Development of generic continuous-flow enzyme immunoassay system for analysis of aminoglycosides in serum

Abstract

A simple generic continuous-flow enzyme immunoassay (CFEIA) for analysis of aminoglycosides in serum has been successfully developed. The developed assay employed a specific monoclonal antibody and β-galactosidase (β-GAL) enzyme as label. The assay involves an off-line competitive binding reaction between the analyte and free labelled analyte for the binding sites of the antibody. After equilibrium is reached, the sample was injected into the flow system. The bound antibody complexes with the analyte and the labelled analyte were trapped in a protein G column, while the unbound free labelled analyte was eluted and detected colorimetrically down-stream, after reaction with chlorophenolic red-β- d-galactopyranoside as a substrate for the β-GAL enzyme. The concentration of the analyte in a sample was quantified by its ability to inhibit the binding of the analyte-enzyme conjugate to the antibody, and the signal was directly proportional to the concentration of the analyte in the original sample. The optimum conditions for the developed CFEIA were investigated and applied to the analysis of tobramycin, as a representative example of the aminoglycosides, in serum samples. The detection limit of the assay was 0.06 μg ml −1. The assay showed good precision; the coefficients of variation were 2.49–4.33 and 3.30–6.82% for intra- and inter-assay precision, respectively. Serum matrix constituents and the endogenous compounds did not interfere with the assay. Analytical recovery of spiked tobramycin, in the concentration range between 0.5 and 8.0 μg ml −1, was 101.55±3.14. The assay results correlated well with those obtained by high-performance liquid chromatography ( r=0.991). All the obtained results strongly demonstrate that the developed CFEIA is a suitable method for a rapid and reliable analysis of aminoglycosides in serum.