Isolation And Purification Of Ag 85 Complex From Mycobacterium Bovis (BCG) And Assessment Of Its Cell Proliferation Response In Whole Blood

Document Type : Original Article


Microbiology Dept., Gilan Medical University, Lahijan, Iran


Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major worldwide health problem. The only TB vaccine currently available is anattenuated strain of mycobacterium bovis termed Bacillus Calmette -Guerin (BCG).The efficacy of BCG remains controversial. Mycobacterium secretory proteins are generally considered important antigens for protection against TB. A major protein component of mycobacterial culture filtrate is Antigen 85 complex which is apromising potential vaccine candidate.
Material and Methods: Antigen 85 complex was purified from Mycobacterium bovis (BCG) culture filtrate by sequential chromatography on phenyl Sepharose 4B, DEAE-Sephacel and Superdex G75. Purification was confirmed by SDS-PAGE and immunoblotting with a specific monoclonal antibody. The in vitro ability of Ag 85 complex to stimulate cell proliferation was compared with that of Purified Protein Derivative (PPD) and the polyclonal T cell mitogen PHA in a whole blood assay in which the target cells were derived from 25 healthy PPD-positive and 25 healthy PPD-negative subjects.
Results: Antigen 85 complex was found to have a molecular weight of 30-32 KDa by SDS-polyacrylamide gel electrophoresis and reacted strongly by immunoblotting with the monoclonal antibody specified against Ag 85. The responses to Ag 85 and PPD were significantly higher in cells of PPD- positive than in cells of PPD-negative donors. Eighty eight percent (22/25) of the PPD- positive cells responded to Ag 85 whereas only 16% (4/25) of the PPD-negative cells responded.
Conclusion: The cell proliferation response to Ag 85 complex is significantly different between cells of skin-test positive and skin- test negative subjects and may suggest an immuno-protective role for Ag 85 complex against M. tuberculosis infection.