Improved methods for immunoassay of mycothiol

Improved enzyme-linked immunosorbent assay (ELISA) methods have been develo ped for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an af finity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-s pecific reagent 3-(N-maleimidopropionyl) biocytin. The MSH-biotin adduct,va s then captured with immobilized avidin and detected with anti-MSH antibody (biotin-capture ELISA) or was captured with immobilized anti-MSH antibody and detected with alkaline phosphatase-labelled avidin (MSH-capture ELISA). The MSH-capture ELISA was the most sensitive method, measuring as little a s 0.3 fmol of MSH. Methods for biotinylating MSH directly from Mycobacteriu m spp. are described. The MSH-capture ELISA was tested for the detection of M. avium seeded in human urine or cerebrospinal fluid samples and for scre ening mutant M. smegmatis strains to detect MSH production.