Back to results
US 4,304,854 AGranted
Alpha-amylase assay and substrates for use therein
AssigneeWorthington Biochemical Corporation
InventorsPaul T. Nix, Rebecca D. Goldfarb, Linda J. Stong, Lorraine E. Sulick, Ramesh C. Trivedi, Stanley W. Morgenstern
Life of this patent
Estimating the term…
Abstract
A maltodextrin phosphorylase limit dextrin in the presence of maltodextrin phosphorylase and inorganic phosphate, is used as a substrate for alpha-amylase, which initiates a series of enzymatic reactions resulting in a chromogen response which can be used to measure the concentration of alpha-amylase in a body fluid. A novel limit dextrin and its preparation also are described.
Claims14
An alpha-amylase assay which comprises:
- (a) reacting maltodextrin phosphorylase limit dextrin with an alpha-amylase specimen in the presence of maltodextrin phosphorylase and inorganic phosphate to form limit dextrin fragments which concomitantly react with said maltodextrin phosphorylase to form glucose-1-phosphate as the initial component of a coupled enzymatic reacting system that is specific thereto and that results in the formation of a chromophore;
- (b) simultaneously performing the reactions of said coupled enzymatic reaction system responsive to the formation of said component;
- and
- (c) measuring the rate of formation of said chromophore;
- wherein the proportions of said limit dextrin, said phosphorylase and said phosphate to said alpha-amylase being such that the amount of said alpha-amylase is rate limiting and wherein said limit dextrin is prepared by the exhaustive treatment of glycogen or starch with maltodextrin phosphorylase or first with .beta.-amylase and then with maltodextrin phosphorylase.
2 more elements
A reagent system for an alpha-amylase assay comprising:
- (a) maltodextrin phosphorylase limit dextrin that is the substrate for said alpha-amylase and that is fragmentable by said alpha-amylase to form limit dextrin fragments;
- (b) maltodextrin phosphorylase;
- (c) inorganic phosphate;
- and
- (d) a coupled enzymatic reaction system for forming a chromophore, which enzymatic reaction system is specific for and responsive to glucose-1-phosphate that is formed by reaction between said phosphorylase and said limit dextrin fragments in the presence of said inorganic phosphate;
- wherein the amounts of said limit dextrin, said phosphorylase, said phosphate and said coupled enzymatic system being such that, when said reagent system is admixed with a specimen containing alpha-amylase, said limit dextrin is fragmented by alpha-amylase to form limit dextrin fragments which react with said phosphorylase to form glucose-1-phosphate, and whereby the amount of alpha-amylase contained in the specimen to be assayed is rate limiting and wherein said limit dextrin is prepared by the exhaustive treatment of glycogen or starch with maltodextrin phosphorylase or first with .beta.-amylase and then with maltodextrin phosphorylase.
3 more elements
A reagent composition for use in an alpha-amylase assay comprising:
- (a) maltodextrin phosphorylase limit dextrin fragmentable by said alpha-amylase to form limit dextrin fragments comprising non-reducing chain terminals;
- (b) maltodextrin phosphorylase in effective amount to react with the chains having said non-reducing terminals comprised in said limit dextrin fragments with resultant formation of glucose-1-phosphate;
- and
- (c) inorganic phosphate and wherein said limit dextrin is prepared by the exhaustive treatment of glycogen or starch with maltodextrin phosphorylase or first with .beta.-amylase and then with maltodextrin phosphorylase.
1 more element
An aqueous composition for use in an alpha-amylase assay comprising:
- ______________________________________ MDP limit dextrin 0.1- 20 mg/ml maltodextrin phosphorylase 1- 10 U/ml phosphoglucomutase 0.1- 10 U/ml glucose-6-phosphate dehydrogenase 0.5- 10 U/ml NAD 0.1- 10 m M phosphate buffer 10- 500 m M glucose-1, 6-diphosphate 0.001- 0.1 m M magnesium chloride 0.2- 100 m M ethylenediamine tetraacetic acid 0.1- 50 m M [pH 5- 8] ______________________________________
- wherein the pH is adjusted with buffer to a value within a range of 5 to 8 and said limit dextrin is prepared by the exhaustive treatment of starch or glycogen with maltodextrin phosphorylase or first with .beta.-amylase and then with maltodextrin phosphorylase.
Dependencies are inferred from the claim text.