Method and kits for determining peroxidatively active catalysts
AssigneeBehringwerke Aktiengesellschaft
InventorsSharat Singh, Arthur C. Switchenko, Cheng-I Lin, Nurith Kurn, Edwin F. Ullman
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Abstract
Methods and compositions are disclosed for determining a peroxidatively active catalyst. The methods comprise the step of detecting a substance formed by the coupling reaction of (a) the product of the peroxidatively active catalyst-catalyzed oxidation of a benzidine with (b) a coupler other than benzidine. The methods have application in a wide variety of systems including assays for analytes. Also disclosed are kits for conducting methods and assays in accordance with the present invention.
Claims13
A method of determining a peroxidase, which method comprises contacting a medium suspected of containing a peroxidase with
- (a) a hydroperoxide
- (b) a benzidine
- wherein at least one amino group of said benzidine is a primary amine and at least one substituent positioned ortho thereto is a hydrogen atom;
- (c) and a coupler selected from the group consisting of ##STR42## with the proviso that said coupler compound has a two electron oxidation potential greater than that of said benzidine;
- and
- examining for the presence or absence of a substance formed by a reaction of a product of a peroxidase catalyzed oxidation of said benzidine and said coupler compound, the presence or absence thereof being related to the presence or absence of said peroxidase.
A kit for examining for the presence or absence of peroxidase activity, said kit comprising in packaged combination:
- (a) a hydroperoxide;
- (b) a benzidine wherein at least one amino group of said benzidine is a primary amine and at least one substituent positioned ortho thereto is a hydrogen atom;
- (c) a coupler selected from the group consisting of ##STR44## (d) a member of a specific binding pair (sbp) conjugated to a peroxidase enzyme.
An assay for an analyte which is a member of a specific binding pair (sbp), which assay comprises:
- combining a medium suspected of containing said analyte, a first sbp member that binds said analyte, and a peroxidase enzyme conjugated to a second sbp member, wherein said second sbp member binds to said analyte or to said first sbp member when said second sbp member is an analyte analog to form a complex, and
- examining for the presence or absence of activity of said peroxidase enzyme in said complex by treating said peroxidase enzyme with a hydroperoxide, a benzidine wherein at least one amino group of said benzidine is a primary amine and at least one substituent positioned ortho thereto is a hydrogen atom, and a coupler selected from the group consisting of:
- ##STR46##
Dependencies are inferred from the claim text.