Improved methods for immunoassay of mycothiol.
Improved enzyme-linked immunosorbent assay (ELISA) methods have been developed for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an affinity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-specific reagent 3-(N-maleimidopropionyl)biocytin. The MSH-biotin adduct was 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 as 0.3 fmol of MSH. Methods for biotinylating MSH directly from Mycobacterium 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 screening mutant M. smegmatis strains to detect MSH production.
Unson
M D
MD
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.
Newton
G L
GL
Arnold
K F
KF
Davis
C E
CE
Fahey
R C
RC
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D019921:Biotinylation; D002555:Cerebrospinal Fluid / Q000382:microbiology; D003545:Cysteine; D004187:Disaccharides / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006020:Glycopeptides; D006801:Humans; D007202:Indicators and Reagents; D007294:Inositol; D009165:Mycobacterium Infections, Nontuberculous / Q000134:cerebrospinal fluid* / Q000175:diagnosis; D020102:Mycobacterium smegmatis / Q000737:chemistry* / Q000254:growth & development / Q000302:isolation & purification; D011720:Pyrazoles; D011817:Rabbits; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014556:Urine / Q000382:microbiology
D000906:Antibodies; D004187:Disaccharides; D006020:Glycopeptides; D007202:Indicators and Reagents; D011720:Pyrazoles; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; D003545:Cysteine