Sero-reactivity of two different antigenic protein fractions of M.tb H37Ra excretory-secretory antigen in pulmonary and extra-pulmonary tuberculosis.

Excretory-secretory proteins of Mycobacterium tuberculosis H37Ra, have been of diagnostic interest in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB). Two different excretory-secretory antigenic proteins of M.tbH37Ra viz., EST-DE1 (a 6% TCA soluble and DEAE anion exchange purified antigen) and ESAS-7 (50% ammonium sulphate solubilized and SDS-PAGE fractionated antigen) were studied in stick-indirect penicillinase ELISA for detecting tuberculous IgG antibodies in serum samples of pulmonary as well as extrapulmonary tuberculosis (tuberculous lymphadenopathy (TBLN), tuberculous meningitis (TBM), bone & joint tuberculosis (B&J TB), abdominal tuberculosis (Abd. TB) patients. The ESAS-7 antigen has shown comparatively better seroreactivity (90%) than that of EST-DE1 antigen in pulmonary tuberculosis cases. The overall specificity of 93.2% using ESAS-7 antigen was also found better compared to 86.4% obtained using EST-DE1 antigen. Further, in extra pulmonary tuberculosis group, using ESAS-7 antigen 84% (21/25) of histopathologically confirmed TBLN cases and 90% (9/10) clinically diagnosed and ATT responded TBM cases showed positive reaction for tuberculous IgG antibody. The per cent positivity using EST-DE1 antigen was however comparatively low in TBLN and TBM cases, (76% and 80% respectively). In histopathologically proven bone and joint tuberculosis and abdominal tuberculosis cases EST-DE1 antigen showed better sensitivity of 75% and 83.3% respectively in IgG antibody detection compared to that of ESAS-7 antigen (50% and 66% respectively). From the present study, it can be envisaged that ESAS-7 antigenic fraction has a good potential in the diagnosis of pulmonary and certain extra-pulmonary tuberculosis infection (TBLN & TBM) whereas EST-DE1 was found to be better in detecting specific antibodies in bone & joint and abdominal tuberculosis.

Purification and characterization of a 31 kDa mycobacterial excretory-secretory antigenic protein with a diagnostic potential in pulmonary tuberculosis.

Proteins secreted into the culture medium by Mycobacterium tuberculosis are shown to be a source of antigens of immunodiagnostic importance. In our earlier study, we had reported a 31-37 kDa seroreactive gel-eluted antigenic fraction (ESAS-7), isolated from culture filtrate proteins of Mycobacterium tuberculosis H37Ra. In this report, we describe further purification of excretory-secretory ESAS-7 antigen fraction by fast protein liquid chromatography (FPLC) on Resource 'S' cation-exchange column and isolation of a more active and purified protein antigen fraction ESAS-7F. ESAS-7F antigen was characterized as a 31 kDa molecular weight glycoprotein containing a metallo-serine protease activity. N-terminal sequence analysis showed the first five amino acids as NTGQS (Asp-Thr-Gly-Glu-Ser). The present study helped in the isolation of a well characterized 31 kDa mycobacterial glycoprotein antigen with protease activity and diagnostic potential in detection of tuberculosis infection.