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A previous nationwide Chinese epidemiological study revealed through isolation of A-B+ Clostridium difficile strains, which produce toxin B (TcdB), but not toxin A TcdA, that the strains are widespread and more frequent in east Asian countries,. The development of a process capable of detecting TcdB is required in microbiological laboratories in order to facilitate the control of the A-B+ C. difficile strains, however, no diagnostic reagents have been developed to date. The aim of the present study was to prepare monoclonal antibodies (mAbs) targeting the receptor binding region of TcdB (CDB3), and to establish a double-antibody sandwich enzyme-linked immunosorbent assay (ds-ELISA), which can be used for the diagnosis of C. difficile infection. The recombinant protein, glutathione S transferase (GST)-CDB3 was expressed and purified using an Escherichia coli system. BALB/c mice were immunized with GST-CDB3 recombinant protein. A hybridoma technique was used for the production of anti-CDB3 mAb. The hybridoma clones were then screened using indirect ELISA, and anti-CDB3 mAb was produced in the ascites of the BALB/c mice. Isotyping of anti-CDB3 mAb was performed using an SBA Clonotyping system/horseradish peroxidase (HRP) ELISA kit. Protein G affinity chromatography was used for purification of anti-CDB3 mAbs, and the titer and specificity of the anti-CDB3 mAbs were assessed using indirect ELISA and western blot analysis, respectively. The ds-ELISA was established using HRP-labeled anti-CDB3 mAbd, which were used to detect TcdB clinically in diarrhea stools. A total of five stable hybridoma cell clones (1E7B, 1F8D3, 2F8A6, 3B6F1 and 4A4G2) producing anti-CDB3 mAb were established. The results of the present study indicated that the immunoglobulin (Ig)G isotype was predominant, as 1E7B2 IgG1 (λ), 2F8A6 IgG2a (κ) and 4A4G2 IgG1 (κ). In addition, the highest titer of anti-CDB3 mAb (2F8A6 and 4A4G2) was 1:51,200. Western blotting revealed that the 2F8A6 and 4A4G2 mAbs recognized the CDB3 protein specifically. Following anti-CDB3 mAb (4A4G2) HRP-labeling, the optimal working concentration was confirmed to be 1:400, and the concentration of coated antibody (2F8A6) was 20 μg/ml. The sensitivity of the ds-ELISA was 73.33% for the A+B+ toxigenic C. difficile strains, and 86.67% for the A-B+ toxigenic C. difficile strains, with a specificity of 100% for all. In conclusion, the present study successfully developed novel mAbs specific to CDB3, and developed a ds-ELISA kit with high specificity and sensitivity for the rapid detection of TcdB. This offers a useful tool for the diagnostic assessment of TcdB. [ABSTRACT FROM AUTHOR]