A Novel ESR Method for Horseradish Peroxidase Activity Using a Combination of p-Acetamidophenol and Hydroxylamine, and Its Application to Enzyme Immunoassays

A new analytical method estimating horseradish peroxidase (HRP) activity for the enzyme immunoassay (EIA) has been developed by using ESR spectroscopy. The optimal conditions sensitively determining HRP activity: buffers, pH, and temperature, have been investigated by using p-acetamidophenol (p-AP) as the substrate of HRP. An appropriate hydroxylamine was used as a trapper of the p-AP radical, which can be converted into a stable nitroxide by a redox reaction with the radical derived from the substrate. The sensitivity of the HRP activity determined by ESR measurement of the nitroxide thus generated is 19 to 25 times higher than those of conventional luminometry and colorimetry assays. Typical applications of the method to Thyroid Stimulating Hormone and glucose assays are also presented to show the usefulness of the method.