Antibody-based arrays for detecting multiple signal transducers in rare 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. An assay for performing a multiplex, high-throughput immunoassay having superior dynamic range, the assay comprising:
(a) incubating a cellular extract with a plurality of dilution series of capture antibodies specific for one or more analytes in the cellular extract to form a plurality of captured analytes, wherein the capture antibodies are restrained on a solid support;
(b) incubating the plurality of captured analytes with detection antibodies specific for the corresponding analytes to form a plurality of detectable captured analytes, wherein the detection antibodies comprise:
(1) a plurality of activation state-independent antibodies labeled with glucose oxidase, and
(2) 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;
(c) incubating the plurality of detectable captured analytes with a second member of the signal amplification pair to generate an amplified signal; and
(d) detecting the amplified signal generated from the first and second members of the signal amplification pair.
2. The assay of claim 1 , wherein the cellular extract comprises an extract of circulating cells of a solid tumor.
3. The assay of claim 2 , wherein the cells are isolated from a patient sample by immunomagnetic separation.
4. The assay 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 assay 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 assay of claim 3 , wherein the isolated cells are i) stimulated in vitro with growth factors; or ii) incubated with an anticancer drug selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, immunosuppressive agent, and combinations thereof, prior to growth factor stimulation; or iii) lysed following growth factor stimulation to produce the cellular extract.
7. The assay of claim 1 , wherein the one or more analytes comprise a plurality of signal transduction molecules.
8. The assay 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 assay of claim 1 , wherein the activation state-independent antibodies further comprise a detectable moiety, wherein said detectable moiety is a fluorophore.
10. The assay of claim 9 , wherein the amount of the detectable moiety is correlative to the amount of one or more of the analytes.
11. The assay of claim 1 , wherein the activation state-independent antibodies are directly labeled with glucose oxidase.
12. The assay of claim 1 , wherein the activation state-independent antibodies are labeled with glucose oxidase via hybridization between an oligonucleotide conjugated to the activation state-independent antibodies and a complementary oligonucleotide conjugated to glucose oxidase.
13. The assay of claim 1 , wherein the activation state-dependent antibodies are directly labeled with the first member of the signal amplification pair.
14. The assay 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 and a second member of the binding pair, wherein the first member of a binding pair is biotin conjugated to the activation state-dependent antibodies and a second member of the binding pair, wherein the second member of a binding pair is streptavidin conjugated to the first member of the signal amplification pair.
15. The assay of claim 1 , wherein the peroxidase is horseradish peroxidase (HRP).
16. The assay of claim 14 , wherein the second member of the signal amplification pair is a tyramide reagent, which is biotin-tyramide.
17. The assay of claim 16 , wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide.
18. The assay of claim 17 , wherein the activated tyramide is directly detected.
19. The assay of claim 17 , wherein the activated tyramide is detected upon the addition of a signal-detecting reagent, which is a streptavidin-labeled fluorophore.
20. The assay of claim 19 , wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent, which is 3,3′,5,5′-tetramethylbenzidine (TMB).