US 7,247,435 B2 · Enzymatic fluorometric assay for cAMP and adenylate cyclase

Abstract

The present invention relates to a method for quickly determining cAMP content or an adenylate cyclase activity in a biological sample containing non-cyclic adenine nucleotides without the use of radioactive agents. Particularly, the present invention provides a method of determining cAMP content or an adenylate cyclase activity in a biological sample containing non-cyclic adenine nucleotides selected from the group consisting of cAMP produced by endogenous adenylate cyclase, and AMP, ATP, ADP and a mixture thereof, which comprises (1) combining a biological sample with effective amounts of apyrase, adenosine deaminase and alkaline phosphatase to enzymatically remove non-cyclic adenine nucleotides other than cAMP, and glucose-6-phosphate in the sample; (2) enzymatically converting cAMP into AMP; (3) determining an amount of AMP without the use of radioactive agents, and a kit to carry out the method.

Claims12

Dependent claims

A method of determining cAMP content or an adenylate cyclase activity in a biological sample comprising the following steps:

Cleaning Reaction:
combining a biological sample with effective amounts of apyrase, alkaline phosphatase and adenosine deaminase without 5′-nucleotidase to enzymatically remove endogenous non-cyclic adenine nucleotides other than cAMP, and endogenous glucose-6-phosphate;
Converting Reaction:
enzymatically converting cAMP in the biological sample into ATP;
and
Detecting Reaction:
enzymatically converting ATP into fructose-6-phosphate which is then enzymatically converted into 6-phosphogluconolactone and NADPH and determining a concentration of NADPH without the use of radioactive agents.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application

10/807,169Filed 24 Mar 2004 · division

Priority

JP 11-07369018 Mar 1999

Published

US 2004/0175750 A19 Sep 2004

Granted

24 Jul 2007

Term ends

Not estimatedThe source records don’t support an estimate.

Classification6 codes

G01N 33/5308
Immunoassay; Biospecific binding assay; Materials therefor: for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
C12Q 1/008
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…
C12Q 1/527
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…
G01N 33/573
Immunoassay; Biospecific binding assay; Materials therefor: for enzymes or isoenzymes

Enzymatic fluorometric assay for cAMP and adenylate cyclase

Life of this patent

Enzymatic fluorometric assay for cAMP and adenylate cyclase

Life of this patent

Abstract

Claims12