METHODS AND COMPOSITIONS FOR COUPLED LUMINESCENT ASSAYS

• US 20080050762A1
• Filed: 05/15/2007
• Published: 02/28/2008
• Est. Priority Date: 02/13/2001
• Status: Abandoned Application

• Current Assignee: Michael Corey, Sumant Dhawan
• Original Assignee: Michael Corey, Sumant Dhawan
• Inventors: Corey, Michael, Dhawan, Sumant
• Application Number:
  US11/748,967
• Publication Number:
  US 20080050762A1
• Time in Patent Office: Days
• US Class Current: 435/15
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2333/918 Carboxylic ester hydrolases (3.1.1)

Abstract

The invention provides methods for measuring the amount or activity of a component in a sample using a luminescent assay comprising a luciferase capable of generating light from a high-energy molecule. In one aspect, the invention provides methods for measuring the amount of NAD+ or the activity of an enzyme or enzyme series that results in the interconversion of NDA+ and NADH. In another aspect, the invention provides methods for measuring the amount of a kinase substrate, free inorganic phosphate, and phosphatase activity. In a further aspect, the invention provides methods for measuring the amount of cAMP in a sample.

Claims

1. A method for measuring the amount of a kinase substrate in a sample, comprising the steps of:

(a) contacting a sample comprising a substrate for an ATP-dependent kinase with (i) an ATP-dependent kinase that phosphorylates the substrate in the presence of ATP and (ii) an ATP-dependent luciferase, under conditions wherein luminance emitted from the sample depends on the initial amount of the substrate in the sample; and

(b) measuring the amount of the substrate in the sample by measuring the emitted luminance.

2. The method of claim 1, where the substrate is acetate and the kinase is acetate kinase or the substrate is choline and the kinase is choline kinase.

3. A method for measuring the activity of acetylcholinesterase in a sample, comprising the step of measuring the activity of acetylcholinesterase in a sample by measuring the amount of acetate or choline in the sample using the method of claim 2, wherein the acetate or choline in the sample is produced in an acetylcholinesterase-catalyzed reaction with acetylcholine.

4. A method for measuring the amount of an inhibitor of acetylcholinesterase in a sample, comprising the steps of:

(a) contacting the sample with an acetylcholinesterase under suitable conditions for producing acetate and choline from acetylcholine; and

(b) measuring the amount of one or more inhibitors of acetylcholinesterase in the sample by measuring the activity of the acetylcholinesterase using the method of claim 3.