Heterologous enzyme immunoassay for puromycin aminonucleoside using beta-D-galactosidase as a label.
A heterologous enzyme immunoassay (EIA) was developed to quantify puromycin aminonucleoside (PA). This double antibody assay was based on the use of anti-puromycin (PU) antibody and used beta-D-galactosidase-labeled PA conjugate prepared via N-(m-maleimidobenzoyloxy)succinimide. The standard curve of the assay ranged from 1 ng to 30 ng, and the lower limit of detection was 22.7 nM (1 ng/tube). The EIA was found to be approximately 20 times more sensitive than the homologous EIA for PA with anti-PA antibody and PA-beta-D-galactosidase conjugate. The heterologous EIA was free from interference by any purine or pyrimidine analogs and drug levels were easily determined in rat tissue following i.v. administration at a dose of 15 mg/kg. The sensitivity and specificity of the EIA should provide a valuable new tool for use in pharmacokinetic and toxicity studies of PA.
D000818:Animals; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D005696:Galactosidases; D007124:Immunoenzyme Techniques; D009401:Nephrosis / Q000139:chemically induced; D011691:Puromycin / Q000031:analogs & derivatives*; D011692:Puromycin Aminonucleoside / Q000276:immunology* / Q000633:toxicity; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution; D001616:beta-Galactosidase
D011691:Puromycin; D011692:Puromycin Aminonucleoside; D005696:Galactosidases; D001616:beta-Galactosidase