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US 2006/0234323 A1Published application
Luminescent ATP detection with extended linearity
OwnerJames Cali, Dongping Mainventors
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Abstract
A luminescent assay capable of detecting a wider range of ATP concentrations is provided.
Claims53
A method to detect the presence or amount of ATP in a sample, comprising:
- a) contacting a sample, a beetle luciferase reaction mixture, and at least two inhibitors of ATP so as to yield a luminogenic reaction mixture, wherein one of the inhibitors is an adenosine derivative which is present in an amount effective to increase the beetle luciferase Km for ATP relative to the luciferase Km for ATP in the absence of the adenosine derivative, and wherein the amount of the inhibitors in the luminogenic reaction mixture provides for a luminogenic reaction that is capable of detecting an ATP concentration beyond the linear range for a corresponding luminogenic reaction that lacks the at least two inhibitors;
- and
- b) detecting the presence or amount of luminescence in the luminogenic reaction mixture.
A method to detect the activity of a non-beetle luciferase ATP consuming enzyme in a sample, comprising:
- a) contacting a sample comprising a reaction mixture for a non-beetle luciferase ATP consuming enzyme, a beetle luciferase reaction mixture, and at least one competitive inhibitor of ATP in a beetle luciferase reaction, so as to yield a luminogenic reaction mixture, wherein the at least one inhibitor is present in an amount that results in the beetle luciferase Km for ATP being the same or greater than the non-beetle luciferase ATP consuming enzyme Km for ATP;
- and
- b) detecting the presence or amount of luminescence in the luminogenic reaction mixture.
A method to detect one or more modulators of a non-beetle luciferase ATP consuming enzyme, comprising:
- a) comparing luminescence from a first luminogenic reaction mixture comprising a reaction mixture for a non-beetle luciferase ATP consuming enzyme which comprises the non-beetle luciferase ATP consuming enzyme and one or more compounds and/or reaction conditions to be tested, and a beetle luciferase reaction mixture comprising at least one inhibitor of ATP, to luminescence from a second luminogenic reaction mixture comprising a reaction mixture for the non-beetle luciferase ATP consuming enzyme which comprises the non-beetle luciferase ATP consuming enzyme but lacks the one or more compounds and/or is subjected to different reaction conditions, and a beetle luciferase reaction mixture comprising the at least one inhibitor, wherein the non-beetle luciferase ATP consuming enzyme Km for ATP is greater than the beetle luciferase Km for ATP in the absence of the at least one inhibitor, wherein the at least one inhibitor of ATP in each luminogenic reaction is present in an amount that results in the luciferase Km for ATP being the same or greater than the non-beetle luciferase ATP consuming enzyme Km for ATP;
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- b) detecting or determining whether the one or more compounds and/or reaction conditions in the first luminogenic reaction mixture alter the luminescence in the first luminogenic reaction mixture relative to the second luminogenic reaction mixture.
A kit comprising:
- a composition comprising at least two inhibitors of ATP in a beetle luciferase reaction, wherein one inhibitor is an adenosine derivative, and
- optionally, one or more of the following reagents:
- isolated beetle luciferase, isolated non-beetle luciferase ATP consuming enzyme, a beetle luciferase substrate and/or a substrate for the non-beetle luciferase ATP consuming enzyme, wherein if present the one or more reagents are optionally separately packaged.
A method to identify a concentration of a divalent cation that alters the Km of a luciferase for ATP, comprising:
- a) providing the Km of one or more luciferases for ATP in a luminogenic reaction at one or more concentrations of a plurality divalent metals, wherein the Km is optionally in the absence of an inhibitor of ATP;
- and
- b) identifying a concentration of at least one divalent metal and at least one luciferase having a selected Km for ATP in the luminogenic reaction in a linear range for the reaction.
Dependencies are inferred from the claim text.