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US 5,707,798 AGranted
Identification of ligands by selective amplification of cells transfected with receptors
AssigneeNovo Nordisk A/S
InventorMark Robert Brann
Life of this patent
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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.
Claims41
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.
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.
1 more element
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.
1 more element
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.
2 more elements
Dependencies are inferred from the claim text.