The mapping of an antibody-binding region on the Mycobacterium tuberculosis 19 kilodalton antigen.
To localize the epitopes of four independently derived murine mAb IT-10, IT-12, IT-16, and IT-19 on the 19-kDa Ag protein of Mycobacterium tuberculosis, expression plasmids were constructed containing deletions of the gene encoding the 19-kDa protein. Reaction of the 4 mAb with Western blots of the truncated recombinant proteins revealed two epitope specificities in the recognition of the 19-kDa protein. IT-10 was found to be dependent only on the presence of amino acids surrounding the first cysteine residue, whereas IT-12, IT-16, and IT-19 all required the presence of both the first and third cysteine residues. These two cysteine residues are separated by 135 amino acids, and are considered to be brought together by tertiary folding of the protein to form an assembled epitope for IT-12, IT-16, and IT-19. These three mAb demonstrated differing sensitivities to the modification of reduced 19-kDa protein using iodoacetamide: a treatment that should have prevented the reformation of disulfide bonds within the protein. This result suggests that, although IT-12, IT-16, and IT-19 appear to be specific for the same epitope, there are probably fine-specificity differences in this recognition. IT-10 was not sensitive to the absence of disulfide bonds within the 19-kDa protein, suggesting that the epitope is not conformationally sensitive, and is likely to be linear in nature.
Ashbridge
K R
KR
Department of Molecular Medicine, School of Medicine, University of Auckland, New Zealand.
Prestidge
R L
RL
Booth
R J
RJ
Watson
J D
JD
D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D004220:Disulfides; D000939:Epitopes; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D011487:Protein Conformation; D011994:Recombinant Proteins; D015183:Restriction Mapping
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004220:Disulfides; D000939:Epitopes; D011994:Recombinant Proteins