β-Amylase determination

Claim 2

A reagent kit for the determination of alphaamylase, comprising: a. a water soluble carboxymethylated starch which is cleaved by alpha-amylase and which is not acted on by an exo-acting amylase, said water soluble carboxymethylated starch being present in an amount effective to determine alpha-amylase by measuring rate of glucose released after cleavage by alpha-amylase and enzymatic release of glucose by an exo-acting amylase; b. an (enzyme or) enzyme mixture including an exo-acting amylase which is free of alpha-amylase and which is capable of and is present in an amount effective for releasing glucose from alpha-amylase cleaved carboxymethylated starch to determine alpha-amylase by measuring the rate of glucose production, said enzyme mixture containing β-amylase and a maltase which is free of alpha-amylase; c. a buffer for maintaining pH for alpha-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. enzymatic reagent for detecting rate of glucose formation, said enzymatic reagent being present in an amount effective for determining alpha-amylase by measuring the rate of glucose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of alpha-amylase.

Claim 3

The K.t of claim 1 wherein component (d) is a glucose hexokinase rate detector, comprising adenosine triphosphate, nicotinamide adenine dinucleotide, hexokinase, and glucose-6-phosphate dehydrogenase.

Claim 4

The K.t of claim 2 wherein component (c) maintains a pH of from 5.0 to 9.0 in the media for α-amylase determination.

Claim 5

The K.t. of claim 3 wherein component (c) is comprised of at least one member selected from the group consisting of imidazole, β-glycerophosphate, 3-(N-morpholino)propane sulfonic acid, and N-2-acetamide-2-aminoethanesulfonic acid.

Claim 6

A reagent kit for the determination of α-amylase, comprising: a. a water soluble carboxymethylated starch which is cleaved by α-amylase and which is not acted on by an exo-acting amylase, said water soluble carboxymethylated starch having a ratio of carboxymethylated glucose to glucose of from 1:4 to 1:16 and being present in an amount effective to determine α-amylase by measuring rate of glucose released after cleavage by α-amylase and enzymatic release of glucose by an exo-acting amylase; b. an enzyme or enzyme mixture including an exo-acting amylase which is free of α-amylase and which is capable of and is present in an amount effective for releasing glucose from α-amylase cleaved carboxymethylated starch to determine α-amylase by measuring the rate of glucose production; c. a buffer for maintaing pH for α-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. enzymatic reagent for detecting rate of glucose formation, said enzymatic reagent being present in an amount effective for determining α-amylase by measuring the rate of glucose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of α-amylase.

Claim 7

A reagent kit for the determination of α-amylase, comprising: a. a water soluble carboxymethylated starch which is cleaved by α-amylase and which is not acted on by an exo-acting amylase, said water soluble carboxymethylated starch having a ratio of carboxymethylated glucose to glucose of from 1:4 to 1:16 and being present in an amount effective to determine α-amylase by measuring rate of glucose released after cleavage by α-amylase and enzymatic release of glucose by an exo-acting amylase; b. an enzyme or enzyme mixture including an exo-acting amylase which is free of α-amylase and which is capable of and is present in an amount effective for releasing glucose from α-amylase cleaved carboxymethylated starch to determine α-amylase by measuring the rate of glucose production; c. a buffer for maintaing pH for α-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. enzymatic reagent for detecting rate of glucose formation, said enzymatic reagent being present in an amount effective for determining α-amylase by measuring the rate of glucose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of α-amylase.

Claim 8

The kit of claim 5 wherein component (b) is glucoamylase which is free of α-amylase.

Claim 9

The kit of claim 6 wherein components (a), (b), (c) and (d) are in the form of a mixture.

Claim 10

The kit of claim 7 wherein the mixture is a dry mixture.

Claim 11

The kit of claim 8 wherein component (d) is a glucose-hexokinase rate detector, comprising adenosine triphosphate, nicotinamide adenine dinucleotide, hexokinase, and glucose-6-phosphate dehydrogenase.

Claim 12

The kit of claim 9 wherein the component (c) maintains a pH of from 5.0 to 9.0 in the media for α-amylase determination.

Claim 13

The kit of claim 10 wherein component (c) is comprised of at least one member selected from the group consisting of imidazole, β-glycerophosphate, 3-(N-morpholino)propane sulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and N-2-acetamide-2-aminoethanesulfonic acid.

Claim 14

The kit of claim 5 wherein component (d) is a glucose oxidase-peroxidase glucose rate detector.

Claim 15

The kit of claim 6 wherein component (d) is a glucose-hexokinase rate detector, comprising adenosine triphosphate, nicotinamide adenine dinucleotide, hexokinase, and glucose-6-phosphate dehydrogenase.

Claim 16

The kit of claim 13 wherein component (c) is comprised of at least one member selected from the group consisting of imidazole, β-glycerophosphate, 3-(N-morpholino)propane sulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and N-2-acetamide-2-aminoethanesulfonic acid.

Claim 17

A reagent package for determination of α-amylase, comprising: a container and a dry mixture in the container, comprising: a. a water soluble carboxymethylated starch which is cleaved by α-amylase and which is not acted on by an exo-acting amylase, said water soluble carboxymethylated starch having a ratio of carboxymethylated glucose to glucose of from 1:4 to 1:16 and being present in an amount effective to determine α-amylase by measuring rate of glucose released after cleavage by α-amylase and enzymatic release of glucose by an exo-acting amylase; b. an enzyme or enzyme mixture including an exo-acting amylase which is free of α-amylase and which is capable of and is present in an amount effective for releasing glucose from α-amylase cleaved carboxymethylated starch to determine α-amylase by measuring the rate of glucose production; c. a buffer for maintaining pH for α-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. enzymatic reagent for detecting rate of glucose formation, said enzymatic reagent being present in an amount effective for determining α-amylase by measuring the rate of glucose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of α-amylase.

Claim 18

A reagent package for determination of α-amylase, comprising: a container and a dry mixture in the container, comprising: a. a water soluble carboxymethylated starch which is cleaved by α-amylase and which is not acted on by an exo-acting amylase, said water soluble carboxymethylated starch having a ratio of carboxymethylated glucose to glucose of from 1:4 to 1:16 and being present in an amount effective to determine α-amylase by measuring rate of glucose released after cleavage by α-amylase and enzymatic release of glucose by an exo-acting amylase; b. an enzyme or enzyme mixture including an exo-acting amylase which is free of α-amylase and which is capable of and is present in an amount effective for releasing glucose from α-amylase cleaved carboxymethylated starch to determine α-amylase by measuring the rate of glucose production; c. a buffer for maintaining pH for α-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. enzymatic reagent for detecting rate of glucose formation, said enzymatic reagent being present in an amount effective for determining α-amylase by measuring the rate of glucose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of α-amylase.

Claim 19

An assay for the determination of α-amylase, comprising: mixing a sample containing α-amylase and the reagent kit containing: a. A water soluble carboxymethylated starch which is cleaved by alpha-amylase and which is not acted on by an exacting amylase, said water soluble carboxymethylated starch being present in an amount effective to determine alpha-amylase by measuring rate of glucose by an oxo-acting amylase; b. An (enzyme or) enzyme mixture including an exacting amylase which is to free of alpha-amylase and which is capable of and is present in an amount effective for releasing glucose form alpha-amylase cleaved carboxymethylated starch to determine alpha-amylase by measuring the rate of glucose production, said enzyme mixture containing α-amylase and a maltase which is free of alpha-amylase: c. A buffer for maintaining pH for alpha-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. Enzymatic reagent of detecting rate of glucose formation, said enzymatic reagent begin present in an amount effective for determining alpha-amylase by measuring the rate of gluycose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of alpha-amylase, incubating the mixture, to unblock the starch by action of α-amylase, release glucose by the action of said enzyme or enzyme mixture and indicate glucose production by the enzymatic reagent; and measuring the rate of glucose production to determine α-amylase in the sample.

Claim 20

An assay for the determination of α-amylase, comprising: mixing a sample containing α-amylase and the reagent kit containing: a. A water soluble carboxymethylated starch which is cleaved by alpha-amylase and which is not acted on by an exacting amylase, said water soluble carboxymethylated starch being present in an amount effective to determine alpha-amylase by measuring rate of glucose by an oxo-acting amylase; b. An (enzyme or) enzyme mixture including an exacting amylase which is to free of alpha-amylase and which is capable of and is present in an amount effective for releasing glucose form alpha-amylase cleaved carboxymethylated starch to determine alpha-amylase by measuring the rate of glucose production, said enzyme mixture containing α-amylase and a maltase which is free of alpha-amylase: c. A buffer for maintaining pH for alpha-amylase determination, said buffer being free of enzyme inhibitor which would interfere with enzymatic activity for releasing glucose; and d. Enzymatic reagent of detecting rate of glucose formation, said enzymatic reagent begin present in an amount effective for determining alpha-amylase by measuring the rate of gluycose production, wherein said components (a), (b), (c) and (d) are present in an amount effective for a rate determination of alpha-amylase, incubating the mixture, to unblock the starch by action of α-amylase, release glucose by the action of said enzyme or enzyme mixture and indicate glucose production by the enzymatic reagent; and measuring the rate of glucose production to determine α-amylase in the sample.

Claim 21

The assay of claim 16 wherein said enzyme for release of glucose is glucoamylase which is free of α-amylase.

Claim 22

The assay of claim 17 wherein the enzymatic glucose rate detector is a glucose-hexokinase rate detector, comprising adenosine triphosphate, nicotinamide adenine dinucleotide, hexokinase, and glucose-6-phosphate dehydrogenase.

Claim 23

The assay of claim 18 wherein the buffer is comprised of at least one member selected from the group consisting of imidazole, β-glycerophosphate, 3-(N-morpholino)propane sulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and N-2-acetamide-2-aminoethanesulfonic acid.