Method for the determination of substrates or enzyme activities

• US 4,168,205 A
• Filed: 04/28/1977
• Issued: 09/18/1979
• Est. Priority Date: 06/09/1976
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Lang, Gunter, Deneke, Ulfert, Roeschlau, Peter, Michal, Gerhard, Danninger, Josef
• Primary Examiner(s): Shapiro, Lionel M.
• Application Number:
  US05/792,073
• Time in Patent Office: 873 Days
• Field of Search: 195/103.5 C, 195/103.5 R, 195/99
• US Class Current: 435/10
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose

Abstract

In a process for the determination of substrates or enzyme activities in a test batch, utilizing a conventional redox reaction as a measurement reaction, the improvement comprising carrying out the process in the presence of ascorbate oxidase; the addition of ascorbate oxidase substantially eliminates the interfering effect of ascorbic acid frequently found in the test sample.

Claims

1. In a process for the determination of substrates or enzyme activities in a test batch, utilizing a redox reaction as a measurement reaction, the improvement comprising carrying out the process in the presence of ascorbate oxidase obtained from small marrow (Curcurbita pepo medullosa).

2. Process as claimed in claim 1 wherein 0.002 to 100 U ascorbate oxidase/ml. test batch are used.

3. Process as claimed in claim 2 wherein 0.01 to 30 U ascorbate oxidase/ml. test batch are used.

4. Process as claimed in claim 1 wherein the ascorbate oxidase is used together with an enzyme forming hydrogen peroxide.

5. Process as claimed in claim 1 wherein the ascorbate oxidase is used together with a tetrazolium salt.

6. Process as claimed in claim 1 wherein the ascorbate oxidase is used together with a phenol capable of being coupled oxidatively with a nucleophilic reagent.

7. Process as claimed in claim 1 wherein it is carried out at a pH value of from 4.0 to 8.5.

8. Process as claimed in claim 7 wherein there is used an ascorbate oxidase obtained from small marrows (Curcurbita pepo medullosa).

9. Reagent for the enzymatic determination of substrates or enzyme activities, comprising a system for the determination of a substrate or enzyme with a redox reaction as measurement reaction, wherein ascorbate oxidase obtained from small marrow (Curcurbita pepo medullosa) is additionally present.

10. Reagent as claimed in claim 9 wherein the system for the determination of a substrate serves for the determination of glucose and comprises peroxidase, glucose oxidase, o-dianisidine or 2-azino-di-3-ethyl-benzthiazoline-6-sulphonate and buffer.

11. Reagent as claimed in claim 9 wherein the system for the determination of a substrate serves for the determination of uric acid and comprises peroxidase, uricase, a phenol, aminoantipyrine and buffer.

12. Reagent as claimed in claim 11 wherein the phenol is 2,4-dichlorophenol.

13. Reagent as claimed in claim 9 wherein the system for the determination of a substrate serves for the determination of glucose and comprises hexokinase, glucose-6-phosphate dehydrogenase, nicotinamide-adenine-dinucleotide phosphate, diaphorase, a tetrazolium salt and buffer.

14. Reagent as claimed in claim 9 wherein the system for the determination of a substrate serves for the determination of glutamate and comprises glutamate dehydrogenase, diaphorase, nicotinamide-adenine-dinucleotide, a tetrazolium salt and buffer.

15. Reagent as claimed in claim 9 wherein the system for the determination of a substrate serves for the determination of tyrosine and comprises tyrosinase, 3-methyl-6-potassium sulphonyl-benzthiazolone-2-hydrazone and buffer.

16. Reagent as claimed in claim 9 wherein the system for the determination of a substrate serves for the determination of pyrocatechol and comprises diphenol oxidase, 3-methyl-6-potassium sulphonyl-benzthiazolone-2-hydrazone and buffer.

17. Reagent as claimed in claim 9 wherein the system for the determination of a substrate and the ascorbate oxidase are impregnated with an absorbent carrier material.

18. Reagent as claimed in claim 17 comprising an absorbent carrier impregnated with glucose oxidase, peroxidase, ascorbate oxidase, a 9-(γ

-dimethylaminopropyl)-6-chloro-3-aminocarbazole salt and buffer.

19. Reagent as claimed in claim 18 comprising an absorbent carrier impregnated with an ethylenediamine-tetraacetic acid salt, dioctyl sodium sulphosuccinate, 2,5-dimethylhexane-2,5-dihydroperoxide, phosphoric acid trimorpholide, ascorbate oxidase, 3,3'"'"',5,5'"'"'-tetramethylbenzidine, phenanthridine and buffer.

20. Reagent as claimed in claim 17 wherein the aborbent carrier is a paper.

21. Reagent as claimed in claim 9 wherein the ascorbate oxidase is impregnated into a water-soluble sheet of carrier material and the system for the determination of a substrate is impregnated into a water-insoluble sheet of absorbent carrier material, the water-soluble and the water-insoluble carrier materials being placed on top of one another.

22. Reagent as claimed in claim 17 comprising ascorbate oxidase, gum guaiac and buffer.