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

New immunoassays for therapeutic drugs digoxin and gentamicin have been des cribed, which involved the separation of free and antibody-bound drug by ca pillary electrophoresis (CE) and the detection by laser-induced fluorescenc e 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 result ed in substantial increases in fluorescence polarization due to the increas e in molecular size. The LIFP detection, capable of measuring vertically an d horizontally polarized fluorescence components simultaneously, provides e nhanced capability for the identification of complex in capillary electroph oretic immunoassays. Proper adjustments of the running buffer pH and the ra tio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin-antibody complex remained stable during CE se paration 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 ru nning buffer of pH 12. The concentration and mass detection limits were 0.0 2 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 cal ibration curves covered a concentration range 0.05-1.0 mu g/ml, with a conc entration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin. (C) 1999 Elsevier Science B.V. All rights reserved.