Identification of ligands by selective amplification of cells transfected with receptors

• US 5,912,132 A
• Filed: 10/20/1997
• Issued: 06/15/1999
• Est. Priority Date: 07/13/1993
• Status: Expired due to Term

• Current Assignee: Acadia Pharmaceuticals Inc.
• Original Assignee: Acadia Pharmaceuticals Inc.
• Inventors: Brann, Mark Robert
• Primary Examiner(s): ULM, JOHN D
• Application Number:
  US08/954,724
• Time in Patent Office: 603 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 435/69.1, 435/172.3, 435/252.3, 435/320.1, 436/501
• US Class Current: 435/7.2
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2334/10 p-Nitrophenol derivatives
• CPC Class Codes: G01N 2333/48 Nerve growth factor [NGF]
• CPC Class Codes: G01N 2333/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• 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 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The invention is directed to a method for identifying substances acting as ligands for transfected receptors by using transfected markers to measure receptor/ligand interactions.

Claims

1. A test kit for detecting a substance capable of acting as a ligand, the kit comprising

(a) a culture of frozen cells which comprise cells transfected with DNA coding for a receptor which increases or decreases cell amplification in response to a ligand, said transfected cells also comprising a transfected marker of cell amplification and which cell culture also comprises non-transfected cells which do not comprise said transfected marker;

(b) a medium for growing cells; and

(c) a reagent for detecting the presence and quantity of the marker.

2. The test kit according to claim 1, wherein the DNA encodes a tyrosine kinase receptor, G-protein coupled receptor, tyrosine kinase activating receptor, tyrosine phosphatase receptor, transcription factor, steroid receptor, oncogene or ligand- or voltage-gated ion channel, or a chimeric receptor composed of the ligand-binding region of one receptor and the signal transducing region of another receptor.

3. The test kit according to claim 1, wherein the DNA encodes a muscarinic receptor.

4. The test kit according to claim 1, which further comprises an agonist of receptor stimulation of cell amplification.

5. The test kit according to claim 1, wherein the marker is the transfected receptor DNA, the transcribed receptor mRNA, an enzyme, a binding protein or an antigen.

6. The test kit according to claim 1, wherein the marker is β

-galactosidase and the reagent for detecting the marker is o-nitrophenyl-β

-D-galactopyranoside.

7. The test kit according to claim 1, wherein the marker is luciferase and the reagent for detecting the marker is luciferin.

8. A test kit for detecting a substance capable of acting as a ligand, the kit comprising,

(a) a culture of frozen cells which comprises cells transfected with DNA coding for a first receptor which increases or decreases cell amplification in response to a ligand, said transfected cells also comprising a transfected marker of cell amplification and which cell culture also comprises non-transfected cells which do not comprise said transfected marker,

(b) a culture of frozen cells which comprises cells transfected with DNA coding for a second receptor which increases or decreases cell amplification in response to a ligand, said transfected cells also comprising a transfected marker of cell amplification and which cell culture also comprises non-transfected cells which do not comprise said transfected marker,

(c) a medium for growing the cells, and

(d) a reagent for detecting the presence and quantity of the marker.

9. The test kit according to claim 8, wherein the DNA encodes a tyrosine kinase receptor, G-protein coupled receptor, tyrosine kinase activating receptor, tyrosine phosphatase receptor, transcription factor, steroid receptor, oncogene or ligand- or voltage-gated ion channel, or a chimeric receptor composed of the ligand-binding region of one receptor and the signal transducing region of another receptor.

10. The test kit according to claim 8, wherein the DNA encodes a muscarinic receptor.

11. The test kit according to claim 8, which further comprises an agonist of receptor stimulation of cell amplification.

12. The test kit according to claim 8, wherein the marker is the transfected receptor DNA, the transcribed receptor mRNA, an enzyme, a binding protein or an antigen.

13. The test kit according to claim 8, wherein the marker is β

-galactosidase and the reagent for detecting the marker is o-nitrophenyl-β

-D-galactopyranoside.

14. The test kit according to claim 8, wherein the marker is luciferase and the reagent for detecting the marker is luciferin.