Methods, compositions and apparatuses for detection of gamma-hydroxybutyric acid (GHB)

A composition for assaying a sample for gamma-hydroxybutyric acid (GHB) comprising the following components:

• 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.

A test support comprising:

• 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.

A kit for assaying a sample for a gamma-hydroxybutyric acid (GHB) source comprising:

• 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.

A screening method for detecting GHB in a sample, comprising:

• 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.

A composition for assaying a sample for gamma-hydroxybutyric acid (GHB) comprising the following components:

• 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.