Antibody-based arrays for detecting multiple signal transducers in rare circulating cells

1. An array having superior dynamic range comprising a plurality of dilution series of capture antibodies specific for one or more analytes in a cellular extract, wherein the capture antibodies are restrained on a solid support; and

a plurality of detection antibodies comprising activation state-independent antibodies and activation state-dependent antibodies specific for one or more analytes, wherein the plurality of detection antibodies comprise: (i) a plurality of activation state-independent antibodies which are labeled with glucose oxidase, and (ii) a plurality of activation state-dependent antibodies labeled with a first member of a signal amplification pair, wherein the first member of the signal amplification pair is a peroxidase, wherein glucose oxidase generates hydrogen peroxide (H 2 O 2 ), which channels to and reacts with the first member of the signal amplification pair when antibodies of (i) and (ii) are in close proximity.

2. The array of claim 1 , wherein the cellular extract comprises an extract of circulating cells of a solid tumor.

3. The array of claim 2 , wherein the cells are isolated from a patient sample by immunomagnetic separation.

4. The array of claim 3 , wherein the patient sample is selected from the group consisting of whole blood, serum, plasma, urine, sputum, bronchial lavage fluid, tears, nipple aspirate, lymph, saliva, fine needle aspirate, and combinations thereof.

5. The array of claim 3 , wherein the isolated cells are selected from the group consisting of circulating tumor cells, circulating endothelial cells, circulating endothelial progenitor cells, cancer stem cells, and combinations thereof.

6. The array of claim 3 , wherein the isolated cells are stimulated in vitro with growth factors.

7. The array of claim 6 , wherein the isolated cells are incubated with an anticancer drug prior to growth factor stimulation.

8. The array of claim 7 , wherein the anticancer drug is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, immunosuppressive agent, and combinations thereof.

9. The array of claim 6 , wherein the isolated cells are lysed following growth factor stimulation to produce the cellular extract.

10. The array of claim 1 , wherein the one or more analytes comprise a plurality of signal transduction molecules.

11. The array of claim 1 , wherein the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, fiber bundles, and combinations thereof.

12. The array of claim 1 , wherein each dilution series comprises at least 3 descending capture antibody concentrations.

13. The array of claim 1 , wherein each dilution series comprises at least 6 descending capture antibody concentrations.

14. The array of claim 1 , wherein the capture antibodies in each dilution series are serially diluted at least 2-fold.

15. The array of claim 1 , wherein the one or more analytes are detected in about 1 cell.

16. The array of claim 1 , wherein the solid support are beads.

17. The array of claim 1 , wherein the one or more analytes in a cellular extract is a member selected from the group consisting of EGFR, VEGFR-1/FLT-1, VEGFR-2/FLK-1/KDR, VEGFR-3/FLT-4, FLT-3/FLK-2, PDGFR, c-KIT/SCFR, INSR, IGF-IR, IGF-IIR, IRR, CSF-1R, FGFR 1-4, HGFR 1-2, CCK4, TRK A-C, MET, RON, EPHA 1-8, EPHB 1-6, AXL, MER, TYRO3, TIE 1-2, TEK, RYK, DDR 1-2, RET, c-ROS, LTK, ALK, ROR 1-2, MUSK, AATYK 1-3, and RTK 106, Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK, Akt, MAPK/ERK, MEK, RAF, PLA2, MEKK, JNKK, JNK, p38, Shc (p66), PI3K, Ras, Rho, Rac1, Cdc42, PLC, PKC, p70 S6 kinase, p53, cyclin D1, STAT1, STAT3, PIP2, PIP3, PDK, mTOR, BAD, p21, p2′7, ROCK, IP3, TSP-1, NOS, PTEN, RSK 1-3, JNK, c-Jun, Rb, CREB, Ki67, paxillin; and combinations thereof.

18. The array of claim 1 , wherein the array further comprises a second member of the signal amplification pair.