Coupled enzyme systems for determination of dissolved substances

• US 4,490,465 A
• Filed: 03/25/1983
• Issued: 12/25/1984
• Est. Priority Date: 03/26/1982
• Status: Expired due to Fees

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Helger, Roland, Limbach, Berthold
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Deck, Randall E.
• Application Number:
  US06/478,765
• Time in Patent Office: 641 Days
• Field of Search: 435/14, 435/26, 435/25, 435/4, 435/805, 435/810
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A test system having an extended range of measurement and a corresponding procedure for the determination of substances in liquids contains several enzymes or enzyme systems together which, independently of one another, react directly or indirectly with the substance to be determined. On adding the test system to the sample solution, various final products are produced which can be distinguished analytically and evaluated by measurement techniques or visually. For example, glucose can be determined over a wide range of concentrations using a combination of an NAD-dependent dehydrogenase and a non-NAD-dependent dehydrogenase.

Claims

1. A method for determining the amount of a substrate in solution, said substrate being capable of direct or indirect conversion catalyzed by a dehydrogenase, comprising the steps of:

(a) admixing a liquid sample containing said substrate with an enzymatic assay system comprising at least a first and second enzyme, to produce an assay solution;

wherein each of said enzymes is independently capable of catalyzing the direct or indirect conversion of said substrate, said conversion in each case resulting in production of a product capable of separate direct or indirect analytical measurement or visualization; and

wherein said first enzyme is a non-pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a first detectable product over a first detection range, and said second enzyme is a pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a second detectable product over a second, different detection range; and

(b) directly or indirectly measuring or visualizing said detectable products in said assay solution.

2. The method of claim 1, wherein said assay system further comprises a first electron donor or acceptor which is oxidized or reduced concomitant with conversion by said non-pyridine-linked dehydrogenase, and a second electron donor or acceptor which is oxidized or reduced concomitant with conversion by said pyridine-linked dehydrogenase;

and wherein said second electron donor or acceptor is reduced or oxidized nicotinamide-adenine dinucleotide (NADH or NAD⁺) or nicotinamide-adenine dinucleotide phosphate (NADPH or NADP⁺).

3. The method of claim 2, wherein said first electron donor or acceptor is the reduced or oxidized form of a cytochrome, a quinone, a pyrroloquinoline-quinone, a flavin nucleotide, hexacyanoferrate, methylene blue, phenazine methosulfate, phenazine ethosulfate, benzoquinone, dichlorophenol-indophenol, dichloroindophenol or trichloroindophenol.

4. The method of claim 2, wherein said substrate is alcohol, glucose, glycerol-3-phosphate, glycine, lactate, malate or mannitol.

5. The method of claim 1, wherein said substrate is converted to an intermediate which is then directly converted in the reaction resulting in production of said product.

6. The method of claim 5, wherein said substrate is at least one triglyceride, and said intermediate is glycerol-3-phosphate.

7. An enzymatic assay reagent system for determining the amount of a substrate in solution, said substrate being capable of direct or indirect conversion catalyzed by a dehydrogenase, comprising an admixture of at least two enzymes, each of which is independently capable of catalyzing the direct or indirect conversion of said substrate, said conversion in each case resulting in production of a product capable of separate direct or indirect analytical measurement or visualization;

wherein said first enzyme is a non-pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a first detectable product over a first detection range, and said second enzyme is a pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a second detectable product over a second, different detection range.

8. The assay reagent system of claim 7, which further comprises a first electron donor or acceptor which is oxidized or reduced concomitant with conversion by said non-pyridine-linked dehydrogenase, and a second electron donor or acceptor which is oxidized or reduced concomitant with conversion by said pyridine-linked dehydrogenase;

and wherein said second electron donor or acceptor is reduced or oxidized nicotinamide-adenine dinucleotide (NADH or NAD⁺) or nicotinamide-adenine dinucleotide phosphate (NADPH or NADP⁺).

9. The assay reagent system of claim 8, wherein said first electron donor or acceptor is the reduced or oxidized form of a cytochrome, a quinone, a pyrroloquinoline-quinone, a flavin nucleotide, hexacyanoferrate, methylene blue, phenazine methosulfate, phenazine ethosulfate, benzoquinone, dichlorophenol-indophenol, dichloroindophenol or trichloroindophenol.

10. The assay reagent system of claim 8, wherein said dehydrogenases are capable of converting alcohol, glucose, glycerol-3-phosphate, glycine, lactate, malate or mannitol.

11. The assay reagent system of claim 10, wherein said second enzyme is a pyridine-linked lactate dehydrogenase;

and said first enzyme is a non-pyridine-linked lactate dehydrogenase.

12. The assay reagent system of claim 8, wherein said second enzyme is a pyridine-linked glucose dehydrogenase from Bacillus megaterium;

and said first enzyme is a non-pyridine-linked glucose dehydrogenase from Acinetobacter calcoaceticus.

13. The assay reagent system of claim 12, wherein said second enzyme is a pyridine-linked alcohol dehydrogenase;

and said first enzyme is a non-pyridine-linked alcohol dehydrogenase.

14. The assay reagent system of claim 8, wherein said first electron acceptor is dichlorophenol-indophenol.

15. The assay reagent system of claim 8, which further comprises a tetrazolium salt and a system for transferring electrons thereto.

16. The assay reagent system of claim 8, which further comprises glucose oxidase and peroxidase.

17. In a kit for use in an enzymatic assay to determine the amount of a substrate in solution, said substrate being capable of direct or indirect conversion catalyzed by a dehydrogenase, said kit comprising an assay reagent system comprising at least one enzyme for converting said substrate, together with coenzymes and auxiliaries necessary for carrying out an enzymatic assay using said assay reagent system, and at least one container for preparing an assay solution,

the improvement wherein said assay reagent system comprises an admixture of at least two enzymes, each of which is independently capable of catalyzing the direct or indirect conversion of said substrate, said conversion in each case resulting in production of a product capable of separate direct or indirect analytical measurement or visualization;

wherein said first enzyme is a non-pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a first detectable product over a first detection range, and said second enzyme is a pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a second detectable product over a second, different detection range.

18. The kit of claim 17, wherein said assay system further comprises diaphorase.

19. In a test strip for use in an enzymatic assay to determine the amount of a substrate in solution, said substrate being capable of direct or indirect conversion catalyzed by a dehydrogenase, said test strip comprising an absorbent material impregnated with a solution of an enzymatic assay reagent system for said substrate, together with coenzymes and auxiliaries necessary for carrying out said assay and visualizing the result,

the improvement wherein said assay reagent system comprises an admixture of at least two enzymes, each of which is independently capable of catalyzing the direct or indirect conversion of said substrate, said conversion in each case resulting in production of a product capable of separate direct or indirect analytical measurement or visualization;

wherein said first enzyme is a non-pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a first detectable product over a first detection range, and second enzyme is a pyridine-linked dehydrogenase which catalyzes conversion and resultant production of a second detectable product over a second, different detection range.