Identification and characterization of individual components of M. tuberculosis have long been a focus of research on tuberculosis. The 30-kDa antigens are major constituents of M. Bovis BCG and M.tuberculosis culture fluids. Because 30-kDa and
32-kDa
antigens are partially identical, these antigens are difficult to purify in large amounts by biochemical techniques. This study was performed to purify 30-kDa antigens to homogenity from the unheated culture filtrate of M. tuberculosis H37Rv. The
30-kDa
and 32-kDa antigen complexes were primarily purified by 50% ammonium sulfate precipitation, hydroxylapatite chromatography and Sephadex G-75 gel filtration. And then further purification for separation of the two antigens was accomplished on
preparative
isoelectric focusing(IEF). Recovery of 30-kDa antigens during above the purification procedures were 28% and 14%, respectively, and 147.0% and 59.8-fold purification were showed, respectively. On silver stained SDS-PAGE gels, the purified 32-kDa
antigen
gave a single band at 32-kDa antigen gave one major band at 30-kDa molecule and faint additional bands at 32-kDa. The pI of 30-kDa antigens were 4.3 and 4.6, respectively. The partial identity between these two antigens was observed through the
same
pattern of reactivity of antigens in the ELISA and immunodiffusion.
To analyse the immunological activity of the purified 30 kDa antigen, we also examined the potential of lymphokines from 30-kDa antigen stimulated mononuclear cells to enhance H2O2 and O2 releasing capacity of human monocyte-derived macrophage
(MDM).
The lymphokines were produced by stimulation of unseparated mononuclear cells or non-adherent mononuclear cells of PPD(+) or PPD(-) persons with 30-kDa antigen. Monocytes were matured into MDM by culture for 4 days and then were activated by the
lymphokines. LK prepared from PPD (+) person enhanced H2O2 and O2 production by MDM in dose-and time-dependent fashion. Especially, MDM incubated with a high dilution (1/64) of PPD(+) Ag-LK demonstrated an enhanced capacity to release H2O2 and
O2.
However, treatment of MDM with control-LK or PPD (-)-LK do not lead to increase in the H2O2 and O2 production.
Considerable variation of H2O2 and O2 releasing capacity of MDM from 9 healthy subjects were observed. MDM activated by rIFN-r showed the highest production and MDM treated by PPD(+) Ag-LK released more H2O2 and O2 than that treated by PPD(+)
MAg-LK.
H2O2 and O2 releasing capacity or of MDM of 5 tuberculosis patients were same or higher than that of healthy subjects, and PPD(-) LK treated MDM also enhanced H2O2 and O2 production.
These results suggest that the 30- and 32-kDa antigens could be effectively purified by the IEF and reactions of partial identity between the two antigens were found However, 30-kDa antigen was the more immunogenic antigen than 32-kDa. The 30-kDa
antigen may be important in the cell mediat3d response to infection with M. tuberculosis, and MDM from tubeculosis patients was able to generate H2O2 and O2 by lymphokines.
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