Identification of ligands by selective amplification of cells transfected with receptors
First Claim
1. A method of detecting a substance which is a ligand of a receptor, said method comprising,(a) incubating a cell culture which comprises cells transfected with DNA coding for said 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 said cell culture also comprises nontransfected cells which do not comprise said transfected marker, with a test substance which is a potential agonist or antagonist of the receptor for a time sufficient to permit amplification of said transfected cells, and(b) determining the level of amplification of said transfected cells by measuring the level of the marker in the cell culture.
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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.
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Citations
41 Claims
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1. A method of detecting a substance which is a ligand of a receptor, said method comprising,
(a) incubating a cell culture which comprises cells transfected with DNA coding for said 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 said cell culture also comprises nontransfected cells which do not comprise said transfected marker, with a test substance which is a potential agonist or antagonist of the receptor for a time sufficient to permit amplification of said transfected cells, and (b) determining the level of amplification of said transfected cells by measuring the level of the marker in the cell culture.
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21. A method of detecting a substance which is a ligand of a receptor, said method comprising
(a) transfecting a cell culture with DNA encoding said receptor which increases or decreases cell amplification in response to a ligand and with DNA encoding a marker enzyme, (b) dividing the cell culture of step (a), the cell culture comprising transfected cells comprising said marker enzyme and nontransfected cells not comprising said marker enzyme, into several identical aliquots, (c) incubating each aliquot with one or more test substances which are potential agonists or antagonists for a period of time sufficient to permit cell amplification, and (d) determining any increase or decrease in cell amplification by measuring marker enzyme activity in each aliquot.
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38. A method of detecting a substance which is a ligand of a receptor, said method comprising
(a) transfecting a cell culture with DNA encoding said receptor which increases or decreases cell amplification in response to a ligand and with DNA encoding a marker enzyme, (b) dividing the cell culture of step (a), the cell culture comprising transfected cells comprising said marker enzyme and nontransfected cells not comprising said marker enzyme, into several identical aliquots, (c) incubating each aliquot with varying concentrations of test substance which is a potential agonist or antagonist for a period of time sufficient to permit cell amplification, and (d) determining any change in cell amplification by measuring marker enzyme activity in each aliquot.
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39. A method for detecting a substance which is a ligand of a receptor, said method comprising
(a) transfecting a cell culture with DNAs encoding two or more distinct receptors, each transfected cell expressing an individual receptor, and with DNA encoding a marker enzyme, (b) dividing the cell culture of step (a), said culture comprising transfected cells comprising said marker enzyme and nontransfected cells not comprising said marker enzyme, into several identical aliquots, (c) incubating each aliquot with one or more test substances which act as potential agonists or antagonists of the receptors for a period of time sufficient to permit cell amplification, (d) determining any change in cell amplification by measuring marker enzyme activity in each aliquot, identifying active ligands by their ability to alter cell amplification, and (e) identifying which receptor is activated by the ligand.
Specification