Fluorescence depolarization assay for quantifying α-amylase in serum and urine

Abstract

We have developed a new method for quantifying α-amylase (EC 3.2.1.1) in serum and urine by fluorescence depolarization. Amylase in the sample catalyzes the hydrolysis of the substrate, a fluorescein-labeled amylose. This results in decreased fluorescence polarization, owing to the increased rate of rotation of the amylose fragment relative to the intact substrate. The TDx amylase assay is calibrated with six human-serum-based pancreatic amylase calibrators. Amylase activities are determined by interpolation from the calibration curve, which is stored in the TDx analyzer's memory. Results correlate well with those by the Du Pont aca assay and the Beckman 'DRI-STAT' assay. Endogenous glucose does not interfere. CVs are <6%, and the reagents are stable in liquid form.