SKIN REACTIVITY AND FIBRONECTIN-BINDING PROPERTY OF TB66 (66-KDA PROTEIN OF MYCOBACTERIUM-TUBERCULOSIS)

A 66-kDa protein (TB66) was purified from culture filtrate (CF) and ce ll sonicate (CS) of Mycobacterium tuberculosis H(37)RV by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NT A) column. TB66 was found to be a fibronectin-binding protein as deter mined by ELISA and could be purified by affinity chromatography with f ibronectin-Sepharose. A similar 66-kDa protein could be isolated also from M. bovis, M. bovis BCG, M. africanum and M. tuberculosis H37Ra by IMAC, but not from any other mycobacteria. The NH2-terminal amino-aci d sequence of TB66 from H(37)Rv and M. bovis 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 try psin-sensitive epitope near the C-terminal end of TB66. This MAb also recognised the 66-kDa protein isolated from the other members of the M . tuberculosis complex. In tests of immunogenicity, TB66 elicited a de layed type hypersensitivity reaction in guinea-pigs immunised with eit her TB66 or with M. tuberculosis H37Rv. TB66 also elicited an antibody response in immunised guinea-pigs and stimulated murine macrophages t o produce tumour necrosis factor.