ناهید مژگانی - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
مهدی بابائی - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
نفیسه شکیبامهر - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
محمد محمدطاهری - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
نادر مصوری - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
آرام قائم پناه - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.
کیومرث سلیمانی بابادی - Reference Laboratory of Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran.

The pathogenesis of Mycobacterium tuberculosis (Mtb) is related to its low molecular weight proteins mainly ESAT۶ and CFP۱۰ 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 ۶۸ °C for ۹۰ min and ۱۰۰ °C for ۳ hrs, respectively. Inactivated cultures were filtered and the proteins in the supernatant fluid precipitated with two rounds of ammonium sulfate, at ۴ °C. The collected precipitates were dialyzed and subjected to gel chromatography (G-۵۰) and the obtained fractions were analyzed for protein concentrations and molecular weight. ESAT۶ and CFP۱۰ 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 ۲۰% ammonium sulfate. SDS-PAGE analysis revealed protein bands of approximately ۱۰-۱۵ kDa. The purity of the proteins was ≥۹۵%, as confirmed by SDS-PAGE. The presence of the ESAT-۶/CFP۱۰ complex was confirmed by Western blot analysis. The purified fractions showed no cross-reaction with BCG or M. avium strain. ESAT-۶/CFP-۱۰ purified by the ammonium sulfate method appeared to be suitable for the development of a diagnostic kit for the detection of Mtb.

Mycobacterium tuberculosis, ESAT-۶/CFP۱۰, Ammonium sulphate, Chromatography, Western blotting