclonal antibody against Clostridium perfringens ε-toxin and establishment of a competitive ELISA
摘要
Abstract
To establish a competitive ELISA method for detecting Clostridium perfringens(Cp)ε-toxin antibodies,the purified C.perfringens ε-toxin was used to immunize the BALB/c mice,then a sta-ble cell line named 6B9 generating anti-C.perfringens ε-toxin monoclonal antibody(mAb)was obtained by hybridoma technology.Using the purified recombinant ε-toxin as the coating antiantigen and mAb 6B9 as the competing antibody,a competitive ELISA for detecting ε-toxin antibodies was developed and optimized with HRP-conjugated goat anti-mouse IgG as the secondary antibody.Optimal conditions were determined as follows:antigen coating at 3.0 μg/mL,with undiluted test serum,mAb dilution ratio of 1∶12 000,secondary antibody dilution ratio of 1∶20 000,and 15 min color development in the dark.The positive and negative cut-off values for ovine serum samples were determined as 27.048%for positive and 19.373%for negative samples,the assay sensitivity reached 1∶1 024 for positive sera.Both intra-and inter-assay coefficients of variation were below 10%.No cross-reactivity was observed with C.perfringens α-,β1-,or β2-toxins,nor with positive sera against C.septicum,Escherichia coli,or Mycoplasma spp.When 126 ovine field sera were examined,the competitive ELISA showed 88.52%positive agreement,96.92%negative agreement,and 92.86%overall agreement with an indirect ELISA.The results indicate that this study successfully obtained a highly specific mAb against C.perfringensε-toxin,and a competitive ELISA detection method was established using mAb 6B9.The system exhibits good repeatability,high specificity,and high sensitivity.This provides technical support for accurate detection of ε-toxin antibodies in animal clinical sera and the evaluation of the immune efficacy of animal Clostridium vaccines.关键词
产气荚膜梭菌/ε毒素重组蛋白/单克隆抗体/竞争ELISA/抗体检测Key words
Clostridium perfringens/ε-toxin recombinant protein/monoclonal antibody/competitive ELISA/antibody detection分类
农业科技引用本文复制引用
狄娜,杨金玲,高鹏程,彭婕,李学瑞,储岳峰..产气荚膜梭菌ε毒素单克隆抗体的制备及其竞争ELISA抗体检测方法的建立[J].中国兽医科学,2026,56(4):496-503,8.基金项目
新疆维吾尔自治区重大专项(2023A02007) （2023A02007）
国家重点研发计划项目(2023YFD1802504-04) （2023YFD1802504-04）
兰州市科技计划项目(2023-114) （2023-114）
中国农业科学院创新工程项目(CAAS-ASTIP-2021-LVRI) （CAAS-ASTIP-2021-LVRI）
