Purification and biological analysis of specific antigens (ESAT6/CFP10) from Mycobacterium tuberculosis

The pathogenesis of Mycobacterium tuberculosis (Mtb) is related to its low molecular weight proteins mainly ESAT6 and CFP10 that are highly specific and potentially useful for the diagnosis of tuberculosis. This research focused on isolation, purification, and characterization of low molecular weight proteins from Mtb. Cultures of Mtb were inactivated by heating at 68 °C for 90 min and 100 °C for 3 hrs, respectively. Inactivated cultures were filtered and the proteins in the supernatant fluid precipitated with two rounds of ammonium sulfate, at 4 °C. The collected precipitates were dialyzed and subjected to gel chromatography (G-50) and the obtained fractions were analyzed for protein concentrations and molecular weight. ESAT6 and CFP10 protein complex in the purified fraction was confirmed by Western blotting. Guinea pig sensitization assay was used for estimating the potency of the purified fraction compared to the standard PPD. The maximum amount of low molecular weight proteins were precipitated by 20% ammonium sulfate. SDS-PAGE analysis revealed protein bands of approximately 10-15 kDa. The purity of the proteins was ≥95%, as confirmed by SDS-PAGE. The presence of the ESAT-6/CFP10 complex was confirmed by Western blot analysis. The purified fractions showed no cross-reaction with BCG or M. avium strain. ESAT-6/CFP-10 purified by the ammonium sulfate method appeared to be suitable for the development of a diagnostic kit for the detection of Mtb.