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.

ABSTRACT

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 125 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.