The Preparation And Preliminary Application Of Monoclonal Antibodies To Clostridium Difficile Toxin B Receptor Binding Region

Abstract:Objective (1) To investigate the best condition to the induced expression of receptor binding region fusion protein of clostridium difficile toxin B (CDB3) and analysis the forms of its expression, the fusion protein was purified by GST Sefinose TM Kit.(2) Preparation of monoclonal antibodies to CDB3and purified the antibodies.(3) Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established and was applied to detect the toxin B in the diarrhea stool. Methods (1) The fusion protein were induced to the single variable of the concentration of bacterial before induction (OD600), the induction temperature, the concentration of inducer IPTG and the induction time, analyzed expression level and the form of its expression by SDS-PAGE and identified by Western blot.(2) The mice BALB/c were immunized with the purified protein CDB3. Anti-CDB3monoclonal antibodies were developed by hybridoma technique. Anti-CDB3mAbs were purified and detection their concentration, molecular weight, potency, subtype, epitope, specificity, relative affinity was analyzed by ELISA, SDS-PAGE and Western blot.(3) Anti-CDB3monoclonal antibodies (4A4G2) labeled with HRP (horse radish peroxidase) through the means of modified sodium periodates oxidation method. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established and optimized through the means of Chessboard titration method and was applied to detect the toxin B in the diarrhea stool. Results (1) The optimal conditions for the induced expression of CDB3fusion protein were the concentration of bacterial before induction (OD600) is0.8, the concentration of IPTG is0.6mmol/L, the induction time is12h and the induction temperature is30℃, and the main forms of expression is the soluble protein, the purity over90%.(2) A total of five positive hybridoma cell lines were found, and the subtype identification results for their were1E7B2IgGl (λ),1F8D3IgM (κ),2F8A6IgG2a (κ),3B6F1IgM (λ) and4A4G2IgGl (κ), Anti-CDB3mAbs were successful purified by bitterness-ammonium sulfate and protein G agarose gel electrophoresis, and the purification effect over90%. Their concentration detection was21.43μg/mL,53.32μg/mL and30.08μg/mL. The molecular weight was approximately160KD, and the relative affinity were4A4G2>2F8A6>1E7B2.2F8A6and4A4G2were act on different epitopes of CDB3.(3) Monoclonal antibody (4A4G2) was successfully labeled, double-antibody sandwich ELISA was established and optimized through the means of Chessboard titration method,15specimens of A+B+-type CD were only7-positive (46.67%) detected by double-antibody sandwich ELISA,15A-B+-type CD were only8-positive (53.33%) and15CD-negative specimens were all negative. Conclusion (1) The fusion protein GST-CDB3was successfully induced, expressed and purified.(2) The anti-CDB3mAbs were successfully prepared and purified,2F8A6and4A4G2have higher concentration and potency and acting on different epitopes of CDB3.(3) Double antibody sandwich ELISA has higher specificity but lower sensitivity, so the detection system should be further improve. Figure26, Table5, reference46.