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US 9,851,309 B2Granted
Ultra-high-sensitive assay of protein and nucleic acid and kit, and novel enzyme substrate
AssigneesSatoshi Watabe, Toshiaki Miura, Teruki Yoshimura
InventorEtsuro Ito
Life of this patent
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Abstract
Provided is a ultra-high-sensitivity assay in which the assay can be made on a commonly used assay apparatus such as an absorptiometer and a plate reader or with naked eyes. The high-sensitivity assay in which the assay can be made on a commonly used assay apparatus or with naked eyes can be provided by combining an enzyme cycling method using thio-NAD(P) as a coenzyme, a labeling enzyme and a substrate for the labeling enzyme optimally, and by amplifying thio-NAD(P)H, which is a signaling substance, exponentially and then quantifying the thio-NAD(P)H colorimetrically.
Claims5
A method of assaying an enzyme activity comprising the steps of:
- (a) providing an antibody-enzyme complex, wherein the enzyme is alkaline phosphatase, glucosidase, galactosidase, fructosidase, mannosidase or peroxidase
- (b) providing a substrate if the enzyme, wherein the substrate is an androsterone derivative represented by the following formula (1) that is a 3α-hydroxy-17β-methoxy-5β-androstane derivative,
- wherein:
- (i) in the 3α-hydroxy-17β-methoxy-5β-androstane derivative, X represents a phosphate group, Y1 represents hydrogen, and Y2 represent a methoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by the formula (1) is used as a substrate of alkaline phosphatase,
- (ii) in the 3α-hydroxy-17β-methoxy-5β-androstane derivative, X represents a sugar moiety, the sugar moiety represents one selected from the group consisting of glucose, galactose, fructose and mannose, and Y1 represents hydrogen, and Y2 represents a methoxy group, when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by the formula (1) is used as a substrate of glucosidase, galactosidase, fructosidase or mannosidase, or
- (iii) in the 3α-hydroxy-17β-methoxy-5β-androstane derivative, X represents —O—CO—R (provided that R represents a C1-6 alkyl group or a phenyl group), Y1 represents hydrogen, and Y2 represents a methoxy group;
- when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by the formula (1) is used as a substrate of peroxidase and
- (c) reacting the enzyme of the antibody-enzyme complex with the androsterone derivative of formula (1) to produce a product;
- and
- (d) quantifying the product of the enzyme reaction (c) by producing thio-NADH and/or thio-NADPH by enzyme cycling reaction using NADH and/or NADPH, thio-NAD and/or thio-NADP, and hydroxysteroid dehydrogenase (HSD), wherein a substrate of the HSD is the product of the enzyme reaction (c), and assaying the amount of the produced thio-NADH and/or thio-NADPH, or measuring the change of the color by the produced thio-NADH and/or thio-NADPH in order to assay the enzyme activity.
7 more elements
A method of assaying a nucleic acid probe comprising the step of
- (a) providing an enzyme-labeled nucleic acid probe, wherein the enzyme is alkaline phosphatase, glucosidase, galactosidase, fructosidase, mannosidase or peroxidase,
- (b) providing the substrate of the enzyme, wherein the substrate is an androsterone derivative represented by the following formula (1) that is a 3α-hydroxy-17β-methoxy-5β-androstane derivative is a substrate of the enzyme,
- wherein
- (i) in the 3α-hydroxy-17β-methoxy-5β-androstane derivative, X represents a phosphate group, Y1 represent hydrogen, and Y2 represents a methoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by the formula (1) is used as a substrate of alkaline phosphatase,
- (ii) X represents a sugar moiety, the sugar moiety represents one selected from the group consisting of glucose, galactose, fructose and mannose, and Y1 represents hydrogen, and Y2 represents a C1 methoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by the formula (1) is used as a substrate of glucosidase, galactosidase, fructosidase or mannodidase, or
- (iii) X represents —O—CO—R (provided that R represents a C1-6 alkyl group or a phenyl group), Y1 represents hydrogen, and Y2 represents a methoxy group when the 3α-hydroxy-17β-methoxy-5β-androstane derivative represented by the formula (1) is used as a substrate of peroxidase, and
- (c) reacting the enzyme of the enzyme-labeled nucleic acid probe with the androsterone derivative of formula (1) to produce a product;
- (d) quantifying the product of the enzyme reaction (c) by producing thio-NADH and/or thio-NADPH by enzyme cycling reaction using NADH and/or NADPH, thio-NAD and/or thio-NADP, and hydroxysteroid dehydrogenase (HSD), wherein a substrate of the HSD is the reaction product of the enzyme of the enzyme-labeled nucleic acid probe and assaying the amount of the produced thio-NADH and/or thio-NADPH, or measuring the change of the color by the produced thio-NADH and/or thio-NADPH.
5 more elements
Dependencies are inferred from the claim text.