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US 2006/0141542 A1Published application
Alpha-amylase assay and uses thereof
OwnerBarb Cohen, Stephen Powers +1inventors
InventorsBarb Cohen, Stephen Powers, Kesuo Fan
Life of this patent
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Abstract
This invention relates to methods and kits for measuring α-amylase activity in samples such as flour, stock, or amylase concentrate.
Claims31
A method for measuring α-amylase activity in a sample, comprising
- forming a reaction mixture by contacting a sample with a detectably labeled starch substrate for a time sufficient for α-amylase in the sample to hydrolyze the starch substrate, thereby releasing soluble detectably labeled starch fragments,
- separating the soluble detectably labeled starch fragments from the reaction mixture, and
- determining the level of hydrolysis of the detectably labeled starch substrate as a measurement of α-amylase activity in the sample.
21-24. (canceled)
A method for measuring α-amylase activity in a sample, comprising
- forming a reaction mixture by contacting a sample with a detectably labeled starch substrate attached to a surface, for a time sufficient for α-amylase in the sample to hydrolyze the starch substrate, thereby releasing soluble detectably labeled starch fragments,
- separating the soluble detectably labeled starch fragments from the reaction mixture, and
- determining the level of hydrolysis of the detectably labeled starch substrate as a measurement of α-amylase activity in the sample.
26-51. (canceled)
A kit for measuring α-amylase activity in a sample, comprising
- a first container containing a detectably labeled starch substrate, a second container containing an α-amylase standard, and instructions for measuring the α-amylase activity in the sample.
53-73. (canceled)
A method of determining amylase in a sample comprising:
- placing about 6 ml incubation buffer in a substrate tube,
- warming the substrate tube to 45° C.,
- adding about 200 mg of the sample to the warmed substrate tube,
- incubating the sample mixture in the substrate tube 10 min at 45° C.,
- adding about 4 ml stop buffer to the sample mixture in the substrate tube,
- filtering the stopped sample mixture into a container,
- determining the fluorescence in the filtrate, and
- optionally converting the fluorescence value into a Falling Number Equivalent value.
5 more elements
75-79. (canceled)
A method of determining amylase in a sample comprising:
- placing an about 3 g sample into a first container,
- adding a sufficient amount of fungal incubation buffer to have the total weight of sample plus buffer equal of about 30 g,
- mixing the solution,
- extracting the solution for 5 minutes at 45° C.,
- adding the about 8 ml of the extract to a substrate tube,
- incubating extract in substrate tube 10 minutes at 45° C.,
- adding about 2 ml stop buffer the tube,
- mixing the contents of the tube,
- filtering the mixture into a second container,
- determining the fluorescence in the filtrate, and
- optionally converting the fluorescence value into an Enzyme Units Equivalent value.
8 more elements
81-87. (canceled)
A method of determining amylase in a sample comprising:
- placing about 200 mg of the sample into a container,
- adding about 20 ml fungal incubation buffer to the sample,
- mixing the sample solution,
- diluting about 2 ml of the solution with 10 ml incubation buffer in a container,
- optionally further diluting the diluted solution to obtain a concentration within range of about 0.1-1.0 SKB unit/ml,
- placing about 8 ml of the diluted sample into a container,
- incubating the about 8 ml diluted sample 10 minutes at 45° C.,
- adding about 2 ml stop buffer to the 8 ml diluted sample,
- filtering the mixture through a filter into a detection container,
- determining the fluorescence in the filtrate, and
- optionally converting the fluorescence value into an Enzyme Units Equivalent value and multiplying by the dilution factor as a measure of the original amylase concentration.
8 more elements
89-95. (canceled)
Dependencies are inferred from the claim text.