Isolation and structural characteristics of a monoclonal antibody-defined cross-reactive phospholipid antigen from Mycobacterium tuberculosis and Mycobacterium leprae.

A low molecular weight antigen of Mycobacterium leprae and other mycobacteria was previously defined in our laboratory by means of IgG2a monoclonal antibody termed L4. The antigen had an apparent molecular mass of 4.5-6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was assumed to be a glycoprotein on the basis of its staining with periodic acid Schiff and sensitivity to periodate treatment. In the present work, the cross-reactive and phospholipidic nature of the antigen, present in Mycobacterium tuberculosis as well as in M. leprae sonicates, was demonstrated and this enabled us to undertake its purification from crude M. tuberculosis phospholipidic extracts. The L4-reactive antigen from M. tuberculosis called L4-PIM, was purified by means of silicic acid high pressure liquid chromatography. Its characterization by gas chromatography and FAB-MS showed the antigen to be the common mycobacterial dimannosylated phosphatidylinositol (PIM2), the structure of which had been previously established by others (Lee, Y. C., and Ballou, C. E. (1964) J. Biol. Chem. 239, 1316-1327; Lee, Y. C., and Ballou, C. E. (1965) J. Biochem. (Tokyo) 4, 1395-1404). Delineation of the L4 epitope on M. tuberculosis L4-PIM revealed the involvement of the axial 2-hydroxyl of the alpha-D-mannosyl residues, without any detectable contribution from the myo-inositol. Consequently, L4 was shown to react with PIM5, the structure of which contains twice the number of epitopes as does PIM2. By using both immunostained thin layer chromatography and indirect enzyme-linked immunosorbent assay, similar L4-PIM epitopes were demonstrated in M. leprae sonicate, thereby explaining the cross-reactive nature of the L4-monoclonal antibody. Antibodies of IgG class directed against M. tuberculosis L4-PIM were detectable in sera from patients with leprosy, but no evidence of T cell reactivity to L4-PIM was obtained. The demonstration of a correlation of anti-L4-PIM IgG and anti-disacharide-conjugated bovine serum albumin IgM antibody titers in the sera of leprosy patients indicates that measurement of antibodies directed against L4-PIM may have the potential to be used as a complementary assay to the disaccharide-conjugated bovine serum albumin test for diagnosis and monitoring of patients undergoing leprosy therapy.