Methods, compositions and apparatuses for detection of gamma-hydroxybutyric acid (GHB)
First Claim
1. A screening method for detecting gamma-hydroxybutyric acid (GHB), comprising:
- providing a sample which is suspected of comprising a source of gamma-hydroxybutyric acid (GHB);
providing a first oxidoreductase comprising an enzymatically active polypeptide that can oxidize GHB to succinic semialdehyde (SSA), said polypeptide selected from the group consisting of (a) a GHB dehydrogenase, (b) an SSA reductase, (c) a glucuronate reductase, and (d) an aflatoxin aldehyde reductase;
providing an oxidized cofactor that can be reduced by said first oxidoreductase in oxidizing GHB to SSA;
a first contacting step wherein the sample is contacted with the first oxidoreductase and the oxidized cofactor under conditions in which the first oxidoreductase can oxidize GHB and reduce the oxidized cofactor to a reduced cofactor to produce a sample suspected of comprising the reduced cofactor;
providing a second oxidoreductase that can oxidize the reduced cofactor;
providing a chromogen or dye that is detectably converted upon oxidation of the reduced cofactor by the second oxidoreductase;
a second contacting step wherein the sample suspected of comprising the reduced cofactor is contacted with the second oxidoreductase and the chromogen or dye under conditions in which the second oxidoreductase can oxidize the reduced cofactor and detectably convert the chromogen or dye; and
determining whether the chromogen or dye has been detectably converted which indicates the presence or absence of GHB in the sample.
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Accused Products
Abstract
Methods, compositions and articles of manufacture for assaying a sample for a GHB source are provided. A sample suspected of containing a GHB source is contacted with a first oxidoreductase selective for GHB and an oxidized cofactor. In the presence of GHB in the sample, the first oxidoreductase oxidizes GHB to succinic semialdehyde and reduces the cofactor. The reduced cofactor thus produced can be detected directly, or a hydride abstractor can be used that abstracts a hydride from the reduced cofactor and produces a detectable change. The hydride abstractor can be a second oxidoreductase that oxidizes the reduced cofactor and produces a detectable change in a chromogen or dye. Preferably a visual change is produced, allowing performance of the assay outside of a laboratory setting. Fusion proteins comprising the first oxidoreductase, polynucleotides encoding such proteins, host cells expressing such proteins, and vectors comprising such polynucleotides are also provided. Stabilized formulations of the first oxidoreductase are also provided. Test supports, devices, and compositions and kits comprising reagents for performing such methods are also provided. Techniques for performing the assay in the presence of ethanol and in the presence of GHB precursors in the sample are also provided.
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Citations
73 Claims
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1. A screening method for detecting gamma-hydroxybutyric acid (GHB), comprising:
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providing a sample which is suspected of comprising a source of gamma-hydroxybutyric acid (GHB);
providing a first oxidoreductase comprising an enzymatically active polypeptide that can oxidize GHB to succinic semialdehyde (SSA), said polypeptide selected from the group consisting of (a) a GHB dehydrogenase, (b) an SSA reductase, (c) a glucuronate reductase, and (d) an aflatoxin aldehyde reductase;
providing an oxidized cofactor that can be reduced by said first oxidoreductase in oxidizing GHB to SSA;
a first contacting step wherein the sample is contacted with the first oxidoreductase and the oxidized cofactor under conditions in which the first oxidoreductase can oxidize GHB and reduce the oxidized cofactor to a reduced cofactor to produce a sample suspected of comprising the reduced cofactor;
providing a second oxidoreductase that can oxidize the reduced cofactor;
providing a chromogen or dye that is detectably converted upon oxidation of the reduced cofactor by the second oxidoreductase;
a second contacting step wherein the sample suspected of comprising the reduced cofactor is contacted with the second oxidoreductase and the chromogen or dye under conditions in which the second oxidoreductase can oxidize the reduced cofactor and detectably convert the chromogen or dye; and
determining whether the chromogen or dye has been detectably converted which indicates the presence or absence of GHB in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 68, 69)
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43. A composition for assaying a sample for gamma-hydroxybutyric acid (GHB) comprising the following components:
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a first oxidoreductase comprising an enzymatically active polypeptide that can oxidize GHB to succinic semialdehyde (SSA), said polypeptide selected from the group consisting of (a) a GHB dehydrogenase, (b) an SSA reductase, (c) a glucuronate reductase, and (d) an aflatoxin aldehyde reductase;
an oxidized cofactor for the first oxidoreductase that is reduced upon oxidation of GHB by the first oxidoreductase;
a second oxidoreductase that can oxidize the reduced cofactor produced by the first oxidoreductase; and
a chromogen or dye that is detectably converted upon oxidation of the reduced cofactor by the second oxidoreductase, wherein the components are present in the composition in forms and amounts effective to produce a detectable change in the chromogen or dye upon contacting the composition with a sample comprising GHB.
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- 44. A fusion protein comprising a catalytically active Ralstonia eutropha succinic semialdehyde reductase and a heterologous peptide.
- 52. A stabilized formulation comprising a stabilizing agent and a catalytically active protein selected from a Ralstonia eutropha SSA reductase, a fusion protein comprising Ralstonia eutropha SSA reductase, a Ralstonia eutropha SSA reductase deletion mutant, and a fusion protein comprising a Ralstonia eutropha SSA reductase deletion mutant.
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57. A test support comprising:
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a support;
a first oxidoreductase operably associated with the support, wherein the first oxidoreductase can oxidize gamma-hydroxybutyrate (GHB) to succinic semialdehyde (SSA);
an oxidized cofactor for the first oxidoreductase operably associated with the support, wherein the oxidized cofactor is reduced to a reduced cofactor by the first oxidoreductase upon oxidation of GHB to SSA;
and a hydride abstractor operably associated with the support that can oxidize the reduced cofactor and produce a detectable color change on the support. - View Dependent Claims (58, 59, 60, 61, 62, 63, 64, 65)
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66. A kit for assaying a sample for a gamma-hydroxybutyric acid (GHB) source comprising:
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a first oxidoreductase that can oxidize GHB to succinic semialdehyde;
an oxidized cofactor for the first oxidoreductase that is reduced upon oxidation of GHB by the first oxidoreductase;
a second oxidoreductase that can oxidize the reduced cofactor produced by the first oxidoreductase;
a chromogen or dye that is detectably converted upon oxidation of the reduced cofactor by the second oxidoreductase;
a case for retaining the first oxidoreductase, the oxidized cofactor, the first oxidoreductase and the chromogen or dye; and
instructions provided with said case that describe how to use the components of the kit to assay a sample for a source of gamma-hydroxybutyric acid. - View Dependent Claims (67)
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72. A screening method for detecting GHB in a sample, comprising:
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providing a sample which is suspected of comprising gamma-hydroxybutyric acid (GHB);
providing a first oxidoreductase comprising an enzymatically active polypeptide from Ralstonia eutropha GHB dehydrogenase;
providing an oxidized nicotinamide cofactor that can be reduced by said first oxidoreductase in oxidizing GHB to SSA;
providing a diaphorase that can oxidize the reduced nicotinamide cofactor;
providing a tetrazolium salt;
contacting the sample with the first oxidoreductase, the oxidized nicotinamide cofactor, the diaphorase and the tetrazolium salt under conditions in which the first oxidoreductase can oxidize GHB and reduce the oxidized nicotinamide cofactor to a reduced nicotinamide cofactor and the diaphorase can oxidize the reduced nicotinamide cofactor and detectably convert the tetrazolium salt to a colored product; and
determining whether the tetrazolium salt has been converted to the colored product which indicates the presence or absence of GHB in the sample.
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73. A composition for assaying a sample for gamma-hydroxybutyric acid (GHB) comprising the following components:
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a first oxidoreductase comprising an enzymatically active polypeptide from Ralstonia eutropha GHB dehydrogenase;
an oxidized nicotinamide cofactor for the first oxidoreductase that is reduced upon oxidation of GHB by the first oxidoreductase;
a diaphorase that can oxidize the reduced nicotinamide cofactor produced by the first oxidoreductase; and
a tetrazolium salt that is detectably converted upon oxidation of the reduced cofactor by the second oxidoreductase, wherein the components are present in the composition in forms and amounts effective to convert the tetrazolium salt to a colored product upon contacting the composition with a sample comprising GHB.
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Specification