Capillary electrophoretic immunoassays for digoxin and gentamicin with laser-induced fluorescence polarization detection

Abstract

New immunoassays for therapeutic drugs digoxin and gentamicin have been described, which involved the separation of free and antibody-bound drug by capillary electrophoresis (CE) and the detection by laser-induced fluorescence polarization (LIFP). While the fluorescein-labeled digoxin and gentamicin (tracers) displayed negligible fluorescence polarization in solution, the complex formation between these small molecules and their antibodies resulted in substantial increases in fluorescence polarization due to the increase in molecular size. The LIFP detection, capable of measuring vertically and horizontally polarized fluorescence components simultaneously, provides enhanced capability for the identification of complex in capillary electrophoretic immunoassays. Proper adjustments of the running buffer pH and the ratio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin–antibody complex remained stable during CE separation with running buffer pH ranging from 9.3 to 12. Calibration curves covering a concentration range of 0.05 to 0.5 ng/ml were obtained with a running buffer of pH 12. The concentration and mass detection limits were 0.02 ng/ml and 26 zmol, respectively. For gentamicin assay, the running buffer pH 10 was used to reduce the adsorption of the tracer while minimizing the dissociation of the antibody–tracer complex during the separation. The calibration curves covered a concentration range 0.05–1.0 μg/ml, with a concentration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin.