isolation and purification of ag 85 complex from mycobacterium bovis (bcg) and assessment of its cell proliferation response in whole blood

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.