Determination of kanamycin concentration in serum by substrate-labeled fluorescent immunoassay

A homogeneous substrate-labeled fluorescent immunoassay was developed for the measurement of kanamycin concentrations in serum. A fluorogenic drug reagent (FDR) (beta-galactosyl-umbelliferone-tobramycin) was prepared that is nonfluorescent under the conditions of the assay but is hydrolyzed upon catalysis by beta-galactosidase to yield a fluorescent product. Binding of the FDR to the antiserum to kanamycin prevented enzyme hydrolysis. The fixed level of FDR in the assay competed with kanamycin in the sample for a limited number of antibody-binding sites. Unbound FDR was hydrolyzed by beta-galactosidase to release a fluorescent product that is proportional to the kanamycin concentration in the sample. The assay exhibited good sensitivity, precision, and accuracy and correlated well with other methods.