Methods and Compositions for Detecting Receptor-Ligand Interactions in Single Cells

Abstract

The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.

1 - 13 . (canceled) 
   
   
       14 . A method for screening for a bioactive agent capable of modulating receptor element clustering comprising:
 a) providing a sample comprising a cell, wherein said cell comprises receptor elements;   b) contacting the cell with the biological agent; and   c) detecting receptor clustering, wherein receptor clustering is indicative of the biological agent being capable of modulating receptor element clustering.   
   
   
       15 . The method of  claim 14 , further comprising contacting said cell with a binding element that binds to at least one of said receptor elements in said receptor clusters, wherein said binding element comprises a label. 
   
   
       16 . The method of  claim 15 , wherein said label of said binding element comprises an antibody conjugated to a fluorophore, and wherein said antibody binds to said binding element. 
   
   
       17 . The method of  claim 14 , further comprising inducing the formation of receptor clusters on said cell, wherein said receptor clusters comprise at least two of said receptor elements. 
   
   
       18 . The method according to  claim 17 , wherein said inducing step activates said receptor elements in said receptor clusters. 
   
   
       19 . The method according to  claim 18 , wherein said method further comprises detecting said activated receptor elements in said receptor clusters by: contacting said cell with an activation-specific antibody that binds to an isoform of said activated receptor elements, wherein said activation-specific antibody comprises an identifying label; and detecting a signal from said identifying label of said activation-specific antibody by flow cytometry to detect said activated receptor elements. 
   
   
       20 . The method according to  claim 19 , wherein multiple activation-specific antibodies are contacted to said cell, wherein each of said multiple activation-specific antibodies comprise an identifying label. 
   
   
       21 . The method according to  claim 19 , wherein said identifying label of said activation-specific antibody is a fluorophore. 
   
   
       22 . The method according to  claim 20 , wherein said identifying label of each of said activation-specific antibodies is a fluorophore. 
   
   
       23 . The method according to  claim 14 , wherein said sample is from a patient. 
   
   
       24 . The method according to  claim 14 , wherein said detecting is by flow cytometry. 
   
   
       25 . The method according to  claim 24 , wherein said detecting further comprises using a FACS machine. 
   
   
       26 . The method according to  claim 24 , wherein said detecting further comprises using a doublet discriminator. 
   
   
       27 . The method according to  claim 14 , wherein said clusters are made up of identical receptor elements. 
   
   
       28 . The method according to  claim 14 , wherein said clusters are made up of different receptor elements. 
   
   
       29 . A method for screening for a bioactive agent capable of modulating receptor element clustering comprising:
 a) providing a sample comprising a cell, wherein said cell comprises receptor elements;   b) contacting the cell with the biological agent;   c) contacting said cell with a binding element that binds to at least one of said receptor elements in said receptor clusters, wherein said binding element comprises a label;   d) detecting a signal from said label on said cell using a flow cytometer equipped with a doublet discriminator;   e) determining the distribution of said label on said single cell based on said signal using said doublet discriminator; and   f) correlating said determined distribution of said label on said cell with said formation of receptor clusters.   
   
   
       30 . The method according to  claim 29 , further comprising: in step c), contacting said cell with a second binding element that binds to at least one of said receptor elements in said receptor clusters, wherein said second binding element comprises an identifying label; and in step d), detecting a signal from said identifying label on said cell by flow cytometry; in step e), determining the distribution of said identifying label on said cell based on said second signal; and in step f), correlating said determined distribution of said second label on said cell with said formation of receptor clusters. 
   
   
       31 . The method according to  claim 29 , wherein said cell comprises second receptor elements, said method further comprising: in step b), inducing the formation of receptor clusters on said cell, wherein said receptor clusters comprise at least two of said second receptor elements; in step c), contacting said cell with a second binding element that binds to at least one of said second receptor elements in said receptor clusters, wherein said second binding element comprises an identifying label; in step d), detecting a second signal from said identifying label on said cell by flow cytometry; in step e), determining the distribution of said identifying label on said cell based on said second signal; and in step f), correlating said determined distribution of said second label on said cell with said formation of receptor clusters.