Antibody-based arrays for detecting multiple signal transducers in rate circulating cells
View Patent ↗The present invention provides antibody-based arrays for detecting the activation state and/or total amount of a plurality of signal transduction molecules in rare circulating cells and methods of use thereof for facilitating cancer prognosis and diagnosis and the design of personalized, targeted therapies.
1. A method for performing a multiplex, high-throughput immunoassay for facilitating a cancer diagnosis, the method comprising:
(a) incubating a cellular extract with a plurality of dilution series of capture antibodies specific for at least one receptor tyrosine kinase in the cellular extract to form a plurality of captured receptor tyrosine kinases, wherein the capture antibodies are restrained on a solid support;
(b) incubating the plurality of captured receptor tyrosine kinases with detection antibodies specific for the at least one receptor tyrosine kinase to form a plurality of detectable captured receptor tyrosine kinases, wherein the detection antibodies comprise:
(1) a plurality of activation state-independent antibodies labeled with a facilitating moiety and specific for the at least one receptor tyrosine kinase, and
(2) a plurality of activation state-dependent antibodies labeled with a first member of a signal amplification pair and specific for the at least one receptor tyrosine kinase,
wherein the facilitating moiety, which is glucose oxidase conjugated to dextran, generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair, which is a peroxidase in proximity;
(c) incubating the plurality of detectable captured receptor tyrosine kinases with a second member of the signal amplification pair to generate an amplified signal;
(d) detecting the amplified signal generated from the first and second members of the signal amplification pair, and
(e) measuring the activation state or level of the at least one receptor tyrosine kinases by monitoring the amplified signal, in the multiplex, high-throughput immunoassay for facilitating the cancer diagnosis, wherein the cancer is selected from the group consisting of lung cancer, digestive cancer, gastrointestinal cancer, esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer, renal cancer, cancer of a central nervous system and a blood cancer.
2. The method of claim 1 , wherein the cellular extract comprises an extract of circulating cells of a solid tumor.
3. The method of claim 2 , wherein the cells are isolated from a patient sample by immunomagnetic separation.
4. The method of claim 1 , wherein the cellular extract is from a patient sample selected from the group consisting of whole blood, serum, plasma, urine, sputum, bronchial lavage fluid, tears, nipple aspirate, lymph, saliva, fine needle aspirate, a tissue sample and combinations thereof.
5. The method of claim 4 , wherein the patient sample contains cells selected from the group consisting of circulating tumor cells, circulating endothelial cells, circulating endothelial progenitor cells, a tumor tissue cell, cancer stem cells, and combinations thereof.
6. The method of claim 3 , wherein the isolated cells are stimulated in vitro with growth factors.
7. The method of claim 6 , wherein the isolated cells are lysed following growth factor stimulation to produce the cellular extract.
8. The method 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.
9. The method of claim 1 , wherein an amount of the amplified signal is correlative to an amount of the at least one receptor tyrosine kinase.
10. The method of claim 1 , wherein the activation state-independent antibodies are directly labeled with the facilitating moiety.
11. The method of claim 1 , wherein the activation state-independent antibodies are labeled with the facilitating moiety via hybridization under stringent hybridization conditions between an oligonucleotide conjugated to the activation state-independent antibodies and a complementary oligonucleotide conjugated to the facilitating moiety.
12. The method of claim 1 , wherein the activation state-dependent antibodies are directly labeled with the first member of the signal amplification pair.
13. The method of claim 1 , 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.
14. The method of claim 13 , wherein the first member of the binding pair is biotin.
15. The method of claim 13 , wherein the second member of the binding pair is streptavidin.
16. The method of claim 1 , wherein the oxidizing agent is hydrogen peroxide (H 2 O 2 ).
17. The method of claim 1 , wherein the peroxidase is horseradish peroxidase (HRP).
18. The method of claim 1 , wherein the second member of the signal amplification pair is a tyramide reagent.
19. The method of claim 18 , wherein the tyramide reagent is biotin-tyramide.
20. The method of claim 19 , wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide.
21. The method of claim 20 , wherein the activated tyramide is directly detected.
22. The method of claim 20 , wherein the activated tyramide is detected upon the addition of a signal-detecting reagent.
23. The method of claim 22 , wherein the signal-detecting reagent is a streptavidin-labeled fluorophore.
24. The method of claim 22 , wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent.
25. The method of claim 24 , wherein the chromogenic reagent is 3,3′,5,5′-tetramethylbenzidine (TMB).
26. The method of claim 1 , wherein the gastrointestinal cancer is a member selected from the group consisting of colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and gastric cancer.
27. The method of claim 1 , wherein the receptor tyrosine kinase is a member selected from the group consisting of HER1, HER2, HER3, HER4, VEGFR-1, VEGFR-2, VEGFR-3, FLT-3, FLK-2, PDGFR, c-KIT, INSR, IGF-IR, IGF-IIR, IRR, CSF-1R, MET and a combination thereof.