腐败梭菌α毒素单克隆抗体的制备及竞争ELISA方法的建立OA

For the convenience of clinical detection of Clostridium septicum(C.septicum)in serum samples and the evaluation of the immunogenic efficacy of C.septicum vaccines in animals,this study utilized an Escherichia coli expression system to induce the expression of recombinant α toxin protein.After purification,BALB/c mice were immunized to prepare monoclonal antibodies against α toxin.Using the screened monoclonal antibody as the capture antibody,the reaction conditions were optimized by a checkerboard titration method to develop a competitive enzyme-linked immunosorbent assay(ELISA)method for detecting antibodies against C.septicum α toxin.The method was applied to determine the positive and negative cut-off values of sheep serum samples and to evaluate its repeatability,sensitivity,specificity,and coincidence rate,as well as to determine the cut-off values for bovine serum samples.The results showed that the target protein of approximately 65 kDa was successfully expressed and purified;through hybridoma technology,a stable hybridoma cell line secreting specific antibody was obtained,and the monoclonal antibody was named 8H2.The optimal antigen coating conditions for the competitive ELISA were determined as follows:coating concentration of 300 ng/well for 16 h,serum dilution of 1∶1,monoclonal antibody dilution of 1∶2 000,blocking with 1%bovine serum albumin(BSA)for 90 min,serum and monoclonal antibody competition for 90 min,secondary antibody dilution of 1∶15 000 incubated for 30 min,and color development time of 15 min.The positive and negative cut-off values for sheep serum samples were determined to be 27.19%and 22.59%,respectively.Both the intra-assay and inter-assay coefficients of variation were below 10%,indicating good repeatability.The standard positive serum remained positive at a dilution of 1∶16.No cross-reactivity was observed with Clostridium perfringens,Brucella spp.,or Salmonella typhimurium,showing high specificity.When the established competitive ELISA method was used to test 100 sheep serum samples,the total coincidence rate with the indirect ELISA was 89.00%(89/100).For bovine serum samples,the positive and negative cut-off values were 20.00%and 16.30%,respectively.In conclusion,one monoclonal antibody specific to C.septicum α toxin was successfully prepared,and a competitive ELISA method for antibody detection was established.This method provides a technical foundation for the early diagnosis of C.septicum infection and for evaluation of vaccine-induced immune efficacy.