Analytical Sciences

A Novel ESR Method for Horseradish Peroxidase ActivityUsing a Combination of p-Acetamidophenol and Hydroxylamine, and ItsApplication to Enzyme Immunoassays

Masaaki AOYAMA*, Masanobu SHIGA**, HiroakiOHYA**, *** and HitoshiKAMADA*

*The Institute for Life Support Technology of Yamagata TechnopolisFoundation, Matsuei, Yamagata 990-2473, Japan
**Dojindo Laboratories, U. S. Office, 3 Bethesda Metro Center Suite700, Bethesda, MD 20814, USA
***Graduate School of Engineering, Yamagata University, Jonan,Yonezawa 992-8510, Japan

A new analytical method estimating horseradishperoxidase (HRP) activity for the enzyme immunoassay (EIA) has beendeveloped by using ESR spectroscopy. The optimal conditions sensitivelydetermining HRP activity: buffers, pH, and temperature, have beeninvestigated by using p-acetamidophenol (p-AP) as thesubstrate of HRP. An appropriate hydroxylamine was used as a trapper of thep-AP radical, which can be converted into a stable nitroxide by aredox reaction with the radical derived from the substrate. The sensitivityof the HRP activity determined by ESR measurement of the nitroxide thusgenerated is 19 to 25 times higher than those of conventional luminometryand colorimetry assays. Typical applications of the method to ThyroidStimulating Hormone and glucose assays are also presented to show theusefulness of the method. (Keywords: Enzyme immunoassay, p-acetamidophenol,hydroxylamine, ESR, redox reaction)

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