US4036697A
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Kinetic assay for alpha-amylase
Assignee: Beckman Coulter Inc
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Filing: 1976-02-13
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Granted: 1977-07-19
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Expires: 1996-02-13
Abstract
Abstract A kinetic assay is provided for measuring alpha-amylase content in aqueous solutions. The assay is based on the following reactions: alpha-1,4 linked glucan .sup.alpha-amylase alpha-maltose alpha-maltose + PO.sub.4 .sup.-.sup.-.sup.- .sup.MP Glucose + beta-D-G-1-P beta-D-G-1-P .sup.beta-PGM G-6-P G-6-P + NAD .sup.G6PDH 6-P-G + NADH the concentration of alpha-amylase is determined by measuring the rate of increase in absorbance associated with the production of NADH which is a measure of the activity of alpha-amylase. A novel reagent system is used with the assay and comprises: A polysaccharide having glucose molecules primarily connected through alpha 1,4 linkage Maltose phosphorylase (MP) Phosphate ions (PO 4 - - - ) beta-nicotinamide-adenine dinucleotide (NAD) or beta-nicotinamide-adenine dinucleotide phosphate Beta-D-phosphoglucomutase (Beta-PGM) Glucose-6-phosphate Dehydrogenase (G6PDH)
Inventors
- 👤 Kenneth Jonas Pierre
- 👤 Ker-Kong Tung
- 👤 Henriette Nadj
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 06:14
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/40; G; G01; G01N; G01N2333/00; G01N2333/90; G01N2333/902; G01N2333/904; G01N2333/91; G01N2333/912; G01N2333/91205; G01N2333/9121; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/853; Y10S435/855; Y10S435/871
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
115 words
An alpha-amylase assay comprising: a. performing simultaneous reactions which comprise: I. reacting a polysaccharide having glucose molecules primarily connected through alpha 1,4 linkages in the presence of an alpha-amylase specimen to form alpha-maltose; ii. reacting alpha-maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and beta-D-glucose-1-phosphate; iii. reacting beta-D-glucose-1-phosphate in the presence of beta-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 beta-nicotinamide-adenine dinucleotide, beta-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 alpha-amylase being measured is rate-limiting.
Claim 2
Dependent Claim
115 words
An alpha-amylase assay comprising: a. performing simultaneous reactions which comprise: I. reacting a polysaccharide having glucose molecules primarily connected through alpha 1,4 linkages in the presence of an alpha-amylase specimen to form alpha-maltose; ii. reacting alpha-maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and beta-D-glucose-1-phosphate; iii. reacting beta-D-glucose-1-phosphate in the presence of beta-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 beta-nicotinamide-adenine dinucleotide, beta-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 alpha-amylase being measured is rate-limiting.
Claim 3
Dependent Claim
21 words
The alpha-amylase assay of claim 1 wherein said beta-D-glucose-1-phosphate is reacted in the presence of beta-D-phosphoglucomutase and glucose-1,6-diphosphate to form glucose-6-phosphate.
Claim 4
Dependent Claim
29 words
The alpha-amylase assay of claim 2 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 5
Dependent Claim
52 words
The alpha-amylase assay of claim 12 wherein said beta-D-glucose-1-phosphate is reacted in the presence of beta-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 6
Dependent Claim
29 words
The alpha-amylase assay of claim 4 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 7
Dependent Claim
31 words
The alpha-amylase assay of claim 5 wherein said cation is selected from the group consisting of Mn + 2 , Mg + 2 , Co + 2 , and mixtures thereof.
Claim 8
Dependent Claim
22 words
The alpha-amylase assay of claim 6 wherein said simultaneous reactions are performed at a pH of from about 6 to about 7.5.
Claim 9
Dependent Claim
12 words
The alpha-amylase assay of claim 7 wherein said pH is about 6.5.
Claim 10
Dependent Claim
22 words
The alpha-amylase assay of claim 1 wherein said simultaneous reactions are performed at a pH of from about 6 to about 7.5.
Claim 11
Dependent Claim
12 words
The alpha-amylase assay of claim 9 wherein said pH is about 6.5.
Claim 12
Dependent Claim
65 words
A reagent system for an alpha-amylase assay comprising: a. a polysaccharide having glucose molecules primarily connected through alpha 1,4 linkages; b. phosphate ions; 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; and f. beta-D-phosphoglucomutase; wherein the above are present in amounts such that the alpha-amylase to be assayed is rate-limiting.
Claim 13
Dependent Claim
65 words
A reagent system for an alpha-amylase assay comprising: a. a polysaccharide having glucose molecules primarily connected through alpha 1,4 linkages; b. phosphate ions; 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; and f. beta-D-phosphoglucomutase; wherein the above are present in amounts such that the alpha-amylase to be assayed is rate-limiting.
Claim 14
Dependent Claim
9 words
The reagent system of claim 11 further comprising glucose-1,6-diphosphate.
Claim 15
Dependent Claim
10 words
The reagent system of claim 12 further comprising 6-phosphogluconate dehydrogenase.
Claim 16
Dependent Claim
39 words
The reagent system of claim 12 further comprising a cation selected from a group consisting of Mn + 2 , Mg + 2 , Co + 2 , Zn + 2 , Ni + 2 , and mixtures thereof.
Claim 17
Dependent Claim
42 words
The reagent system of claim 12 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 18
Dependent Claim
30 words
The reagent system of claim 15 wherein said cation is selected from the group consisting of Mg + 2 , Mn + 2 , Co + 2, and mixtures thereof.
Claim 19
Dependent Claim
16 words
The reagent system of claim 16 wherein said glucose-1,6-diphosphate consists essentially of the beta form thereof.
Claim 20
Dependent Claim
13 words
The reagent system of claim 17 wherein said polysaccharide is an alpha-1,4 glucan.
Claim 21
Dependent Claim
12 words
The reagent system of claim 18 wherein said alpha-1,4 glucan is starch.
Claim 22
Dependent Claim
103 words
A reagent system for an alpha-amylase assay wherein the alpha-amylase to be assayed is rate-limiting comprising per liter of reagent: a. at least about 1 gram of a polysaccharide having glucose molecules primarily connected through alpha 1,4 linkages; b. from about 0.01 to about 0.1 molar concentration of phosphate ions; c. at least about 200 International Units of maltose phosphorylase; d. at least about 1 millimole of a co-enzyme selected from a group consisting of beta-nicotinamide-adenine dinucleotide, beta-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof; e. at least about 500 International Units of glucose-6-phosphate dehydrogenase; and f. at least about 100 International Units of beta-D-phosphoglucomutase.
Claim 23
Dependent Claim
103 words
A reagent system for an alpha-amylase assay wherein the alpha-amylase to be assayed is rate-limiting comprising per liter of reagent: a. at least about 1 gram of a polysaccharide having glucose molecules primarily connected through alpha 1,4 linkages; b. from about 0.01 to about 0.1 molar concentration of phosphate ions; c. at least about 200 International Units of maltose phosphorylase; d. at least about 1 millimole of a co-enzyme selected from a group consisting of beta-nicotinamide-adenine dinucleotide, beta-nicotinamide-adenine dinucleotide phosphate, and mixtures thereof; e. at least about 500 International Units of glucose-6-phosphate dehydrogenase; and f. at least about 100 International Units of beta-D-phosphoglucomutase.
Claim 24
Dependent Claim
15 words
The reagent system of claim 20 further comprising at least about 0.1 grams of glucose-1,6-diphosphate.
Claim 25
Dependent Claim
17 words
The reagent system of claim 21 further comprising at least about 200 International Units of 6-phosphogluconate dehydrogenase.
Claim 26
Dependent Claim
44 words
The reagent system of claim 21 further comprising at least one millimole of a cation selected from a group consisting of Mn + 2 , Mg + 2 , Co + 2 , Zn + 2 , Ni + 2 , and mixtures thereof.
Claim 27
Dependent Claim
70 words
The reagent system of claim 20 comprising per liter of reagent: a. about 5 grams of starch; b. about 0.025 moles of phosphate ions; c. about 1000 International Units of maltose phosphorylase; d. about 2.5 millimole of said co-enzyme; e. about 5000 International Units of glucose-6-phosphate dehydrogenase; f. about 500 International Units of beta-phosphoglucomutase; g. about 0.075 grams of beta-glucose-1,6-diphosphate; h. about 1.7 grams of MgCl 2 .6H 2 O.
Claim 28
Dependent Claim
15 words
The reagent system of claim 24 further comprising about 700 International Units of phosphogluconate dehydrogenase.
Claim 29
Dependent Claim
23 words
A freeze dried reagent system for an alpha-amylase assay, said freeze dried reagent prepared from the reagent system set forth in claim 11.
Claim 30
Dependent Claim
23 words
A powdered reagent system for an alpha-amylase assay which when solubilized in water will produce the reagent system set forth in claim 11.
Claim 31
Dependent Claim
22 words
A kit comprising a plurality of reagents which when mixed together will produce the reagent system as set forth in claim 11.
Claim 32
Dependent Claim
12 words
The reagent system of claim 11 wherein said polysaccharide is a maltooligosaccharide.
Claim 33
Dependent Claim
19 words
The reagent system of claim 29 wherein said maltooligosaccharide is selected from a group consisting of maltotetraose and maltopentaose.
Claim 34
Dependent Claim
11 words
The reagent system of claim 30 wherein said maltooligosaccharide is maltotetraose.
Claim 35
Dependent Claim
12 words
The reagent system of claim 17 wherein said polysaccharide is a maltooligosaccharide.
Claim 36
Dependent Claim
19 words
The reagent system of claim 32 wherein said maltooligosaccharide is selected from a group consisting of maltotetraose and maltopentaose.
Claim 37
Dependent Claim
11 words
The reagent system of claim 33 wherein said maltooligosaccharide is maltotetraose.
Claim 38
Dependent Claim
12 words
The reagent system of claim 20 wherein said polysaccharide is a maltooligosaccharide.
Claim 39
Dependent Claim
19 words
The reagent system of claim 35 wherein said maltooligosaccharide is selected from a group consisting of maltotetraose and maltopentaose.
Claim 40
Dependent Claim
11 words
The reagent system of claim 36 wherein said maltooligosaccharide is maltotetraose.
References Cited (Backward Citations)
US3879263A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1980-07-18 | 1981-01-17 | 1981-07-19 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-07-18 | 1985-01-17 | 1985-07-19 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-07-18 | 1989-01-17 | 1989-07-19 | $7700.00 | Window Closed / Lapsed |