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.

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.

Mycobacterial ES-31 Serine Protease - A Biomarker for Mycobacterium tuberculosis - A preliminary Report.

There is a need for simple and reliable method to identify Mycobacterium tuberculosis from AFB smear positive cases. Utility of mycobacterial ES-31 serine protease as a marker to detect Mycobacterium tuberculosis bacilli was explored using Fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. The presence of ES-31 serine protease in bacilli was indicated by green fluorescence on the cell surface. Green fluorescence was observed with M.tb. H37Ra bacilli and M.tb. H37Rv bacilli while no Fluorescence was observed with M. chelonae, Nocardia farcinicum as well as in E. coli showing the usefulness of ES-31 serine protease as a marker for identification of mycobacterium tubercle bacilli in cultures.