Bioluminescent protease assay
AssigneePromega Corporation
InventorsMartha O'Brien, Keith V. Wood, Dieter Klaubert, William Daily
Life of this patent
Estimating the term…
Abstract
A sensitive bioluminescent assay to detect proteases including caspases, trypsin and tryptase is provided.
Claims77
A luminescent assay method to detect one or more caspases, comprising:
- a) contacting a sample suspected of having one or more caspases with a mixture comprising luciferase and an amino-modified aminoluciferin or a carboxy-terminal protected derivative thereof, wherein the modification is the covalent linkage of a substrate for the caspase to the amino group of aminoluciferin or the derivative thereof via a peptide bond, and wherein the caspase cleaves the substrate at the peptide bond;
- and
- b) detecting luminescence in the sample, wherein the luminescent assay is more sensitive than a corresponding assay with a conjugate comprising a fluorophore covalently linked to the substrate or a functional equivalent thereof.
A luminescent assay method to detect a protease that specifically cleaves a substrate comprising aspartate, comprising:
- a) contacting a sample suspected of having one or more aspartate-specific proteases with a mixture comprising luciferase and an amino-modified aminoluciferin or a carboxy-terminal protected derivative thereof, wherein the modification is the covalent linkage of a substrate comprising aspartate to the amino group of aminoluciferin or the derivative thereof via a peptide bond, and wherein the protease cleaves the substrate at the peptide bond;
- and
- b) detecting luminescence in the sample, wherein the luminescent assay is more sensitive than a corresponding assay with a conjugate comprising a fluorophore covalently linked to the substrate or a functional equivalent thereof.
A compound comprising aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a substrate for a caspase.
A compound comprising aminoluciferin or a carboxy-terminal protected derivative thereof covalently linked via a peptide bond to a substrate that specifically cleaves a substrate comprising aspartate.
A method to prepare a carboxy-terminal protected derivative of aminoluciferin, comprising protecting the corresponding acid with a suitable carboxy-protecting group.
A compound comprising aminoluciferin coupled via the carboxyl group to a solid support.
A compound comprising N-protected aminoluciferin coupled via the carboxyl group to a solid support.
A compound of formula (I):
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- wherein R is a peptide that is a substrate for caspase, which is linked to the remainder of the compound of formula (I) through its C-terminus forming a peptide (amide) bond;
- and R′ is H or a suitable carboxy protecting group, or a suitable salt thereof.
A compound of formula (I):
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- wherein R is a peptide that is linked to the remainder of the compound of formula (I) through an aspartate group at the C-terminus of the peptide forming a peptide bond;
- and R′ is H or a suitable carboxy protecting group, or a suitable salt thereof.
A method to prepare a compound of formula (I):
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- wherein R is a peptide that is a substrate for caspase, which is linked to the remainder of the compound of formula (I) through its C-terminus forming a peptide (amide) bond;
- and R′ is a suitable carboxy protecting group;
- comprising protecting the corresponding acid, wherein R′ is hydrogen, with a suitable carboxy-protecting group.
A method to prepare a compound of formula (I):
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- wherein R is a peptide that is linked to the remainder of the compound of formula (I) through an aspartate group at the C-terminus of the peptide forming a peptide bond;
- and R′ is a suitable carboxy protecting group;
- comprising protecting the corresponding acid, wherein R′ is hydrogen, with a suitable carboxy-protecting group.
A compound of formula (I):
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- wherein R is a peptide that is a substrate for caspase, which is linked to the remainder of the compound of formula (I) through its C-terminus forming a peptide (amide) bond;
- and R′ is a solid support.
A compound of formula (I):
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- wherein R is a peptide that is linked to the remainder of the compound of formula (I) through an aspartate group at the C-terminus of the peptide forming a peptide bond;
- and R′ is a solid support.
Dependencies are inferred from the claim text.