Kinetic assay for acid phosphotase and composition therefore

Claim 2

A reagent system for an acid phosphotase assay comprising: (a) maltose; (b) an organic phosphate selected from a group consisting of β-glycerophosphate, phenol phosphate, p-nitrophenol phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phenolphthalein monophosphate; (c) maltose phosphorylase; (d) a co-enzyme selected from a group consisting of beta-nicotinamide-adenine dinucleotide, beta-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof; (e) glucose-6-phosphate dehydrogenase; (f) beta-D-phosphoglucomutase; wherein the above are present in the amount such that the acid phosphatase to be assayed is rate-limiting, and (g) a non-phosphate containing buffer having a pH from about 4.5 to about 6.

Claim 3

The reagent system of claim 1 wherein said buffer is selected from a group consisting of sodium citrate, sodium hydrogen maleate, and sodium cacodylate.

Claim 4

The reagent system of claim 1 further comprising glucose-1,6-diphosphate.

Claim 5

The reagent system of claim 3 further comprising 6-phosphogluconate dehydrogenase.

Claim 6

The reagent system of claim 3 further comprising a cation selected from a group consisting of Mn +2 , Mg +2 , Co +2 , Zn +2 , Ni +2 , and mixtures thereof.

Claim 7

The reagent system of claim 3 further comprising 6-phosphogluconate dehydrogenase and a cation selected from a group consisting of Mn +2 , Mg +2 , Co +2 , Zn +2 , Ni +2 , and mixtures thereof.

Claim 8

The reagent system of claim 6 wherein said cation is selected from the group consisting of Mg +2 , Mn +2 , Co +2 , and mixtures thereof.

Claim 9

The reagent system of claim 7 wherein said glucose-1,6-diphosphate consists essentially of the beta formed thereof.

Claim 10

A reagent system for an acid phosphatase assay wherein the acid phosphotase to be assayed is rate-limiting comprising per liter of reagent: (a) at least about 2 millimoles of maltose; (b) at least about 0.5 millimoles of organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate, p-nitrophenyl phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phenolphthalein monophosphate; (c) at least about 500 International Units of maltose phosphorylase; (d) at least about 0.1 millimole of a co-enzyme selected from a group consisting of β-nicotinamide-adenine dinucleotide, β-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof; (e) at least about 1000 International Units of glucose-6-phosphate dehydrogenase; (f) at least about 100 International Units of β-D-phosphoglucomutase; and (g) at least about 0.01 moles of a non-phosphate buffer.

Claim 11

A reagent system for an acid phosphatase assay wherein the acid phosphotase to be assayed is rate-limiting comprising per liter of reagent: (a) at least about 2 millimoles of maltose; (b) at least about 0.5 millimoles of organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate, p-nitrophenyl phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phenolphthalein monophosphate; (c) at least about 500 International Units of maltose phosphorylase; (d) at least about 0.1 millimole of a co-enzyme selected from a group consisting of β-nicotinamide-adenine dinucleotide, β-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof; (e) at least about 1000 International Units of glucose-6-phosphate dehydrogenase; (f) at least about 100 International Units of β-D-phosphoglucomutase; and (g) at least about 0.01 moles of a non-phosphate buffer.

Claim 12

The reagent system of claim 9 further comprising from about 0.02 to about 0.2 millimoles of glucose-1,6-diphosphate.

Claim 13

The reagent system of claim 10 further comprising from about 1 to about 5 millimoles of a cation selected from a group consisting of Mn +2 , Mg +2 , Co +2 , Zn +2 , Ni +2 , and mixtures thereof.

Claim 14

An acid phosphatase assay comprising: (a) performing simultaneous reactions at a pH of from about 4.5 to about 6 which comprise: (i) reacting an organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate, p-nitrophenyl phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phenolphthalein monophosphate in the presence of acid phosphatase to release inorganic phosphate; (ii) reacting maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and β-D-glucose-1-phosphate; (iii) reacting β-D-glucose-1-phosphate in the presence of β-D-phosphoglucomutase to form glucose-6-phosphate; and (iv) reacting glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogenase and a co-enzyme selected from a group consisting of β-nicotinamide-adenine dinucleotide, β-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof to form the reduced form of said co-enzyme and 6-phosphogluconate; and (b) measuring the rate of production of said reduced co-enzyme, wherein the acid phosphatase being measured is rate-limiting and wherein a non-phosphate buffer is used to control said pH.

Claim 15

An acid phosphatase assay comprising: (a) performing simultaneous reactions at a pH of from about 4.5 to about 6 which comprise: (i) reacting an organic phosphate selected from a group consisting of beta-glycerophosphate, phenyl phosphate, p-nitrophenyl phosphate, alpha-naphthyl phosphate, adenosine-3'-monophosphate, thymolphthalein monophosphate, and phenolphthalein monophosphate in the presence of acid phosphatase to release inorganic phosphate; (ii) reacting maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and β-D-glucose-1-phosphate; (iii) reacting β-D-glucose-1-phosphate in the presence of β-D-phosphoglucomutase to form glucose-6-phosphate; and (iv) reacting glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogenase and a co-enzyme selected from a group consisting of β-nicotinamide-adenine dinucleotide, β-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof to form the reduced form of said co-enzyme and 6-phosphogluconate; and (b) measuring the rate of production of said reduced co-enzyme, wherein the acid phosphatase being measured is rate-limiting and wherein a non-phosphate buffer is used to control said pH.

Claim 16

The acid phosphotase assay of claim 11 wherein said β-D-glucose-1-phosphate is reacted in the presence of β-D-phosphoglucomutase and glucose-1-6-diphosphate to form glucose-6-phosphate.

Claim 17

The acid phosphatase assay of claim 13 further comprising: reacting 6-phosphogluconate in the presence of said co-enzyme and 6-phosphogluconate dehydrogenase to form the reduced form of said co-enzyme and ribulose-5-phosphate.

Claim 18

The acid phosphatase assay of claim 13 wherein said β-D-glucose-1-phosphate is reacted in the presence of β-D-phosphoglucomutase, glucose-1,6-diphosphate, and a cation selected from a group consisting of Mn +2 , Mg +2 , Co +2 , Zn +2 , Ni +2 , and mixtures thereof to form glucose-6-phosphate.

Claim 19

The acid phosphatase assay of claim 15 further comprising: reacting 6-phosphogluconate in the presence of said co-enzyme and 6-phosphogluconate dehydrogenase to form the reduced form of said co-enzyme and ribulose-5-phosphate.

Claim 20

The acid phosphatase assay of claim 16 wherein said cation is selected from the group consisting of Mn +2 , Mg +2 , and mixtures thereof.

Claim 21

The acid phosphatase assay of claim 17 wherein said simultaneous reactions are performed at a pH of from about 5.0 to about 6.

Claim 22

The acid phosphatase assay of claim 12 wherein said simultaneous reactions are performed at a pH of from about 5.0 to about 6.