Claims
- 1. A method for determining a dehydrogenase or its substrate in a dehydrogenase reaction, comprising the steps of:
- (a) mixing of dehydrogenase and its substrate in the presence of an electron carrier or a combination of a coenzyme and an electron carrier, such that H.sub.2 O.sub.2 is formed through transfer of electrons derived from the substrate by the dehydrogenase reaction to O.sub.2 via the electron carrier or via the coenzyme and electron carrier;
- (b) adding a stopping reagent to the mixture of step (a) such that the dehydrogenase reaction is terminated by the addition of said reagent;
- (c) adding a peroxidase and a chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase to the mixture of step (b ) such that the H.sub.2 O.sub.2 oxidizes the chormogen to be oxidized with H.sub.2 O.sub.2 to produce a dye and wherein step (c) is performed simultaneously or subsequently to step (b); and
- (d) measuring said dye and correlating said dye measurement to the present of said dehydrogenase or its substrate.
- 2. The method according to claim 1, wherein the dehydrogenase reaction is stopped by deactivating the dehydrogenase.
- 3. The method according to claim 2, wherein the dehydrogenase is deactivated by adding an acid or an alkali according to step (b).
- 4. The method according to claim,2, wherein the dehydrogenase is deactivated by changing the pH value of the reaction according to step (b) with a buffer solution having a different pH value.
- 5. The method according to claim 2, wherein the dehydrogenase is deactivated by adding a surfactant to the reaction according to step (b).
- 6. The method according to claim 2, wherein the dehydrogenase is deactivated by adding a dehydrogenase inhibitor to the reaction according to step (b).
- 7. The method according to claim 5, wherein the surfactant is sodium dodecyl sulfate.
- 8. The method according to claim 5, wherein the surfactant is sodium dodecylbenzene sulfonate.
- 9. The method according to claim 1, wherein the coenzyme is a member selected from the group consisting of NAD.sup.+, NADP.sup.+ and flavins.
- 10. The method according to claim 1, wherein the dehydrogenase is a member selected from the group consisting of glucose .alpha.-dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, 3-.alpha.-hydroxysteroid dehydrogenase, lactate dehydrogenase, L-glutamate dehydrogenase, leucine dehydrogenase, sarcosine dehydrogenase, amine dehydrogenase, succinate dehydrogenase, choline dehydrogenase, fructose dehydrogenase and sorbitol dehydrogenase.
- 11. The method according to claim 1, wherein the electron carrier is a member selected from the group consisting of phenazine methosulfate, 1-methoxy-5-methylphenazium methylsulfate and dimethylaminobenzo-.alpha.-phenazoxonium chloride.
- 12. The method according to claim 1, wherein the chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase is a color-forming pair combination selected from the group consisting of 4-aminoantipyrine/aniline derivatives, 4-aminoantipyrine/phenol derivatives and benzothiazolinone hydrazone derivative/aniline derivatives.
- 13. The method of claim 1, further comprising using a reagent system consisting essentially of (i) the dehydrogenase or the substrate for the dehydrogenase, (ii) the electron carrier or the coenzyme and the electron carrier, (iii) the stopping reagent for the dehydrogenase reaction, (iv) the peroxidase and (v) the chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of the peroxidase.
- 14. A reagent system for determining a substrate of a dehydrogenase, comprising a first reagent containing a combination of (i) the dehydrogenase and (ii) an electron carrier or a combination of a coenzyme and an electron carrier; and a second reagent containing (iii) a stopping reagent for the dehydrogenase reaction; and a third reagent containing a combination of (iv) a peroxidase and (v) a chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase.
- 15. A reagent system for determining a dehydrogenase, comprising a first reagent containing a combination of (i) a substrate of the dehydrogenase and (ii) and electron carrier or a combination of a coenzyme and an electron carrier; and a second reagent containing (iii) a stopping reagent for the dehydrogenase reaction; and a third reagent containing a combination of (iv) a peroxidase and (v) a chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase.
- 16. The reagent system for determining the dehydrogenase according to claim 15, which comprises (i) the substrate of the dehydrogenase, (ii) the electron carrier, (iii) the stopping reagent for the dehydrogenase reaction, (iv) the peroxidase and (v) the chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase.
- 17. The reagent system for determining the dehydrogenase according to claim 15, which comprises (i) the substrate of the dehydrogenase, (ii) the coenzyme and the electron carrier, (iii) the stopping reagent for the dehydrogenase reaction, (iv) the peroxidase and (v) the chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase.
- 18. A reagent system for determining a substrate of a dehydrogenase, comprising a first reagent containing a combination of (i) the dehydrogenase and (ii) and electron carrier or a combination of a coenzyme and an electron carrier; and a second reagent containing a combination of (iii) a stopping reagent for the dehydrogenase reaction, (iv) a peroxidase and (v) a chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase.
- 19. A reagent system for determining a dehydrogenase, comprising a first reagent containing a combination of (i) a substrate of the dehydrogenase and (ii) an electron carrier or a combination of a coenzyme and an electron carrier; and a second reagent containing a combination of (iii) a stopping solution for the dehydrogenase reaction, (iv) a peroxidase and (v) a chromogen which is oxidized to develop color with H.sub.2 O.sub.2 in the presence of peroxidase.
Priority Claims (1)
Number |
Date |
Country |
Kind |
62-82277 |
Apr 1987 |
JPX |
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Parent Case Info
This application is a continuation of U.S. application Ser. No. 07/177,354, now abandoned, filed Apr. 1, 1988.
US Referenced Citations (4)
Number |
Name |
Date |
Kind |
3985621 |
Maruyama et al. |
Oct 1976 |
|
4394444 |
Cameron et al. |
Jul 1983 |
|
4622296 |
Yamanishi et al. |
Nov 1986 |
|
4629697 |
Limbach et al. |
Dec 1986 |
|
Foreign Referenced Citations (3)
Number |
Date |
Country |
29104 |
May 1981 |
EPX |
0124909 |
Nov 1984 |
EPX |
0113181 |
Jul 1983 |
JPX |
Non-Patent Literature Citations (1)
Entry |
Chemical Abstracts, vol. 106, No. 13, p. 374 Abstract No. 99215r, Mar. 1987. |
Continuations (1)
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Number |
Date |
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Parent |
177354 |
Apr 1988 |
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