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OA

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.