Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays

Immunoassays using capillary electrophoresis with laser-induced fluorescenc e detection (CE-LIF) is a powerful approach to the determination of trace a mounts of analytes in a complex biological matrix. However, its applicabili ty is limited by the requirement that the free and bound tracer (fluorescen tly labeled compound) be resolved for their identification and quantitation . Here we show that replacing LIF with laser-induced fluorescence polarizat ion (LIFP) permits ultrasensitive immunoassays to be performed with or with out the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstra te both homogeneous and heterogeneous immunoassay approaches. In the homoge neous scheme where the free and bound tracer were not separated, the fluore scence polarization of the mixture was a quantitative measure of the antibo dy-bound tracer. The concentration and mass detection limits for CBA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10(-18) mol), respectively. The heterogeneous assay involved a nearly baseline sep aration of the free and bound tracer using CE with a phosphate running buff er of pH 7.0. The complex of the tracer with the antibody had a fluorescenc e polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte con centration, and the fluorescence intensity of either the free or bound trac er was used for quantitation. Results from both assays suggest that the CE- LIFP approaches may have a wider application than the immunoassays based on either CE-LIF or fluorescence polarization alone. (C) 1999 Elsevier Scienc e B.V. All rights reserved.