Pierce Biotechnology, Inc.

Pierce Chemical Company

M Dean Savage

Laura L Sykaluk

Edward K Fujimoto

Timothy Jon McCauley

Sherri Z Millis

An assay is disclosed for measuring activity of enzymes, such as kinases, phosphatases, and proteases. Measurements of enzymatic activity are accomplished in a homogenous assay format utilizing a fluorescence quenching technique employing paramagnetic metal ions.

Homogenous assay for enzymatic activity

A precursor for the construction of chelated metal conjugates which demonstrate improved assay performance and utility in minimizing non-specific binding while maintaining specificity for target molecules is disclosed. The precursor has tridentate functionality towards multivalent ions such as iron and nickel and contains a diacetyl glycine group covalently linked via an amide to a molecule such as a proteinaceous molecule providing a primary amide group for amide bond formation. The precursor is preferably prepared in monomeric form by reacting nitrilotriacetic acid or a salt thereof in an aqueous medium at an alkaline pH of at least 8 with a proteinaceous molecule containing a primary amine group in the presence of a carbodiimide. The proteinaceous molecule may be bovine serum albumin or an enzyme such as alkaline phosphatase or horseradish peroxidase.

Preparing monomeric metal ion chelator containing diacetyl glycine group linked to proteinaceous molecule

A method is disclosed for preparing a fluorogenic phenolic compound with improved optical qualities for use in formulating a substrate solution for assay of peroxidase or peroxide activity. The method involves forming a solution under anoxic conditions which contains the phenolic compound and an aminopolycarboxylic acid or aminopolyphosphonic acid, or salt thereof, metal chelating agent and, while the solution is maintained under anoxic conditions, recovering the compound from the solution in an optically enhanced condition. A composition of matter is also disclosed which includes the fluorogenic phenolic compound in crystal form and a trace quantity of the metal chelating agent.

Process for the preparation of fluorogenic phenolic compounds

An improved method is disclosed for assaying peroxidase or peroxide activity utilizing a substrate solution containing a fluorogenic phenolic compound, hydrogen peroxide and a metal chelating compound. The improvement inclosed including within the assay, in an amount sufficient to enhance the effective working range of the assay, a boron acid or salt thereof, or a phosphine-based or hydride-based reducing agent.

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Rockford, IL, United States of America

M Dean Savage