Serum theophylline determination by fluorescence polarization immunoassay utilizing an umbelliferone derivative as a fluorescent label

Abstract

We report the development of a non-radioisotopic homogeneous immunoassay to determine serum theophylline concentrations using fluorescence polarization. Theophylline is a therapeutic drug which is used in the treatment of asthma. Monitoring this drug in serum is necessary to ensure effective dosage and to avoid toxicity. The fluorescence polarization immunoassay is based on labeling the analyte with a fluorescent probe and monitoring the changes in the probe's fluorescence polarization occurring in the hapten-antibody reaction. Previous reported fluorescence polarization immunoassays invariably involved the use of fluorescein as a fluorescent label. The existence of polymorphic forms in fluorescein and the presence of contaminants in commercial preparations of fluorescein have led us to use an alternative fluorophore, namely, an umbelliferone derivative, in the polarization immunoassay. This fluoreceent probe is characterized by a high quantum yield, a large extinction coefficient, a large Stokes shift, and the absence of nonspecific interaction with normal human serum. The spectroscopic properties of this label including both the excitation and emission polarization spectra are presented, and the procedure to obtain the optimized fluorescence polarization standard curve is described. Using the polarization immunoassay technique, the theophylline concentrations of 25 clinical serum samples were determined. The values obtained correlated well with values determined by high-pressure liquid chromatography (equation of linear regression analysis y = 0.97 x + 1.33; and correlation coefficient, 0.93).