Methods and Compositions for Detecting Receptor Clustering and Activation Events 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 . A method of detecting the clustering of receptor elements in single cells, said method comprising:
 a) providing a sample comprising a cell, wherein said cell comprises receptor elements;   b) contacting said cell with a binding element that binds to at least one of said receptor elements to activate the formation of clusters, wherein said binding element comprises a label, wherein said clusters comprise
 i) at least two of said receptor elements; 
 ii) said binding element bound to at least one of said receptor elements of said cluster; and, 
 c) detecting said label. 
   
     
     
         2 . A method of simultaneously detecting multiple clusters in single cells, said method comprising:
 a) providing a sample comprising a cell wherein said cell comprises a plurality of different receptor elements;   b) contacting said cell with a plurality of different binding elements that bind to at least one of said different receptor elements to activate the formation of multiple clusters, wherein each of said different binding elements comprise an identifying label, wherein said clusters comprise
 i) at least two of said receptor elements; 
 ii) at least one of said binding elements bound to at least one of said receptor elements of said cluster; and 
 c) detecting said label. 
   
     
     
         3 . A method of simultaneously detecting the activation state and clustering of receptor elements in single cells, said method comprising:
 a) providing a sample comprising a cell wherein said cell comprises at least first receptor elements;   b) contacting said cell with at least first activation-specific antibodies that binds to an isoform of said first receptor elements, wherein each of said first activation-specific antibodies comprise an identifying label;   c) contacting said cell with at least first binding elements that bind to at least one of said receptor elements to activate the formation of clusters, wherein each of said first binding elements comprise an identifying label, wherein said clusters comprise
 i) at least two receptor elements; 
 ii) at least one of said first binding elements bound to at least one of said receptor elements of said cluster; and 
   d) simultaneously detecting said identifying labels of said first activation-specific antibodies and said identifying label of each of said clusters.   
     
     
         4 . The method according to  claim 1 , wherein the binding of said binding elements to said receptor elements is activated by providing said cell with an activator. 
     
     
         5 . The method according to  claim 1 , wherein said label of said binding element comprises an antibody conjugated to a fluorophore, and wherein said antibody binds to said binding element. 
     
     
         6 . The method according to  claim 1 , wherein said clustering activates said receptor elements in said clusters. 
     
     
         7 . The method according to  claim 1 , wherein said signal is proportional to the number of said clusters. 
     
     
         8 . The method according to  claim 1 , wherein said detecting is by flow cytometry. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . The method according to  claim 1 , wherein said sample is from a patient. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The method according to  claim 2 , wherein the binding of said binding elements to said receptor elements is activated by providing said cell with an activator. 
     
     
         15 . The method according to  claim 2 , wherein said label of said binding element comprises an antibody conjugated to a fluorophore, and wherein said antibody binds to said binding element. 
     
     
         16 . The method according to  claim 2 , wherein said clustering activates said receptor elements in said clusters. 
     
     
         17 . The method according to  claim 2 , wherein said signal is proportional to the number of said clusters. 
     
     
         18 . The method according to  claim 2 , wherein said detecting is by flow cytometry. 
     
     
         19 . The method according to  claim 2 , wherein said sample is from a patient. 
     
     
         20 . The method according to  claim 3 , wherein the binding of said binding elements to said receptor elements is activated by providing said cell with an activator. 
     
     
         21 . The method according to  claim 3 , wherein said label of said binding element comprises an antibody conjugated to a fluorophore, and wherein said antibody binds to said binding element. 
     
     
         22 . The method according to  claim 3 , wherein said clustering activates said receptor elements in said clusters. 
     
     
         23 . The method according to  claim 3 , wherein said signal is proportional to the number of said clusters. 
     
     
         24 . The method according to  claim 3 , wherein said detecting is by flow cytometry. 
     
     
         25 . The method according to  claim 3 , wherein said sample is from a patient.