COLLABORATIVE ENZYME ENHANCED REACTIVE (CEER) IMMUNOASSAY USING FLOW CYTOMETRY
The present invention provides systems, methods, devices and kits for the rapid identification and quantification of an analyte such as a protein biomarker. In the multiplexed immunoassay of the present invention, a plurality of capture antibodies are attached to a bead or a plurality of beads to capture and retain a biomarker from a sample such as a cell lysate. The bead, with a particular capture antibody attached thereto, can identify the biomarker captured from the cell lysate.
1 . A method for performing a multiplexed immunoassay on a sample, the method comprising:
(a) contacting blood, plasma or a cell lysate with a plurality of capture antibodies attached to a bead, wherein said bead is specific for at least one analyte to form a plurality of captured analytes;
(b) contacting the plurality of captured analytes with at least one type of detection antibody specific for the at least one analyte, wherein the at least one type of detection antibody has a first member of a signal amplification pair, to form a plurality of detectable captured analytes;
(c) contacting 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 method of claim 1 , wherein said bead is labeled with a fluorochrome or detectable tag.
3 . The method of claim 1 , wherein the 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 (FNA), cerebral spinal fluid, and combinations thereof.
4 . The method of claim 1 , wherein the at least one analyte comprises at least one signal transduction molecule, a cytokine, a metabolite, a pathological molecule, or an autoantibody.
5 . The method of claim 1 , wherein the at least one type of detection antibody is at least two types of detection antibodies, which comprise:
(i) a plurality of activation state-independent antibodies labeled with a facilitating moiety; and
(ii) a plurality of activation state-dependent antibodies labeled with the first member of a signal amplification pair.
6 . The method of claim 5 , wherein the facilitating moiety generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair.
7 . The method of claim 1 , wherein the detection antibody comprises one type of detection antibody, which is a plurality of detection antibodies labeled with a first member of a signal amplification pair, specific for the at least one analyte.
8 . The method of claim 7 , wherein a facilitating moiety generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair.
9 . The method of claim 5 , wherein the facilitating moiety is glucose oxidase.
10 . The method of claim 6 , wherein the oxidizing agent is hydrogen peroxide (H 2 O 2 ).
11 . The method of claim 1 , wherein the first member of the signal amplification pair is a peroxidase.
12 . The method of claim 11 , wherein the peroxidase is horseradish peroxidase (HRP).
13 . The method of claim 1 , wherein the second member of the signal amplification pair is a tyramide reagent.
14 . The method of claim 13 , wherein the tyramide reagent is directly labeled with a fluorophore.
15 . The method of claim 13 , wherein the tyramide reagent is biotin-tyramide.
16 . The method of claim 1 , wherein the amplified signal is generated by peroxidase oxidation of the tyramide to produce an activated tyramide.
17 . The method of claim 16 , wherein the activated tyramide is directly detected.
18 . The method of claim 16 , wherein the activated tyramide is detected upon the addition of a signal-detecting reagent.
19 . The method of claim 18 , wherein the signal-detecting reagent is a streptavidin-labeled fluorophore.
20 . The method of claim 18 , wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent, wherein the chromogenic reagent is 3,3′,5,5′-tetramethylbenzidine (TMB).
21 . The method of claim 1 , wherein the method is used as a human disease diagnostic test, wherein the disease is a metabolic disease, a brain and cognitive disease, a gastrointestinal disease or a cancer.
22 . The method of claim 21 , wherein the cancer is a member selected from the group consisting of breast cancer, colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, prostate cancer, and gastric cancer.