Substrate-labeled fluorescent immunoassay for amikacin in human serum.
A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.
D000583:Amikacin / Q000097:blood*; D001667:Binding, Competitive; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007612:Kanamycin / Q000031:analogs & derivatives*; D011863:Radioimmunoassay / Q000379:methods*; D013379:Substrate Specificity
D007106:Immune Sera; D007612:Kanamycin; D000583:Amikacin