Competitive immunoassay for cyclosporine using capillary electrophoresis with laser induced fluorescence polarization detection

Abstract

Frequent monitoring of immunosuppressive drug cyclosporine A (CsA) in blood samples of tissue transplant patients is required in clinical practice because of the narrow therapeutic range between the immunosuppressive effect and the toxic effect of this drug. We describe a competitive immunoassay capillary electrophoresis (CE) with laser induced fluorescence polarization detection method, which is rapid and sensitive for the determination of CsA. The method is based on the competitive immunochemical reaction between the analyte and fluorescent hapten (CsA *) with the antibody, CE separation of the antibody bound and free fluorescent CsA *, followed by the laser induced fluorescence polarization detection (LIFP) of the fluorescent species. The method detection limit is governed by the stability of the antibody–CsA * complex rather than by the detector noise. The use of post-column sheath flow cuvette LIFP detection resulted in excellent detection limit, typically 0.9 n M (or 9·10 −19 mol for 1 nl injection) of CsA. CsA in whole blood samples from organ transplant patients were measured and results agreed well with those obtained by using a standard fluorescence polarization immunoassay. Each determination took less than 3 min. The CsA metabolites AM9 and AM19 were also determined by using this technique, and their cross-reactivities with the antibody were 13% and 2%, respectively.