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US 5,350,678 AGranted
Method of differential assay for .alpha.-amylase isozymes and a kit for the same
AssigneeWako Pure Chemical Industries, Ltd.
InventorsTokuji Ikenaka, Kaoru Omichi
Life of this patent
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Abstract
Differential assay of human pancreatic .alpha.-amylase and human salivary .alpha.-amylase can be made extremely easily with good accuracy using a reducing-end modified maltooligosaccharide derivative as a substrate, by acting coupled enzymes having different substrate specificities on the degradation products formed by the hydrolytic action of .alpha.-amylases and measuring the resulting reaction products.
Claims14
A method for the differential assay of human pancreatic .alpha.-amylase and human salivary .alpha.-amylase isozymes in a sample containing at least one of said isozymes comprising:
- a) i) preparing a maltooligosaccharide derivative in which the hydroxy group at the 1-position of the reducing-end glucose residue is substituted with --OR.sup.1, wherein R.sup.1 represents an organic residue, selected from the group consisting of compounds represented by formulae (I), (II), and (III), ##STR3## wherein R.sup.3 to R.sup.6 may be the same or different and are selected from the group consisting of hydrogen, a lower alkoxy group, a nitro group, a carboxyl group, a sulfonic acid group or an halogen, and R.sup.3 together with R.sup.5, and R.sup.4 together with R.sup.6, may be bonded to each other to form an aromatic ring, and R.sup.7 represents hydrogen, a lower alkoxy group, an halogen or a nitro group, R.sup.8 represents hydrogen, a methyl group or a trifluoromethyl group, and R.sup.9 represents hydrogen or an halogen;
- and
- (ii) preparing a solution of said maltooligosaccharide derivative and a first enzyme selected from the group consisting of isomaltase, .beta.-glucosidase and .beta.-amylase;
- wherein the first enzyme specifically acts on one of reducing-end modified degradation products produced from said derivative by the hydrolytic action of .alpha.-amylase(s) in said sample and in which the hydroxy group at the 1-position of the reducing-end glucose residue thereof is substituted with said --OR.sup.1 to release a compound R.sup.1 OH;
- (iii) adding to said solution said sample containing .alpha.-amylase isozymes;
- and
- (iv) measuring a quantity A by an optical method, the quantity A being the rate of increase in optical change corresponding to the appearance of R.sup.1 OH as a result of the action of said first enzyme on one of the reducing-end modified degredation products produced by the action of .alpha.-amylase on said maltooligosaccharide
- (v) adding at least one second enzyme that non-specifically acts on said reducing-end modified degredation products to release the compound R.sup.1 OH;
- (vi) measuring a quantity B by an optical method, the quantity B being the rate of increase in optical change corresponding to the appearance of R.sup.1 OH as a result of the action of said second enzyme on the reducing-end modified degradation products produced by the action of .alpha.-amylase(s) on said maltooligosaccharide derivative;
- and
- c) determining the ratio of human pancreatic .alpha.-amylases and human salivary .alpha.-amylase in said sample from the ratio A/B using a standard curve.
8 more elements
A method for the differential assay of human pancreatic .alpha.-amylase and human salivary .alpha.-amylase isozymes in a sample containing at least one of said isozymes comprising:
- (i) preparing a first solution comprising a maltooligosaccharide derivative in which the hydroxy group at the 1-position of the reducing-end glucose residue is substituted with --OR.sup.1, wherein R.sup.1 represents an organic residue, selected from the group consisting of compounds represented by formulae (I), (II), and (III), ##STR5## wherein R.sup.3 to R.sup.6 may be the same or different and are selected from the group consisting of hydrogen, a lower alkoxy group, a nitro group, a carboxyl group, a sulfonic acid group or a halogen, and R.sup.3 together with R.sup.5, and R.sup.4 together with R.sup.6, may be bonded to each other to form an aromatic ring, and R.sup.7 represents hydrogen, a lower alkoxy group, an halogen or a nitro group, R.sup.8 presents hydrogen, a methyl group or a trifluoromethyl group, and R.sup.9 presents hydrogen, a methyl group or a trifluoromethyl group, and R.sup.9 represents hydrogen or an halogen, and with a first enzyme from the group consisting of isomaltase, .beta.-glucosidase and .beta.-amylase that specifically acts on one of the degradation products produced from said derivative by the hydrolytic action of .alpha.-amylase(s) in said sample;
- .beta.-glucosidase and a second solution comprising with or without said derivative at least one coupled enzyme that non-specifically acts on the degradation products produced from said maltooligosaccharide derivative by the action of .alpha.-amylase(s) in said sample;
- (ii) contacting said sample with said first solution and then with said second solution without said derivative, or contracting said sample with said first solution and with said second solution with said derivative, respectively;
- (iii) measuring by an optical method a quantity A of the reaction product per unit of the appearance of R.sup.1 OH formed by the action of said first coupled enzyme in said first solution from the degradation products of .alpha.-amylase(s) in said sample;
- and measuring by an optical method a quality B of the reaction product per unit time formed by the action of said second coupled enzyme in said second solution from the degradation products of .alpha.-amylase(s) in said sample;
- and
- (iv) determining the ratio of human pancreatic .alpha.-amylase and human salivary .alpha.-amylase in the sample from the ratio A/B using a standard curve.
4 more elements
Dependencies are inferred from the claim text.