Production and characterization of monoclonal antibody against Mycobacterium tuberculosis RpfB domain

RpfB, one of the five resuscitation-promoting factors (Rpfs) produced by Mycobacterium tuberculosis (MTB), plays an important role in the resuscitation and growth of the dormant MTB. RpfB is likely the target antigen recognized by the host immune system. Studies have shown that Rpf genes exist in many bacteria and their encoded proteins all contain Rpf-like domain. It is likely that this domain has biological characteristics and immunogenicity similar to that of the complete Rpf protein. Therefore, RpfB domain protein from M. tuberculosis was selected for this study. Mice were subcutaneously immunized three times over 2-week intervals. Mice splenocytes were then isolated and fused with SP20 cells. Hybridoma colonies were screened for monoclonal antibody (MAb) against RpfB domain. ELISA was used to examine the titer, specificity, and relative affinity of the antibody. The ability of produced anti-RpfB monoclonal antibody to recognize other proteins in the Rpf family and to inhibit the growth of Mycobacterium tuberculosis and Micrococcus luteus was examined. Our results showed that three anti-RpfB MAbs were successfully generated. All three MAbs can recognize RpfB domain specifically and can effectively inhibit the promoting effect of RpfB domain on the growth of MTB H37Ra strain and M. luteus at 1:1000 dilution, indicating that anti-RpfB domain MAbs may inhibit the reactivation of dormant or latent MTB in vivo. Therefore, they may be able to prevent the recurrence of the occult infection. The production of anti-RpfB domain MAbs provides a powerful experimental tool to further study the biological and immunological characteristics of the RpfB domain and to evaluate the possibility of using RpfB domain as a candidate component for tuberculosis subunit vaccine.