ELISA protocol for rapid screening of potential antitubercular drugs based on antigenic reactivity of mycobacterial ES-31 serine protease - a drug target supported by axenic culture of Mycobacterium tuberculosis H37 Ra strain in the presence of inhibitor.

Background: Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs. Aim: To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity. Material and Methods: Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor. Results: Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5μg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4μg/well, while orlistat showed inhibition of 60% at 0.5μg/well. Inhibition of Mtb H37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4μg/well and orlistat at 0.5μg/well were observed respectively on bacterial growth. Conclusion: Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

Inhibitory effect of isoniazid and orlistat combination on mycobacterial ES-31 serine protease in vitro and on the growth of M.tb bacilli in axenic culture.

Background: Isoniazid and orlistat were reported to have inhibitory effect on mycobacterial ES-31 serine protease in vitro and bacterial cell growth in axenic culture. Aim: To study the cumulative effect and understand drug - drug interaction, if any, when isoniazid and orlistat used in combination. Material and Methods: Inhibition of mycobacterial ES-31 serine protease by different combinations of orlistat and isoniazid together and individually were studied using azocasein assay. Inhibition of secretion of excretory secretory ES- 31 antigen in Sautan culture medium was studied under axenic condition and growth of M.tuberculosis H37Ra bacilli by CFU count on LJ-medium. Results: Orlistat and isoniazid both showed inhibitory activity of ES-31 serine protease in in vitro as well as in vivo. Individually, isoniazid showed 90% inhibition at 200 ng/ml while orlistat at 250 ng/ml showed 65% inhibition of mycobacterial ES-31 serine protease in vitro. A combination of orlistat (250 ng/ml) and isoniazid (200 ng/ml) showed 86% inhibition in vitro while 73% inhibition was observed by orlistat (25 ng/ml) and isoniazid (200 ng/ml) on bacterial growth in axenic culture. Conclusion: Significant inhibition by orlistat suggests that it could be tried in patients with intolerance to isoniazid or in those already developed isoniazid resistance. It may also be explored in the suspected TB patients as initial medication in place of antibiotics for clinical relief.

Multi-antigen and antibody assays (Seva TB Elisa) for the diagnosis of tuberculous pleural effusion.

Objective: Prospective evaluation of inhouse developed SEVA TB ELISA using cocktail of Mycobacterial antigens ES-31 and EST-6(containing ES-38 and ES-41) and their specific antibodies in the diagnosis of Tuberculous pleural effusion was done in a tertiary care hospital. Methods: Detection of circulating free and immune-complexed (IC) antigens and antibody by sandwich and indirect peroxidase ELISA respectively was done in pleural fluid and sera specimens. Total 33 patients with pleural effusion, including 24 patients diagnosed as tuberculous pleural effusion based on clinico-radiological, microbiological and biochemical profile (protein, LDH and ADA) of pleural effusion and nine patients with non-tuberculous pleural effusion, were studied. Results: Pleural fluid showing either antigen or immune-complexed antigen or antibody positive was considered as ELISA positive for tuberculous pleural effusion. Multi antigen and antibody assay (SEVATB ELISA) showed 100% specificity and 83% sensitivity in pleural fluid while 78% specificity and 92% sensitivity in serum of tuberculous pleuritis patients. Conclusion: This study showed usefulness of SEVATB ELISA, using cocktail of ES-31 and EST-6 antigens and their antibodies for antibody and antigen detection respectively in analysis of either sera or pleural fluid samples of suspected tuberculous pleuritis patients as an adjunct test to clinical diagnosis.

A sensitive and specific ES-31 antigen detection based fluorometric assay for confirmation of Mycobacterium tuberculosis in cell culture.

Confirmation of presence of M. tuberculosis bacilli on microscopic examination is very important in diagnosis of tuberculosis. The present study was undertaken to find the usefulness of mycobacterial ES-31 serine protease as a marker to detect tuberculosis bacilli using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. This immunofluorescence method was compared with Ziehl-Neelsen and auramine-O staining methods for detection of tuberculosis bacilli. Slides were prepared for each serially diluted tuberculosis H37Ra bacilli (1×107 bacilli/ml to 5 bacilli/ml). Slides for each dilution group were stained by ZN method, auramine-O and immunostaining methods using fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. ZN staining method showed efficacy for detection of M. tuberculosis H37Ra upto 1×104 bacilli/ml while auramine-O method showed upto 1×102 bacilli/ml. The presence of bacilli was indicated by green fluorescence on immunostaining using anti-ES-31 antibody conjugate and this method was effective upto 10 bacilli/ml. The slides which were negative for ZN (1×103 cells/ml) and auramine-O (100 cells/ml) method showed positivity on restaining with immunofluorescent staining method. The results of this preliminary study showed that immunofluorescent staining method using specific anti-ES-31 antibody conjugate was more sensitive for detection of tuberculosis bacilli than ZN and auramine-O methods in samples of laboratory strain. The utility of this method will be studied further in clinical specimens.

Effect of HIV protease inhibitors and orlistat on mycobacterial ES-31 serine protease, a potential drug target in mycobacterium tuberculosis.

Background: Mycobacterial excretory secretory-31 (SEVA TB ES-31) antigen is shown to possess protease and lipase activities. Aim: To study the effect of commonly used HIV-protease inhibitors and lipase inhibitor Orlistat if any on mycobacterial ES-31 serine protease in vitro enzyme activity and on the growth of M.tb H37Ra bacilli in axenic culture. Methods: Effect of HIV-protease inhibitors namely Ritonavir, Lopinavir and Indinavir and Orlistat on protease activity of ES-31 was assessed using azocasein assay and on bacillary growth in axenic culture of Mycobacterium tuberculosis H37Ra. The concentration of ES-31 antigen in culture filtrate was determined by sandwich peroxidase ELISA using anti ES-31 antibody and the growth of bacilli by CFU count. Results: HIV-protease inhibitors such as Ritonavir, Lopinavir and Indinavir and lipase inhibitor Orlistat inhibited serine protease activity by 41.3 - 69.7% in vitro. These inhibitors also showed decreased bacterial growth in axenic culture and further confirmed by decreased concentration of ES-31 serine protease secretion in the culture fluid. Ritonavir showed maximum inhibition of 77% on the growth of the bacilli in axenic culture while anti obesity drug Orlistat showed 61% inhibition. Conclusion: SEVA TB ES-31 with serine protease and lipase activities may be a potential drug target in tuberculosis management.