Cell-signaling assays

• US 8,568,973 B2
• Filed: 02/25/2010
• Issued: 10/29/2013
• Est. Priority Date: 06/09/1999
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Sportsman, J. Richard, Kauvar, Lawrence M.
• Primary Examiner(s): Emch, Gregory S
• Application Number:
  US12/713,107
• Publication Number:
  US 20100151481A1
• Time in Patent Office: 1,342 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/0405 manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
• CPC Class Codes: G01N 2035/0425 Stacks, magazines or elevators for plates
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1011 Control of the position or alignment of the transfer device
• CPC Class Codes: G01N 35/1074 arranged in a two-dimensional array

Abstract

Assays for detecting the presence and activity of cell-signaling components. These assays include luminescence polarization assays for detecting cell-signaling nucleotides and modulators of receptors and enzymes related to the generation and activity of such nucleotides.

Claims

1. A method of testing a compound for modulation of a receptor or enzyme involved in generation of a cyclic nucleotide, the method comprising:

contacting a sample with a tracer comprising a cyclic nucleotide coupled to a luminophore, and with an opposite member of a specific binding pair to the cyclic nucleotide;

illuminating the sample with polarized light, wherein the light is capable of inducing emission of polarized light from the luminophore;

measuring the extent of polarization of light emitted from the luminophore in the presence and in the absence of a compound; and

identifying the compound as a modulator of the receptor or enzyme if the extent of polarization changes in the presence relative to the absence of the compound.

2. The method of claim 1, wherein the step of identifying includes a step of identifying the compound as an inhibitor of the receptor or enzyme if the extent of polarization increases in the presence relative to the absence of the compound.

3. The method of claim 1, wherein the step of identifying includes a step of identifying the compound as an activator of the receptor or enzyme if the extent of polarization decreases in the presence relative to the absence of the compound.

4. The method of claim 1, wherein the step of measuring the extent of polarization of light comprises a step of evaluating a function selected from the group consisting of polarization and anisotropy.

5. The method of claim 1, wherein the opposite member of a specific binding pair is an antibody.

6. The method of claim 1, wherein the nucleotide is cyclic AMP (cAMP) or cyclic GMP (cGMP).

7. The method of claim 6, wherein the nucleotide is cyclic AMP (cAMP).

8. The method of claim 6, wherein the nucleotide is cyclic GMP (cGMP).

9. The method of claim 1, wherein the step of contacting includes a step of contacting whole cells or a cell lysate with the compound.

10. The method of claim 1, wherein the step of identifying includes a step of identifying the compound as an inhibitor or activator of a G-protein-coupled receptor.

11. The method of claim 1, wherein the step of identifying includes a step of identifying the compound as an inhibitor or activator of adenylyl cyclase.

12. The method of claim 1, wherein the step of identifying includes a step of identifying the compound as an inhibitor or activator of guanylyl cyclase.

13. The method of claim 1, further comprising a step of providing the tracer in a lysis buffer.