Method and reagent for the enzymatic determination of enzyme substrates

• US 4,517,287 A
• Filed: 03/25/1980
• Issued: 05/14/1985
• Est. Priority Date: 04/04/1979
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Klose, Sigmar, Bernt, Erich, Scheibe, Peter
• Primary Examiner(s): Warden, Robert J.
• Application Number:
  US06/133,722
• Time in Patent Office: 1,876 Days
• Field of Search: 435/14, 435/4, 435/10, 435/11, 435/20, 435/28, 435/16, 435/22, 435/15, 435/26
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Q 1/60 involving cholesterol
• CPC Class Codes: C12Q 1/62 involving uric acid

Abstract

The present invention provides a reagent for the enzymatic determination of an enzyme substrate, containing a system for the determination of the substrate, which comprises at least one enzyme, at least one buffer substance and at least one indicator substance and optionally contains adjuvants, wherein the reagent contains a definite amount of the enzyme substrate in an insufficient amount with regard to the substrate determination capacity of the system for the determination of this substrate.

The present invention also provides a process for the enzymatic determination of an enzyme substrate in the presence of disturbing substances which can react with the substrate itself or with an intermediate or end product of the indicator reaction, wherein, before the addition of the sample to be investigated, a definite amount of the substrate is first reacted with the reagent until reaction is complete, whereafter the sample to be determined is added.

Claims

1. In a reagent for the enzymatic determination of an enzyme substrate, containing a substrate determination system including at least one enzyme, at least one buffer substance and at least one indicator substance, the improvement comprising

including in said reagent a defined amount of the enzyme substrate to be determined, which amount is less than the substrate determination capacity of the said system but is sufficient to react with interfering reagent ingredients.

2. Reagent as claimed in claim 1 wherein the substrate is glucose and said substrate determination system comprises glucose oxidase, peroxidase, ABTS and buffer.

3. Reagent as claimed in claim 1 wherein the substrate is uric acid and said substrate determination system comprises uricase, peroxidase, a 2,4-dichlorophenolate, 4-aminoantipyrine and buffer.

4. Reagent as claimed in claim 1 wherein the substrate is cholesterol and said substrate determination system comprises cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, phenol and buffer.

5. Reagent as claimed in claim 1 wherein the substrate is glucose and said substrate determination system comprises glucose oxidase, peroxidase, 4-aminophenazone, phenol and buffer.

6. Reagent as claimed in claim 1 also containing at least one adjuvant.

7. Method for the enzymatic determination of an enzyme substrate with a reagent which contains a system for determining the substrate and interfering substances which can react with the substrate or an intermediate or end product of the indicator reaction, which method comprises reacting, before the addition of the sample to be analyzed, a defined amount of the substrate with said reagent until reaction is complete, and then contacting the sample to be determined with said reagent.

8. Method as claimed in claim 7 wherein the enzyme substrate is glucose and the system for determining the substrate comprises glucose oxidase, peroxidase, ABTS and buffer.

9. Method as claimed in claim 7 wherein the substrate is uric acid and the system for determining the substrate comprises uricase, peroxidase, a 2,4-dichlorophenolate, 4-aminoantipyrine and buffer.

10. Method as claimed in claim 7 wherein the substrate is cholesterol and the system for determining the substrate comprises cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, phenol and buffer.

11. Method as claimed in claim 7 wherein the substrate is glucose and the system for determining the substrate comprises glucose oxidase, peroxidase, 4-aminophenazone, phenol and buffer.