A tubulin-based fluorescent polarization assay for paclitaxel

Abstract

This paper reports the development, characterization, and application of a separation-free, homogeneous, and simple to perform fluorescence polarization bioassay for paclitaxel. The bioassay is based on the binding interaction of paclitaxel to tubulin, the receptor protein on which this drug acts. The bioassay was carried out in a competitive format where the paclitaxel and a synthetic fluorescent-labeled paclitaxel (Rh-Tx) competed for tubulin binding, causing a change in fluorescence polarization, which was an inverse function of the paclitaxel concentration. The bioassay had a dynamic range from 0.03 to 0.35 μM, which falls in the therapeutic range (0.01–10 μM), and a limit of detection of 23 nM. The bioassay was selective against other naturally occurring taxanes such as baccatin III and 10-deacetylbaccatin III. However, there was interference from cephalomannine. The excellent sensitivity, accuracy, reproducibility, and simplicity make this analytical technique suitable for routine and high-throughput analyses and might be helpful in providing better care for patients. The bioassay was successfully applied to the determination of paclitaxel in human plasma.