No abstract

A homogeneous substrate-labeled fluorescent immunoassay was developed for the measurement of kanamycin concentrations in serum. A fluorogenic drug reagent (FDR) (i-galactosyl-umbelliferone-tobramycin) was prepared that is nonfluorescent under the conditions of the assay but is hydrolyzed upon catalysis by l-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 ,3-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.Kanamycin is an aminoglycoside antibiotic which is effective in the treatment of severe infections caused by gramnegative bacteria (3). It has pharmacokinetic properties very similar to amikacin (4,8). It has been used to treat infections caused by Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and Proteus spp., as well as other bacteria. Since kanamycin has a narrow therapeutic range, proper dosage administration is necessary to achieve maximum therapeutic efficacy and to avoid adverse effects, including nephrotoxicity and ototoxicity (5). Interpatient variability is also evident in response to a given dose of an aminoglycoside antibiotic. Therefore, therapeutic drug monitoring with a fast and accurate method is the most effective way of ensuring adequate therapy (6, 7, 10). We developed a homogeneous substrate-labeled fluorescent immunoassay (SLFIA) for the determination of total kanamycin levels in serum. The principle of the SLFIA, described previously (2,12,13), is based on the following findings. The kanamycin fluorogenic drug reagent (FDR), which is actually the cross-reacting compound ,-galactosyl-umbelliferone-tobramycin, is nonfluorescent under the conditions of the assay. (The FDR compound was used for convenience, since it had previously been synthesized for use in an SLFIA for the detection of tobramycin.) However, hydrolysis catalyzed by P-galactosidase (E. coli 3D-galactoside galactohydrolase; EC 3.2.1.23) yields a fluorescent product. When antiserum to kanamycin binds to the FDR, it is inactive as a substrate for ,-galactosidase, and fluorescence is inhibited. Kanamycin in the clinical sample competes with a constant amount of FDR for a limiting number of antibody-binding sites so that a fluorescent response is generated as a function of the kanamycin concentration.The performance of the kanamycin assay was evaluated for accuracy and precision, standard curve reproducibility, time dependence, sensitivity and cross-reactivity. Comparison of the assay with radioimmunoassays (RIA), highperformance liquid chromatography (HPLC), and gravimetric values is shown. Enzyme. 1-Galactosidase from E. coli (Sigma Chemical Co., St. Louis, Mo.) was assayed at 25°C in 50 mM Bicine (N,N-bis[2-hydroxyethyl]-glycine)...

The current substrate-labeled fluorescent immunoassay for gentamicin has been modified by substituting monoclonal antibodies for conventional antiserum to gentamicin. The standard curve generated with gentamicin monoclonal antibody was essentially linear from 0 to 12 p,g/ml, could detect 0.4 .g of gentamicin per ml, and had overall intra-and interassay precision values (coefficients of variation) of 1.9 and 5.0o, respectively. The substrate-labeled fluorescent immunoassay produced with gentamicin monoclonal antibody gave results with clinical specimens comparable (r = 0.991) to those obtained with the commercially available substrate-labeled fluorescent immunoassay and also with an enzyme immunoassay, a radioimmunoassay, and a fluorescent immunoassay. This technologically state-of-the-art assay employs both a nonisotopic label and monoclonal antibodies. It offers excellent precision, sensitivity, lot-to-lot reproducibility, linearity, and reagent stability.A substrate-labeled fluorescent immunoassay (SLFIA) for measuring gentamicin levels in human serum or plasma that employs a conventional antiserum was described previously (1, 2). Sisomicin, which is structurally similar to gentamicin, is labeled with ,-galactosyl-umbeliiferone to form a ,B-galactosyl-umbelUiferone-sisomicin conjugate. Hereafter this conjugate, a fluorogenic substrate for the enzyme 1-galactosidase (EC 3.2.1.23), is denoted as the fluorogenic drug reagent (FDR). The FDR is nonfluorescent under normal assay conditions unless it is hydrolyzed by the enzyme to yield a fluorescent product. When the FDR is bound by antibody to gentamicin, enzymatic hydrolysis is inhibited.This inhibition is relieved upon the addition of gentamicin to the reaction because it competes with the FDR for the limiting number of antibody-binding sites. The amount of unbound FDR available for reaction with the enzyme is proportional to the concentration of gentamicin in the sample.Since conventional antiserum is a heterogeneous mixture of antibodies varying in specificity and affinity, immunoassay performance characteristics are influenced by animal-to-animal and within-animal variation (9). By design, monoclonal antibodies produced via the hybridoma methodology are uniformly specific, but not necessarily monospecific, and are homogeneous with respect to antigen binding properties (3, 4). Monoclonal antibodies are advantageous for use in immunoassays because they are homogeneous, standardized reagents and can be obtained in virtually limitless quantities (9, 10). We have obtained a murine monoclonal antibody to gentamicin (8;

We have developed a homogeneous substrate-labeled fluorescent immunoassay for the measurement of dibekacin concentrations in serum and plasma. The fluorogenic enzyme substrate ,B-galactosyl-umbelUiferone was covalently attached to tobramycin, an aminoglycoside structurally similar to dibekacin, to prepare a fluorogenic drug reagent (FDR). The FDR is nonfluorescent under assay conditions, but fluoresces upon hydrolysis by P-galactosidase. However, binding of the FDR by antiserum to the cross-reactive drug kanamycin prevents enzyme hydrolysis. The fixed level of FDR competes with dibekacin within the sample for the limiting number of antibody-binding sites in the reaction mixture. Unbound FDR is hydrolyzed by ,B-galactosidase to release a fluorescent product that is proportional to the dibekacin concentration in the sample. The assay exhibits good precision, standard curve reproducibility, recovery, sensitivity, and correlation with a comparative method. Additionally, the substrate-labeled fluorescent immunoassay is rapid and easy to perform. Dibekacin (3',4'-dideoxykanamycin B) is a semisynthetic aminoglycoside antibiotic useful in the treatment of severe gram-negative bacterial infections (6, 9, 10). Since dibekacin has a rather narrow therapeutic range and may be ototoxic or nephrotoxic at high concentrations in the blood, therapeutic drug monitoring is indicated (3, 4,7,8

Splenocytes from BALB/c mice immunized with a gentamicin-hemocyanin conjugate were fused with X63-Ag8.653 murine myeloma cells to produce hybridomas that secreted monoclonal antibody to gentamicin. Sixteen positive clones were obtained. The monoclonal antibody chosen for a gentamicin immunoassay has been characterized with respect to class, subclass, type of light chain, electrophoretic homogeneity, and binding affinity. Gentamicin monoclonal antibody purified from mouse ascites fluid was analyzed by immunoelectrophoresis, double immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and polyacrylamide gel electrophoresis (PAGE). The results show that the antibody is an IgG2a (kappa). Two bands were detected when the purified antibody was electrophoresed on polyacrylamide gels: a major band and a less mobile minor component (5.6% of the major band). Both were IgG2a (kappa). The major band contained antibody which bound 2.1 moles of the substrate-labeled gentamicin derivative, beta-galactosyl-umbelliferone sisomicin, per mole of IgG, whereas one mole of the minor band bound only 0.095 moles of the substrate-labeled conjugate. The antibody has an affinity constant of 1.22 X 10(10) M-1.