A 66-kDa protein (TB66) was purified from culture filtrate (CF) and cell sonicate (CS) of HRv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NTA) column. TB66 was found to be a fibronectin-binding protein as determined by ELISA and could be purified by affinity chromatography with fibronectin-Sepharose. A similar 66-kDa protein could be isolated also from BCG, and HRa by IMAC, but not from any other mycobacteria. The NH-terminal amino-acid sequence of TB66 from HRv and was identical and showed 85% homology with the N-terminal sequence of bovine serum albumin (BSA). A monoclonal antibody (MAb) OD4AG3 recognised a heat-stable and trypsin-sensitive epitope near the C-terminal end of TB66. This MAb also recognised the 66-kDa protein isolated from the other members of the complex. In tests of immunogenicity, TB66 elicited a delayed type hypersensitivity reaction in guinea-pigs immunised with either TB66 or with H37Rv. TB66 also elicited an antibody response in immunised guinea-pigs and stimulated murine macrophages to produce tumour necrosis factor.


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