Preparation of anti-Helicobacter pylori UreB monoclonal antibody and the detection of the ability of inhibition the urease activity

Objective To prepare the anti-UreB monoclonal antibody (mAb), and detect the inhibition ability of the anti-UreB mAb on urease activity. Methods mAb was prepared by hybridoma technique using recombined UreB (rUreB) as immunogen. The titer and affinity constant of the mAb were determined by ELISA and the specificity of the mAbs was analyzed by Western blotting. The mAb was preincubated with the urease fraction, and then the buffer containing urea and phenol red was added into the culture solution. The inhibition ability of mAb on urease activity was detected at 550 nm. Results Five hybridoma cell lines of 1D5, 9E1, 3A2, 1D6, 6E6 were screened, which could secrete the anti-UreB mAb stably. The 5 mAbs have isotypes of IgG1/IgG1/IgG1/IgG2a/IgG2a, respectively, and affinity constant of 4×108, 4.6× 108, 2.8×108, 2×109, 3×109 L/mol, respectively. Each of the 5 antibodies could bind with rUreB or the UreB antigen specifically, and could not cross-react with bacteria protein in intestinal tract. Among 5 mAbs, only 6E6 could inhibit the activity of urease in a dose-dependent manner. Conclusion Five hybridoma cell strains are prepared, which can secrete high-titer and high-specific monoclonal antibodies against UreB stably, and one mAb (6E6) of them can inhibit the urease activity. The result provides a basis for the mechanism research of urease and affords a valuable tool for UreB purification as well as Hp diagnose and clinical treatment.