Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein of Mycobacterium tuberculosis H37Ra.

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction (Mtb EST -6) of purified Mycobacterium tuberculosis H37Ra and affinity purified polyclonal antibodies against Mtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system using Mtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified anti Mtb EST antibodies detected circulating antigen in 83% and 61 % of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91 % respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility of Mtb EST -6 as a diagnostic marker of pulmonary tuberculosis

Fractionation, analysis and diagnostic utility of Mycobacterium tuberculosis H37Ra excretory-secretory antigen in pulmonary tuberculosis.

Excretory-secretory antigen of Mycobacterium tuberculosis H37Ra (Mtb ES antigen) isolated from culture filtrate was partially purified by 6% trichloroacetic acid precipitation. The TCA supernatant fraction (Mtb EST antigen) was examined for its diagnostic use in the detection of tuberculous IgG antibody in human sera by stick Indirect ELISA. Using Mtb EST antigen, tuberculous IgG antibody was detected in 90% of smear positive and 71% of smear negative pulmonary tuberculosis cases and 10% of healthy and disease controls. Further fractionation of Mtb EST antigen by SDS-PAGE yielded four active antigenic fractions viz., Mtb EST-3,4,6 and 10. Diagnostic evaluation of these fractions showed Mtb EST-6 antigen fraction to be useful in detection of both smear positive and smear negative pulmonary tuberculosis cases with sensitivities of 94% and 78% respectively and specificity of 88%.