Preparation and application of monoclonal antibodies specific for Helicobacter hepatic us urease B subunit

摘要

Helicobacter hepaticus(Hh) and Helicobacter bilis(Hb) infect mice and cause hepatitis and colitis. The current diagnosis method for Helicobacter infection in mice is PCR using genus-specific or species-specific primers. However, the procedures of PCR-based diagnosis are complicated and time-consuming, and non-specific reactions are problematic. Therefore, we produced monoclonal antibodies against Hh in order to apply to fecal Hh antigen detection test. Urease B subunit (UreB) protein of Hh was expressed in E.coli from cloned UreB gene and used for immunization of mice to produce monoclonal antibodies. Hybridomas producing monoclonal antibodies were screened by the ELISA using UreB proteins of Hh and Hb as antigens. Then, specificity of the selected monoclonal antibodies was analyzed by the immunoblot assay using bacterial lysates of Hh, Hb, and H.muridarum(Hm) as antigens. Among the murine Helicobacter species, Hh, Hb, and Hm are positive for urease which is one of the major proteins they produce. Homology of the UreB amino acid sequences between Hh and Hb was 82%, implying that the existence of species-specific epitopes on the UreB protein. As expected, monoclonal antibodies specific for Hh UreB protein were generated. In addition, several monoclonal antibodies cross-reacted with UreB of Hb and Hm. By using monoclonal antibodies produced in this study, we are now examining sandwich ELISA method for specific detection of Hh UreB antigens in feces of mice.