Comparison of radioimmunoassay with a new immunofluorescent method (FIAX) for measuring tobramycin in serum

Abstract

A new immunofluorescent method (FIAX) was compared with a radioimmunoassay procedure for the determination of tobramycin serum concentrations. When assaying three tobramycin control sera repeatedly, within-run and run-to-run variations by the FIAX method were all within acceptable limits, and no statistically significant differences were found. Results (3,9,11,14). Numerous methods have been described for assaying tobramycin serum concentrations including: bioassay (8, 10, 12), enzymatic assays (13), gas-liquid chromatography (6, 7), high-pressure liquid chromatography (1), and radioimmunoassay (RIA) (2).In this study, the FIAX immunofluorescent assay method was compared with RIA to determine the following: (i) within-run precision; (ii) run-to-run reproducibility; (iii) accuracy of determinations by comparison with expected values; and (iv) the reliability of the FIAX method in determining tobramycin levels in patient sera. MATERIALS AND METHODSThis study employed 155 selected serum samples from patients who had been under treatment with tobramycin. Specimens that could not be assayed on the day they were obtained were frozen and stored at -20°C until tested.RIA. Tobramycin RIA kits were obtained from Diagnostic Products Corp., Los Angeles, Calif. All samples were assayed according to their recommended protocol.FIAX. All reagents used for the FIAX assay procedure were supplied by International Diagnostic Technology, Santa Clara, Calif.The solid-phase immunofluorescence method primarily involves competition between a fluoresceinlabeled and an unlabeled antigen for a fixed amount of antibody immobilized on a reactive surface. In this case, specific anti-tobramycin antibody was immobilized on a defined polymeric surface such as that which is present on the StiQ sampler. This sampler was immersed sequentially into a mixture of fluorescein-labeled tobramycin and a sample (standards, controls, or unknown) containing unlabeled tobramycin. Thus, the amount of fluorescein-labeled tobramycin that attaches to the specific anti-tobramycin antibody is inversely proportional to the concentration of unlabeled tobramycin in the sample.The following procedure outlines the assay protocol used in this study: 700 gul of fluorescein-labeled tobramycin was dispensed into 12-by 75-mm glass tubes to which 10 ,ul of sample (standard, control, or unknown) was added; each sample was tested in duplicate. All tubes were shaken briefly by hand. StiQ samplers were placed into these tubes and then mixed on a mechanical shaker for 25 min at room temperature. The tubes and StiQ samplers were removed from the shaker and allowed to equilibrate for an additional 5 min before reading. Fluorescence is measured on a fluorometer set at 995 nm (excitation) and 530 nm (emission). The degree of fluorescence was expressed in fluorescence signal units. The fluorometer was adjusted to read 190 fluorescence signal units with a reference solution containing 1 ,ug of tobramycin per ml.A standard curve was constructed with tobramycin standards (included in t...

Analytical Methods for the Determination of Aminoglycosides Antibiotics by Chromatrographic Technique

“…Additionally, different agar pH needs to be used for the analysis of various kinds of aminoglycosides molecules. Although microbiological methods are useful, simple and relatively cheap but looks like they are inaccurate and subject to interferences caused by nonspecific inhibitors or other antimicrobial drugs [7].…”