title: Elisa protocol for rapid screening of potential anti-tubercular 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.
  
  
  OBJECTIVE
  To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity.
  
  
  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 5 microg/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 0.4 microg/well, while orlistat showed inhibition of 60% at 0.5 microg/well. Inhibition of Mtb H,37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4 microg/well and orlistat at 0.5 microg/well were observed respectively on bacterial growth.
  
  
  CONCLUSIONS
  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.
authors.affiliation: JB Tropical Disease Research Centre, MGIMS, Sevagram, India.
authors.forename: Vinita
authors.initials: V
authors.lastname: Hutke
authors.forename: Gauri
authors.initials: G
authors.lastname: Wankhade
authors.forename: Pranita J
authors.initials: PJ
authors.lastname: Waghmare
authors.forename: B C
authors.initials: BC
authors.lastname: Harinath
mesh_terms: D000995:Antitubercular Agents / Q000493:pharmacokinetics; D061245:Axenic Culture; D001426:Bacterial Proteins / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007538:Isoniazid / Q000493:pharmacokinetics*; D007783:Lactones / Q000493:pharmacokinetics*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000378:metabolism; D000077403:Orlistat; D057057:Serine Proteases / Q000378:metabolism*; D014376:Tuberculosis / Q000188:drug therapy
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D007783:Lactones; D000077403:Orlistat; D057057:Serine Proteases; D007538:Isoniazid