A coupled enzyme assay for measurement of sialidase activity

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.