Development of Mouse Monoclonal Antibody Anti-Ag85B Protein of Mycobacterium tuberculosis

One of the abundant biomarkers in tuberculosis (TB) infected human serum is the Ag85B antigen.The quest for protein biomarkers has been conducted to develop a diagnostic kit and vaccine for TB.Numerous studies have demonstrated that the development of monoclonal antibodies (mAbs) that recognize a specific repertoire of Mycobacterium tuberculosis antigens and the tests based on monoclonal antibodies have proved helpful in TB detection.Unfortunately, numerous mAbs currently used for TB serodiagnosis were generated elsewhere.Improving the ability to produce mouse monoclonal antibodies, specifically against Ag85B, is imperative as Indonesia is the second prevalent TB country.Therefore, a synthetic plasmid expressing Ag85B-HA fusion protein was expressed in the mammalian system.The expression of Ag85B protein in the 293T cells lysates was confirmed by western blot analysis.A specific protein band around 36 kDa appeared against antibody anti-HA.The lysate of recombinant mammalian cells was used as an inducer to generate B-cells expressing mouse monoclonal antibodies anti-Ag85B.By fusing 'PAI' mouse myeloma cells and Bcells from immunized mice, hybridoma mAb-producing cells were obtained.As many as 43 hybridomas cells were tested against recombinant Ag85B antigen by immunofluorescence assay (IFA).We found two (2) hybridomas cells specifically secreting monoclonal antibody anti-Ag85B.One hybridoma, 3C12 clone, was confirmed to have the ability to recognize Ag85B protein validated by western blot analysis.Similar to previous research, our western blot analysis shows that our monoclonal antibody can identify the antigen Ag85 at a protein size of around 30 kDa.Further analysis and characterization should be conducted to explore the potency of those monoclonal antibodies for developing TB diagnostic kits.