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