B-CELL EPITOPES AND QUANTIFICATION OF THE ESAT-6 PROTEIN OF MYCOBACTERIUM-TUBERCULOSIS

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis, In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides sp anning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted wit h two peptides in the N-terminal region of the molecule. Assays with s ynthetic truncated peptides allowed a precise mapping of the epitope t o the residues EQQWNFAGIEAAA at positions 3 to 15, Hydrophilicity plot s revealed one hydrophilic area at the N terminus and two additional a reas further along the polypeptide chain, Antipeptide antibodies were generated by immunization,with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin, Prolonged i mmunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native E SAT-6, A double-antibody ELISA was then developed with monoclonal anti body HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suit able for quantification of ESAT-6 in M. tuberculosis antigen preparati ons, showing no reactivity with M, bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8, This capture ELISA permitted the identif ication of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.