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1. A method for determining the sensitivity of a test cell to a compound that modulates HER2 activity, the method comprising: (a) contacting the test cell with the compound;(b) lysing the test cell to produce a cellular extract;(c) determining the activation level of HER2 and p95HER2 in the cellular extract produced from the test cell using a proximity immunoassay comprising capture antibodies, activation state-dependent antibodies, and activation state-independent antibodies, wherein the activation state-independent antibodies are labeled with a facilit...
1. A method for determining the sensitivity of a test cell to a compound that modulates HER2 activity, the method comprising: (a) contacting the test cell with the compound;(b) lysing the test cell to produce a cellular extract;(c) determining the activation level of HER2 and p95HER2 in the cellular extract produced from the test cell using a proximity immunoassay comprising capture antibodies, activation state-dependent antibodies, and activation state-independent antibodies, 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, thereby allowing the first member of the signal amplification pair to generate an amplified signal when incubated with a second member of the signal amplification pair;(d) determining a first reference activation level of HER2 and p95HER2 in a cellular extract produced from a compound-sensitive cell that is treated with the compound and a second reference activation level of HER2 and p95HER2 in a cellular extract produced from a compound-resistant cell that is treated with the compound using the proximity immunoassay, wherein the level of p95HER2 activation in the compound-resistant cell is at least 5-fold higher than the level of p95HER2 activation in the compound-sensitive cell; and(e) determining that the test cell is sensitive to the compound if the activation level of HER2 and p95HER2 in the cellular extract produced from the test cell is equal to or at least about 1.5-fold lower compared to the first reference activation level of HER2 and p95HER2 or determining that the test cell is resistant to the compound if the activation level of HER2 and p95HER2 in the cellular extract produced from the test cell is equal to or at least about 1.5-fold higher compared to the second reference activation level of HER2 and p95HER2. 2. The method of claim 1, wherein the compound inhibits HER2 activity. 3. The method of claim 2, wherein the compound is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, and combinations thereof. 4. The method of claim 3, wherein the monoclonal antibody is selected from the group consisting of trastuzumab (Herceptin®), pertuzumab (2C4), and combinations thereof. 5. The method of claim 3, wherein the tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, pilitinib, canertinib, lapatinib, and combinations thereof. 6. The method of claim 1, wherein the compound-sensitive cell is a BT-474 cell. 7. The method of claim 1, wherein the level of HER2 activation in the compound-resistant cell is at least 2 to 3-fold higher than the level of HER2 activation in the compound-sensitive cell. 8. The method of claim 1, wherein the activation level of HER2 and p95HER2 comprises a phosphorylation level of HER2 and p95HER2. 9. The method of claim 1, further comprising determining the activation level of one or more additional signal transduction molecules in the cellular extract produced from the test cell. 10. The method of claim 9, wherein the one or more additional signal transduction molecules is selected from the group consisting of EGFR (HER1), HER3, HER4, PI3K, AKT, MEK, PTEN, SGK3, 4E-BP1, ERK2 (MAPK1), ERK1 (MAPK3), PDK1, P70S6K, GSK-3β, Shc, IGF-1R, c-MET, c-KIT, VEGFR1, VEGFR2, VEGFR3, a receptor dimer, and combinations thereof. 11. The method of claim 10, wherein the presence of a similar or higher level of HER3 activation in the cellular extract produced from the test cell compared to a second reference activation level of HER3 from the compound-resistant cell indicates that the test cell is resistant to the compound. 12. The method of claim 11, wherein the level of HER3 activation in the compound-resistant cell is at least 2 to 3-fold higher than the level of HER3 activation in the compound-sensitive cell. 13. The method of claim 10, wherein the presence of a similar or higher level of PI3K activation in the cellular extract produced from the test cell compared to a second reference activation level of PI3K in the compound-resistant cell indicates that the test cell is resistant to the compound. 14. The method of claim 10, wherein the receptor dimer is selected from the group consisting of a p95HER2/HER3 heterodimer, HER2/HER2 homodimer, HER2/HER3 heterodimer, HER1/HER2 heterodimer, HER2/HER3 heterodimer, and combinations thereof. 15. The method of claim 14, wherein the presence of a similar or higher level of p95HER2/HER3 heterodimer activation in the cellular extract produced from the test cell compared to a second reference activation level of p95HER2/HER3 heterodimer in the compound-resistant cell indicates that the test cell is resistant to the compound. 16. The method of claim 10, wherein the presence of a similar or lower level of HER3 activation in the cellular extract produced from the test cell compared to a first reference activation level of HER3 from the compound-sensitive cell indicates that the test cell is sensitive to the compound. 17. The method of claim 10, wherein the presence of a similar or lower level of PI3K activation in the cellular extract produced from the test cell compared to a first reference activation level of PI3K in the compound-sensitive cell indicates that the test cell is sensitive to the compound. 18. The method of claim 14, wherein the presence of a similar or lower level of p95HER2/HER3 heterodimer activation in the cellular extract produced from the test cell compared to a first reference activation level of p95HER2/HER3 heterodimer in the compound-sensitive cell indicates that the test cell is sensitive to the compound. 19. The method of claim 1, wherein the test cell is a tumor cell. 20. The method of claim 19, wherein the tumor cell is a circulating tumor cell or a fine needle aspirate (FNA) cell obtained from a tumor. 21. The method of claim 19, wherein the tumor cell is a breast cancer cell. 22. The method of claim 1, wherein the test cell is isolated from a sample. 23. The method of claim 22, wherein the sample is obtained from a subject with breast cancer. 24. The method of claim 22, wherein the sample is a whole blood, serum, plasma, or tumor tissue sample. 25. The method of claim 1, further comprising a step of providing the result of the determination obtained in step (e) to a user in a readable format. 26. The method of claim 1, wherein determining the activation level of HER2 and p95HER2 in step (c) comprises detecting a phosphorylation level of HER2 and p95HER2 in the cellular extract produced from the test cell with an antibody specific for phosphorylated HER2 and p95HER2. 27. The method of claim 1, wherein determining the activation level of HER2 in step (c) comprises: (i) incubating the cellular extract produced from the test cell with a dilution series of capture antibodies specific for HER2 to form a plurality of captured receptors, wherein the capture antibodies are restrained on a solid support;(ii) incubating the plurality of captured receptors with detection antibodies comprising activation state-independent antibodies and activation state-dependent antibodies specific for HER2 to form a plurality of detectable captured receptors,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;(iii) incubating the plurality of detectable captured receptors with a second member of the signal amplification pair to generate an amplified signal; and(iv) detecting the amplified signal generated from the first and second members of the signal amplification pair. 28. The method of claim 1, wherein determining the activation level of p95HER2 in step (c) comprises: (i) incubating the cellular extract produced from the test cell with a plurality of beads specific for an extracellular domain (ECD) binding region of full-length HER2;(ii) removing the plurality of beads from the cellular extract produced from the test cell, thereby removing full-length HER2 to form a cellular extract produced from the test cell devoid of full-length HER2;(iii) incubating the cellular extract produced from the test cell devoid of full-length HER2 with a dilution series of capture antibodies specific for an intracellular domain (ICD) binding region of full-length HER2 to form a plurality of captured receptors, wherein the capture antibodies are restrained on a solid support;(iv) incubating the plurality of captured receptors with detection antibodies comprising activation state-independent antibodies and activation state-dependent antibodies specific for an ICD binding region of full-length HER2 to form a plurality of detectable captured receptors, 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;(v) incubating the plurality of detectable captured receptors with a second member of the signal amplification pair to generate an amplified signal; and(vi) detecting the amplified signal generated from the first and second members of the signal amplification pair. 29. The method of claim 28, wherein the plurality of beads specific for an ECD binding region comprises a streptavidin-biotin pair, wherein the streptavidin is attached to the bead and the biotin is attached to an antibody. 30. The method of claim 29, wherein the antibody is specific for the ECD binding region of full-length HER2. 31. The method of claim 28, wherein the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, fiber bundles, and combinations thereof. 32. The method of claim 28, wherein the capture antibodies are restrained on the solid support in an addressable array. 33. The method of claim 28, wherein the activation state-independent antibodies are directly labeled with the facilitating moiety. 34. The method of claim 28, wherein the activation state-dependent antibodies are directly labeled with the first member of the signal amplification pair. 35. The method of claim 28, wherein the activation state-dependent antibodies are labeled with the first member of the signal amplification pair via binding between a first member of a binding pair conjugated to the activation state-dependent antibodies and a second member of the binding pair conjugated to the first member of the signal amplification pair. 36. The method of claim 35, wherein the first member of the binding pair is biotin. 37. The method of claim 35, wherein the second member of the binding pair is streptavidin. 38. The method of claim 28, wherein the facilitating moiety is glucose oxidase. 39. The method of claim 38, wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule. 40. The method of claim 39, wherein the sulfhydryl-activated dextran molecule has a molecular weight of about 500 kDa. 41. The method of claim 38, wherein the oxidizing agent is hydrogen peroxide (H2O2). 42. The method of claim 41, wherein the first member of the signal amplification pair is a peroxidase. 43. The method of claim 42, wherein the peroxidase is horseradish peroxidase (HRP). 44. The method of claim 42, wherein the second member of the signal amplification pair is a tyramide reagent. 45. The method of claim 44, wherein the tyramide reagent is biotin-tyramide. 46. The method of claim 45, wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide. 47. The method of claim 46, wherein the activated tyramide is directly detected. 48. The method of claim 46, wherein the activated tyramide is detected upon the addition of a signal-detecting reagent. 49. The method of claim 48, wherein the signal-detecting reagent is a streptavidin-labeled fluorophore. 50. The method of claim 48, wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent. 51. The method of claim 50, wherein the chromogenic reagent is 3,3′,5,5′-tetramethylbenzidine (TMB). 52. The method of claim 1, wherein the compound-resistant cell is a BT/R cell. 53. The method of claim 1, further comprising determining the expression level of HER2 and/or p95HER2 in the cellular extract produced from the test cell.
