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Patent US4649108A successfully refreshed from Google Patents.
US4649108A
Expired / Lapsed
US
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Alpha amylase assay
Assignee: Genzyme Corp
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Filing: 1984-07-26
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Granted: 1987-03-10
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Expires: 2004-07-26
Abstract
Abstract Method of measuring the amount of α-amylase in a liquid sample, including the steps of providing an oligosaccharide substrate for α-amylase, the substrate being characterized in that it contains at least 3 glucose units, its reducing-end glucose unit is bonded, via a bond cleavable by α- or β-glucosidase, to a label which exhibits an optically measurable change upon cleavage of the bond, and its terminal glucose unit is bonded to a blocking substituent which inhibits cleavage by exo-enzymes of the bond between the terminal glucose unit and the adjacent glucose unit; contacting the sample with the oligosaccharide substrate and with a first exo-enzyme capable of cleaving the bond between the reducing-end glucose unit and the label, and measuring the optically measurable change as a measure of α-amylase in the sample.
Inventors
- 👤 Henry E. Blair
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 15:06
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/40; C12Q2334/00; C12Q2334/10
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
160 words
A method of measuring the amount of α-amylase in a liquid sample comprising the steps of providing an oligosaccharide substrate for α-amylase, said substrate being characterized in that it contains at least 3 glucose units, the reducing-end glucose unit is bonded, via a bond cleavable by α- or β-glucosidase, to a label which exhibits an optically measurable change upon cleavage of said bond, and the terminal glucose unit is bonded to a chemical blocking substituent which inhibits cleavage by exo-enzymes of the bond between said terminal glucose unit and the adjacent glucose unit, contacting said sample with said oligosaccharide substrate and with a first added exo-enzyme capable of cleaving the bond between said reducing-end glucose unit and said label, and a second added exo-enzyme, capable of cleaving bonds between glucose units, to form a mixture comprising said substrate, said first exo-enzyme, and said second exo-enzyme and measuring said optically measurable change as a measure of said α-amylase in said sample.
Claim 2
Independent Claim
160 words
A method of measuring the amount of α-amylase in a liquid sample comprising the steps of providing an oligosaccharide substrate for α-amylase, said substrate being characterized in that it contains at least 3 glucose units, the reducing-end glucose unit is bonded, via a bond cleavable by α- or β-glucosidase, to a label which exhibits an optically measurable change upon cleavage of said bond, and the terminal glucose unit is bonded to a chemical blocking substituent which inhibits cleavage by exo-enzymes of the bond between said terminal glucose unit and the adjacent glucose unit, contacting said sample with said oligosaccharide substrate and with a first added exo-enzyme capable of cleaving the bond between said reducing-end glucose unit and said label, and a second added exo-enzyme, capable of cleaving bonds between glucose units, to form a mixture comprising said substrate, said first exo-enzyme, and said second exo-enzyme and measuring said optically measurable change as a measure of said α-amylase in said sample.
Claim 3
Dependent Claim
20 words
The method of claim 1 wherein said label is a chromophore, a fluorophore, a chemiluminescent substituent, or a bioluminescent substituent.
Claim 4
Dependent Claim
12 words
The method of claim 2 wherein said chromophore comprises p-nitrophenol or o-nitrophenol.
Claim 5
Dependent Claim
12 words
The method of claim 2 wherein said fluorophore comprises a coumarin derivative.
Claim 6
Dependent Claim
11 words
The method of claim 4 wherein said coumarin derivative comprises 4-methylumbelliferone.
Claim 7
Dependent Claim
11 words
The method of claim 1 wherein said first exo-enzyme comprises α-glucosidase.
Claim 8
Dependent Claim
11 words
The method of claim 1 wherein said first exo-enzyme comprises α-glucosidase.
Claim 9
Dependent Claim
16 words
The method of claim 1 wherein said first exo-enzyme comprises a mixture of α-glucosidase and β-glucosidase.
Claim 10
Dependent Claim
38 words
The method of claim 1 wherein the activity of said second exo-enzyme is independent of substrate chain length and wherein said second enzyme is not capable of cleaving said bond between said label and said reducing-end glucose unit.
Claim 11
Dependent Claim
11 words
The method of claim 1 wherein said second exo-enzyme comprises glucoamylase.
Claim 12
Dependent Claim
21 words
The method of claim 1 wherein said first and said second exo-enzymes are contacted with said sample at the same time.
Claim 13
Dependent Claim
19 words
The method of claim 1 wherein said first exo-enzyme comprises α- or β-glucosidase and said secon exo-enzyme comprises glucoamylase.
Claim 14
Dependent Claim
10 words
The method of claim 1 wherein said label comprises luciferin.
Claim 15
Dependent Claim
11 words
The method of claim 1 wherein said blocking substituent comprises benzylidene.
Claim 16
Dependent Claim
14 words
The method of claim 1 wherein said substrate contains eight or fewer glucose units.
Claim 17
Dependent Claim
12 words
The method of claim 15 wherein said substrate contains six glucose units.
Claim 18
Dependent Claim
12 words
The method of claim 15 wherein said substrate contains seven glucose units.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1990-03-09 | 1990-09-08 | 1991-03-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-03-09 | 1994-09-08 | 1995-03-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-03-09 | 1998-09-08 | 1999-03-10 | $7700.00 | Window Closed / Lapsed |