Multiplex cell signalling assays

Abstract

Disclosed are methods useful in multiplex cell-based assays for compound screening employing imaging instrumentation. The methods described herein offer high content information relating to the biological potency of test agents, off-target effects and cellular toxicity of potential drug candidates.

What is claimed is: 
     
         1 - 15 . (canceled) 
     
     
         16 . A method for measuring at least one intracellular event and a cell-associated analyte in a single population of living cells wherein said at least one intracellular event and said cell-associated analyte are each components of a concerted biochemical process operating in said cells, the method comprising the steps of:
 a) providing a sample containing a single population of living cells;   b) contacting at least one living cell in said single population of cells with a test agent causing or suspected of causing said at least one cell to produce a cell-associated analyte;   c) measuring a change in a physical property in said at least one cell as a measure of at least one intracellular event;   d) washing the cells to remove extracellular fluids;   e) measuring the presence, amount or activity of said cell-associated analyte;   f) correlating the change in said at least one intracellular event in said at least one cell with the presence, amount or activity of said cell-associated analyte; and   g) comparing the change in said at least one intracellular event and a change in the presence, amount or activity of said cell-associated analyte with control values for each of the said at least one intracellular event and the presence, amount or activity of said cell-associated analyte in the absence of the test agent.   
     
     
         17 . The method according to  claim 16 , wherein said control value is stored electronically in a database or other electronic format. 
     
     
         18 . The method according to  claim 17 , wherein in step a) each cell in said population of cells comprises a first reporter gene construct comprising a nucleic acid sequence encoding a first detectable reporter molecule operably linked to and under the control of an expression control element; and wherein said contacting step b) is performed under conditions permitting expression of said first reporter gene construct. 
     
     
         19 . The method according to  claim 18 , wherein said first reporter gene construct comprises a nucleic acid sequence encoding a fluorescent protein. 
     
     
         20 . The method according to  claim 19 , wherein the fluorescent protein is a Green Fluorescent Protein (GFP) or a functional GFP analogue. 
     
     
         21 . The method according to  claim 18 , wherein said first reporter gene construct comprises a nucleic acid sequence encoding an enzyme. 
     
     
         22 . The method according to  claim 21 , wherein said enzyme is selected from the group consisting of a luciferase, β-galactosidase, alkaline phosphatase and nitroreductase. 
     
     
         23 . The method according to  claim 16 , wherein said intracellular event is an increase in ion concentration and/or an increase in gene expression. 
     
     
         24 . The method according to  claim 16 , wherein in step c) said at least one intracellular event is measured by a change in fluorescence emitted by the cell. 
     
     
         25 . The method according to  claim 16 , wherein in step c) said at least one intracellular event is measured by an optical imaging method. 
     
     
         26 . The method according to  claim 16 , wherein the presence, amount or activity of said cell-associated analyte is measured by an immunochemical method. 
     
     
         27 . The method according to  claim 16 , wherein the presence, amount or activity of said cell-associated analyte is measured by an optical imaging method. 
     
     
         28 . The method according to  claim 16 , wherein the population of cells consist of eukaryotic cells. 
     
     
         29 . The method according to  claim 28 , wherein said cell is selected from the group consisting of mammalian cells, yeast cells and insect cells. 
     
     
         30 . The method according to  16 , wherein said test agent is a chemical entity selected from the group consisting of a drug, a food dye, a hormone, a toxin, an alkylating agent, an oxidising agent and a carcinogen. 
     
     
         31 . The method according to  16 , wherein said test agent is a physical agent selected from the group consisting of electromagnetic radiation, β −  radiation and heat.