Methods for detecting truncated receptors using antibody-based arrays
View Patent ↗The present invention provides compositions and methods for detecting the activation states of components of signal transduction pathways in tumor cells. Information on the activation states of components of signal transduction pathways derived from use of the invention can be used for cancer diagnosis, prognosis, and in the design of cancer treatments.
1. A method for detecting the presence of a truncated receptor, said method comprising:
(a) incubating a cellular extract with a plurality of beads specific for an extracellular domain (ECD) binding region of a full-length receptor and incubating said cellular extract with a plurality of capture antibodies, wherein said plurality of capture antibodies is specific for an intracellular domain (ICD) binding region of said truncated receptor, and wherein said plurality of capture antibodies is restrained on a solid support in a planar array to form a plurality of captured truncated receptors;
(b) incubating the plurality of captured truncated receptors with detection antibodies specific for the corresponding truncated receptors to form a plurality of detectable captured truncated receptors;
(c) incubating the plurality of detectable captured truncated receptors with first and second members of a signal amplification pair to generate an amplified signal; and
(d) detecting an amplified signal generated from the first and second members of the signal amplification pair.
2. The method of claim 1 , wherein the detection antibodies specific for the corresponding truncated receptors comprise a plurality of activation state-independent antibodies and a plurality of activation state-dependent antibodies.
3. The method of claim 2 , wherein the activation state-independent antibodies are labeled with a facilitating moiety, the activation state-dependent antibodies are labeled with a first member of a signal amplification pair, and the facilitating moiety generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair.
4. The method of claim 3 , wherein the plurality of detectable captured truncated receptors is incubated with a second member of the signal amplification pair to generate an amplified signal.
5. The method of claim 1 , wherein said plurality of beads specific for an extracellular domain (ECD) binding region of a full-length receptor comprise a streptavidin-biotin pair, wherein the streptavidin is attached to the bead and the biotin is attached to an antibody.
6. The method of claim 1 , wherein the cellular extract is produced by lysing circulating cells of a breast tumor or by lysing cells isolated from tumor tissue.
7. The method of claim 6 , wherein the tumor tissue is primary tumor tissue or metastatic tumor tissue.
8. The method of claim 6 , wherein the cells are isolated from tumor tissue as a fine needle aspirate sample.
9. The method of claim 1 , wherein an activation state of said plurality of detectable captured truncated receptors is interrogated.
10. The method of claim 9 , wherein said activation state is selected from the group consisting of a phosphorylation state, ubiquitination state, complexation state, and combinations thereof.
11. The method of claim 3 , wherein the facilitating moiety is glucose oxidase.
12. The method of claim 11 , wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule.
13. The method of claim 12 , wherein the sulfhydryl-activated dextran molecule has a molecular weight of 500 kDa.
14. The method of claim 3 , wherein the oxidizing agent is hydrogen peroxide (H 2 O 2 ).
15. The method of claim 1 , wherein said truncated receptor is p95ErbB2.
16. The method of claim 15 , wherein said full-length receptor is ErbB2 (HER-2).
17. The method of claim 1 , wherein said full-length receptor is ErbB2 (HER-2).
18. The method of claim 1 , wherein the first member of the signal amplification pair is a peroxidase.
19. The method of claim 18 , wherein the peroxidase is horseradish peroxidase (HRP).
20. The method of claim 18 , wherein the second member of the signal amplification pair is a tyramide reagent.