Method and kits for determining peroxidatively active catalysts

• US 5,532,138 A
• Filed: 06/21/1994
• Issued: 07/02/1996
• Est. Priority Date: 04/26/1990
• Status: Expired due to Fees

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Behringwerke Aktiengesellschaft (Sanofi )
• Inventors: Singh, Sharat, Kurn, Nurith, Ullman, Edwin F., Lin, Cheng-I, Switchenko, Arthur C.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US08/263,164
• Time in Patent Office: 742 Days
• Field of Search: 435/7.9-7.95, 435/28, 435/810, 435/975, 436/135
• US Class Current: 435/7.93
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: C12Q 2326/10 Benzidines
• CPC Class Codes: C12Q 2326/50 Phenols; Naphthols; Catechols
• CPC Class Codes: Y10S 435/975 Kit

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.

Claims

1. 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.

2. The method of claim 1 wherein said peroxidase forms part of a signal producing system in an assay for the detection of an analyte.

3. The method of claim 1 wherein said benzidine is selected from the group consisting of ##STR43##

4. The method of claim 1 wherein said substance examined for is a condensation product of said product and said coupler.

5. The method of claim 1 wherein said substance examined for is a chromophor.

6. 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.

7. The kit of claim 6 wherein said benzidine is selected from the group consisting of:

##STR45##

8. A kit of claim 6 wherein said peroxidase enzyme is horseradish peroxidase.

9. A kit of claim 6 wherein said sbp member is a ligand or a receptor.

10. A kit of claim 6 wherein said sbp member is an antibody, an antigen, or a ligand analog.

11. 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##

12. The assay of claim 11 wherein said first sbp member is insolubilized and is a receptor for said analyte and said second sbp member is a receptor for said analyte.

13. The assay of claim 11 wherein said second sbp member is an analyte analog and said first sbp member is a receptor for said analyte and said analyte analog.