Stabilized liquid enzyme composition for glucose determination

Claim 2

A homogeneous liquid enzyme reagent with a long shelf like for use for the quantitative determination of glucose in a glucose assay using adenosine triphosphate and nicotinamide-adenine dinucleotide as coenzymes, the coenzymes being in excess, the enzyme reagent when initially prepared comprising: (a) at least about 60% v/v of water; (b) a water-miscible polyol organic solvent in an amount of from about 20 to about 40% v/v; (c) hexokinase enzyme; (d) glucose-6-phosphate dehydrogenase enzyme; (e) a stabilizer system comprising a heavy metal ion chelating agent in a sufficient amount of at least about 0.5 mM such that the enzyme reagent has a shelf life of at least two years when stored at a temperature in the range of from about 2 to about 8 °C.

Claim 3

The enzyme reagent of claim 1 wherein the chelating agent is ethylene diamine tetraacetic acid.

Claim 4

The enzyme reagent of claim 1 or claim 2 wherein the stabilizer system also comprises an antioxidant.

Claim 5

The enzyme reagent of claim 3 wherein the antioxidant is bovine serum albumin in an amount of at least 2 q per liter.

Claim 6

The enzyme reagent of claim 3 wherein the antioxidant comprises polyvinylpyrrolidone-40 in an amount of at least about 2 q per liter, and N-acetyl cysteine in an amount of at least about 0.4 g per liter.

Claim 7

The enzyme reagent of any preceding claim wherein the stabilizer system also comprises a microbio-control agent.

Claim 8

The enzyme reagent of claim 6 wherein the microbio­control agent is sodium azide in an amount of at least about 0.25 g per liter.

Claim 9

The enzyme reagent of claim 4 wherein the polyol solvent is glycerol, the enzyme reagent comprising: (a) hexokinase enzyme in an amount of from about 6 to about 80 KIU per liter; (b) glucose-6-phosphate dehydrogenase enzyme in an amount of from about 3 to about 60 KIU per liter; (c) bovine serum albumin, in an amount of from about 2 to about 8 g per liter. (d) ethylene diamine tetraacetic acid in an amount of from about 0.5 to about 5 mM; (e) sodium azide in an amount of from about 0.25 to about 1.0 g per liter; and (f) TRIS-HCl buffer, in an amount of from about 0.05 to about 0.2 mM; wherein the pH of the enzyme reagent is adjusted to about 7-1/2.

Claim 10

The enzyme reagent of claim 5 wherein the polyol solvent is glycerol, the enzyme reagent comprising: (a) hexokinase enzyme in an amount of from about 6 to about 80 KIU per liter; (b) glucose-6-phosphate dehydrogenase enzyme in an amount of from about 3 to about 60 KIU per liter; (c) polyvinylpyrrolidone-40 in an amount of from about 2 to about 8 gm per liter, and N-acetyl cysteine in an amount of from about 0.4 to about 1.6 g per liter; (d) ethylene diamine tetraacetic acid, in an amount of from about 0.5 to about 5 mM; (e) sodium azide, in an amount of from about 0.25 to about 1.0 g per liter; and (f) TRIS-HCl buffer, in an amount of from about 0.05 to about 0.2 mM; wherein the pH of the enzyme reagent is adjusted to about 7-1/2.

Claim 11

The enzyme reagent of claim 8 or claim 9 further characterized in that, during its shelf life, when it is used in a glucose assay procedure and mixed with a liquid coenzyme reagent containing magnesium ions and the coenzymes adenosine triphosphate and nicotinamide-adenine dinucleotide, and with a test sample containing glucose to form an assay reaction mixture, the volume ratio of enzyme reagent:coenzyme reagent:test sample being 100:1000:11, the glucose concentration in the test sample being between about 10 to about 500 mg per deciliter, the coenzymes in the reaction mixture being in substantial excess for the glucose assay relative to the glucose concentration in the same, and the chelating agent concentration in the reaction mixture being no more than about 50% of the magnesium ion concentration in the same, the end point for the assay can be reached in no more than about ten minutes.

Claim 12

The enzyme reagent of claim 10 wherein hexokinase is in an amount of from about 15 to about 35 KIU per liter, glucose-6-phosphate dehydrogenase is in an amount of from about 20 to about 35 KIU per liter, and wherein the molar ratio of adenosine triphosphate to glucose is more than about 3:1 in the reaction mixture, and the molar ratio of nicotinamide adenine dinucleotide to glucose in the reaction mixture is more than about 5:1, and the end point for the assay can be reached in no more than about two minutes.

Claim 13

The enzyme reagent of claim 8 or claim 9 further characterized in that, during its shelf life, when it is used in a glucose assay procedure and mixed with a liquid coenzyme reagent containing magnesium ions and the coenzymes adenosine triphosphate and nicotinamide-adenine dinucleotide, and with a test sample containing glucose to form an assay reaction mixture, the volume ratio of enzyme reagent:coenzyme reagent:test sample being 100:1000:11, the glucose concentration in the test sample being between about 10 to about 500 mg per deciliter, the coenzymes in the reaction mixture being in substantial excess for the glucose asay relative to the glucose concentration in the same, and the chelating agent concentration in the reaction mixture being no more than about 50% of the magnesium ion concentration in the same, the time of one-half reaction for the glucose assay is no more than about 1.5 times the time of one-half reaction for the identical assay when the enzyme reagent was initially prepared.

Claim 14

A kit of reagents for use for the quantitative determination of glucose in a glucose assay, the kit comprising: (a) a homogeneous liquid enzyme reagent with a long shelf life, in accordance with any preceding claim; (b) a homogeneous liquid coenzyme reagent comprising: (i) at least about 80 v/v of water; (ii) a water-miscible polyol organic solvent in an amount of from about 5 to about 20% v/v; (iii) adenosine triphosphate coenzyme; (iv) nicotinamide-adenine dinucleotide coenzyme; and (v) magnesium ions; the enzyme and coenzyme reagents being formulated such that when they are mixed together to form a combined reagent suitable for performing the glucose assay, in the combined reagent the amount of chelating agent from the enzyme reagent is no more than about half of the amount of magnesium ions from the coenzyme reagent, on a molar basis.

Claim 15

The kit of claim 13 wherein the combined reagent is formed by mixing 1:10 v/v of the enzyme and coenzyme reagents, and wherein in the combined reagent the amount of chelating agent from the enzyme reagent is no more than about one-tenth the amount of magnesium ions from the coenzyme reagent.

Claim 17

The method of claim 15 wherein the mixing steps comprising first mixing 1:10 v/v of the enzyme and coenzyme reagents to form a combined reagent and wherein in the combined reagent the amount of chelating agent from the enzyme reagent is no more than about one-tenth the amount of magnesium ions from the coenzyme reagent.