A low molecular weight ES-20 protein released in vivo and in vitro with diagnostic potential in lymph node tuberculosis.

PURPOSE: To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB). METHODS: In vivo released circulating tuberculosis antigen (CTA) was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES) antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analysed for antigen by ELISA. RESULTS: Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20) antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. CONCLUSIONS: Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB.

Study of M. tuberculosis ES-31 and ES-20 antigen levels in different pathogenic grades of lymph node tuberculosis.

OBJECTIVE: To study tuberculous excretory-secretory (ES) 31 and ES-20 antigens in different pathogenic grades of lymph node tuberculosis (TB). DESIGN: The study group included lymph node TB patients showing granuloma with mature epithelioid cells based on cytology findings (strong immune response group, SI) and patients showing no granuloma formation and acellular necrosis (weak immune response group, WI). Sandwich ELISA was performed using affinity purified antibodies against Mycobacterium tuberculosis ES-31 and ES-20 antigens to assay free and immune complexed antigen levels in the serum of these patients. RESULTS: Higher levels of immune complexed ES-31 (geometric mean titre [GMT] 848) and ES-20 (GMT 1818) antigens than free ES-31 (GMT 462) and ES-20 (GMT 647) were observed in WI patients. There were higher levels of immune complexed ES-20 antigen levels (GMT 1818) in WI patients than in SI lymph node TB patients; the difference was significant (P < 0.05). CONCLUSION: Elevated levels of immune complexed ES-20 antigen in patient's serum may be a useful immunological marker for weak immune response patients in lymph node TB.

Detection of free and immune-complexed serine protease and its antibody in TB patients with and without HIV co-infection.

OBJECTIVE: To understand the usefulness of detecting tuberculous IgG antibodies against mycobacterial excretory-secretory 31 kDa serine protease antigen (SEVA TB ES-31) and circulating free and circulating immune-complexed (CIC) serine protease in TB patients with and without HIV infection. DESIGN: Serum was collected from 144 individuals: patients with TB, with TB-HIV co-infection and HIV infection only, and ill and healthy controls. SEVA TB ES-31 antigen, a serine protease isolated from Mycobacterium tuberculosis H37Ra culture fluid, was used in indirect penicillinase ELISA to detect tuberculous antibodies. Similarly, affinity purified anti-ES-31 antibody was used in sandwich ELISA to detect circulating free and CIC serine protease. RESULTS: There was less sensitivity for tuberculous antibody in HIV-infected TB patients (46%) than in those with TB alone (87%) using mycobacterial serine protease. However, the sensitivity of detection of TB in the presence of HIV increased to 87% by concomitant detection of circulating free and CIC serine protease antigen. CONCLUSION: Detection of free and CIC tuberculous serine protease antigen along with antibody is more useful for detecting TB in the presence of HIV co-infection.