JP2016527495A - Immunoassay with coordinated enzyme-enhanced reaction (CEER) using microfluidics - Google Patents

Abstract

The present invention diagnoses a disease state of a patient by detecting the presence / absence, expression level and / or activity level of an analyte component of interest in a patient sample using a proximity dual detection assay for microcarriers. Providing a method for The present invention also provides an assay device for performing the methods described herein. [Selection figure] None

Description

  [0001] Traditional epidemiological studies involving large cohorts of patients do not take into account the genetic diversity of each individual belonging to a given population. As a result, certain drugs or treatments are very effective for some individuals, while not very effective for others. The purpose of personalized medicine is to ensure that a given drug is administered to the correct individual at the correct dose.

  [0002] Strategies for optimizing treatment can be developed using various biomarkers, such as genes, SNPs, enzyme activity, protein expression and activity, as well as their impact on metabolic pathways. Once the relevant biomarkers are identified, diagnostic assays useful for understanding drug efficacy and risk of side effects can be developed. To optimize patient response and minimize patient risk, it may be very important to select an appropriate dose or schedule.

  [0003] In developing diagnostic assays and kits for personalized medicine, identifying appropriate biomarkers is the first important step. Once a biomarker has been identified, it can be verified using a diagnostic test and then quantified or measured. In certain examples, a point of care approach is used. In this approach, the assay is performed at or near the location where the patient is treated where medical examination and / or treatment can be performed. For example, the location for point of care includes, but is not limited to, a hospital, a patient's home, a clinic, or a location in an ambulance or where an emergency response is required.

  [0004] The effects associated with rapidly diagnosing a medical condition, selecting an appropriate treatment, or optimizing a point-of-care dose have numerous advantages. Individualizing drugs, doses and treatment plans means that the patient has the best opportunity to benefit from modern medicine. There is a need in the art for new methods to identify and measure biomarkers for patient clinical effects using point-of-care devices. The present invention fulfills this and other needs.

[0005] In one embodiment, the present invention provides an in vitro method for performing a multiplexed immunoassay, such as a microfluidic collaborative enzyme enhanced reactive (CEER) immunoassay, on a sample. To provide such a method. This method
(A) contacting a cell lysate with a plurality of capture antibodies that are specific for at least one analyte component and immobilized on an encoded microcarrier to produce a plurality of captured analyte components Generating,
(B) contacting a plurality of captured analyte components with at least two detection antibodies specific for the corresponding analyte components to produce a plurality of captured analyte components that are detectable (where At least one detection antibody has a first member of a signal amplification pair);
(C) contacting a detectable plurality of captured analyte components with a second member of a signal amplification pair to produce an amplified signal;
(D) detecting the amplified signal generated from the first and second members of the signal amplification pair.

[0006] In some embodiments, the encoded microcarriers are latex, polystyrene, cross-linked dextran, polymethylstyrene, polycarbonate, polypropylene, cellulose, polyacrylamide, polydimethylacrylamide, fluorinated ethylene-propylene, glass, SiO _2, silicon, PMMA (polymethylmethacrylate), gold, silver, aluminum, is made from a material selected from the group consisting of steel, and SU-8. In one embodiment, the encoded microcarrier is made of silicon.

  [0007] In some embodiments, the encoded microcarrier is formed into a shape selected from the group consisting of a sphere, a wafer, a square, a disk, a rectangle, a circle, a triangle, and a hexagon. In one embodiment, the encoded microcarrier is formed into a wafer.

  [0008] In some embodiments, the encoded microcarrier includes a plurality of microcarriers.

  [0009] In some embodiments, the plurality of microcarriers has at least two groups. In some examples, each of the at least two groups of encoded microcarriers has a different function. In one example, the specific function can be identified by a code.

  [0010] In some embodiments, the encoded microcarrier has a function determined by reading a code. In some examples, the code is in the form of a hole that traverses the microcarrier. In another example, the specific function is the identification of multiple capture antibodies.

  [0011] In some embodiments, the sample is whole blood, serum, plasma, urine, sputum, bronchial lavage, tears, nipple aspirate, lymph, saliva, cerebrospinal fluid (CSF), fine needle aspirate (FNA) ), And combinations thereof.

[0012] In another embodiment, the detection antibody is:
(I) a plurality of antibodies labeled with a facilitating moiety and independent of activation state;
(Ii) a plurality of antibodies that are labeled with the first member of the signal amplification pair and depend on the activation state.

[0013] In some embodiments, the facilitating moiety produces an oxidizing agent that channels and reacts to the first member of the signal amplification pair. In certain instances, the facilitating moiety is glucose oxidase. In certain embodiments, the oxidant is hydrogen peroxide (H ₂ O ₂ ). In some embodiments, the first member of the signal amplification pair is peroxidase. In certain examples, the peroxidase is horseradish peroxidase (HRP).

  [0014] In some embodiments, the second member of the signal amplification pair is a tyramide formulation. In certain examples, the tyramide agent is biotin-tyramide.

  [0015] In some embodiments, the amplified signal is generated by biotin-tyramide being oxidized by peroxidase to produce activated tyramide. In some embodiments, activated tyramide is detected directly. In other embodiments, activated tyramide is detected by the addition of a signal detection agent. In some examples, the signal detection agent is a streptavidin labeled fluorophore. In another example, the signal detection agent is a combination of streptavidin-labeled peroxidase and a color former. In some embodiments, the color former is 3,3 ', 5,5'-tetramethylbenzidine (TMB).

  [0016] In some embodiments, the methods provided herein are used as a diagnostic test for human disease. In some aspects, the disease is a metabolic disease, brain and cognitive disorder, brain disorder, cognitive disorder, gastrointestinal disease, or cancer.

  [0017] In some aspects, the metabolic disease is diabetes, including pre-diabetes, type 1 diabetes, type 2 diabetes, other forms of diabetes, and diabetes related diseases.

  [0018] In some embodiments, the brain and cognitive disorder is Alzheimer's disease.

  [0019] In some examples, the cancer consists of breast cancer, colorectal cancer, gastrointestinal stromal tumor, gastrointestinal carcinoid, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, prostate cancer, and gastric cancer It is selected from the group.

  [0020] In some embodiments, the at least one analyte component comprises at least one signaling molecule, pathological protein aggregate, or autoantibody.

  [0021] In some examples, the at least one signaling molecule is AMP-activated protein kinase (AMPK), AMPK kinase (AMPKK), calmodulin-dependent protein kinase kinase (CAMKK), LKB1, transforming growth factor − Selected from the group consisting of β-activated kinase 1 (TAK1), AKT, adiponectin, leptin, glucose, other AMPK modulators, and combinations thereof.

  [0022] In other examples, the at least one pathological protein aggregate is tau, activated (phosphorylated) tau, amyloid beta (Aβ) peptide, soluble amyloid precursor protein (APPβ), fragments thereof, These complexes are selected from the group consisting of these oligomers.

  [0023] In other examples, the at least one autoantibody is a GAD65 autoantibody, an insulinoma antigen 2 (IA-2) autoantibody, an insulin autoantibody (IAA), a zinc transporter protein 8 (ZnT8) autoantibody, and Selected from the group consisting of these combinations.

  [0024] In yet another example, the at least one signaling molecule is a receptor tyrosine kinase. In certain embodiments, the receptor tyrosine kinase is HER1, HER2, HER3, HER4, VEGFR-1, VEGFR-2, VEGFR-3, FLT-3, FLK-2, PDGFR, c-KIT, INSR, IGF. -A member selected from the group consisting of IR, IGF-IIR, IRR, CSF-1R, cMET, and combinations thereof. In other embodiments, the at least one receptor tyrosine kinase further comprises a non-receptor tyrosine kinase.

  [0025] In some embodiments, the sample flows through a microchannel that includes encoded microcarriers. In some embodiments, the microchannel is transparent at least on one side.

[0026] In some embodiments, the amplified signal is associated with an encoded microcarrier identity. In some examples, the amplified signal is related to a kinetic binding parameter. These parameters, for example, association _{rate, k on;} dissociation _{rate, k off;} or affinity constant, such as _{K a,} include detailed binding kinetic parameters.

[0027] The methods described herein are preferably in vitro methods. In one embodiment, the present invention provides an in vitro method for performing a multiplexed immunoassay, such as a microfluidic coordinated enzyme enhanced reaction (CEER) immunoassay, on a sample, the method comprising:
(A) contacting a cell lysate with a plurality of capture antibodies that are specific for at least one analyte component and immobilized on an encoded microcarrier to produce a plurality of captured analyte components Generating,
(B) contacting a plurality of captured analyte components with at least two detection antibodies specific for the corresponding analyte components to produce a plurality of captured analyte components that are detectable (where At least one detection antibody has a first member of a signal amplification pair);
(C) contacting a detectable plurality of captured analyte components with a second member of a signal amplification pair to produce an amplified signal;
(D) detecting the amplified signal generated from the first and second members of the signal amplification pair.

  [0028] In some embodiments of the method, detecting the amplified signal is performed using an optical sensor array coupled to optical means, wherein the sensor is a CCD or C-MOS photosensor. It is.

[0029] In another embodiment, the present invention provides a point-of-care diagnostic system. The system:
(A) an assay device comprising a reaction chamber, wherein the reaction chamber comprises a microchannel;
(B) i) a first group of individually encoded microcarriers having a function and ii) a kit comprising at least two detection antibodies;
The first group of individually encoded microcarriers has a first plurality of capture antibodies bound to the microcarriers, and the at least two detection antibodies are:
a) a plurality of antibodies labeled with a facilitating moiety and independent of activation state;
b) a plurality of antibodies that are labeled with the first member of the signal amplification pair and depend on the activation state.

  [0030] In some embodiments of the system, the apparatus includes means for restricting movement of the microcarriers in the longitudinal direction of the microchannel while still allowing fluid to flow. In other embodiments, the kit further comprises a second group of individually encoded microcarriers, wherein the second group of individually encoded microcarriers has a second plurality of capture antibodies. In another embodiment, the first group of individually encoded microcarriers has a function and the second group of individually encoded microcarriers has a different function.

  [0031] In some embodiments, the function is the identification of multiple capture antibodies. In other aspects, the function can be specified by a code. In some embodiments, the code is configured to form a hole across the microcarrier.

[0032] In some embodiments, the encoded microcarrier is latex, polystyrene, cross-linked dextran, polymethylstyrene, polycarbonate, polypropylene, cellulose, polyacrylamide, polydimethylacrylamide, fluorinated ethylene-propylene, glass, SiO _2, silicon, PMMA (polymethylmethacrylate), gold, silver, aluminum, is made from a material selected from the group consisting of steel, and SU-8. In one embodiment, the encoded microcarrier is made of silicon.

  [0033] In some embodiments, the function of the encoded microcarrier is determined by reading the code.

  [0034] In some embodiments, the encoded microcarriers are formed into a shape selected from the group consisting of a sphere, a wafer, a square, a disk, a rectangle, a circle, a triangle, and a hexagon. In one embodiment, the encoded microcarrier is formed into a wafer.

  [0035] These and other objects, features and advantages of the present invention will become more apparent to those skilled in the art by reference to the following detailed description and drawings.

FIG. 1 shows the results obtained from an exemplary embodiment of the present invention. FIG. 1A shows the dilution series fluorescence of BT474 cell lysate when biotin-tyramide (BT-Tyr) was used at high concentration (1: 500). FIG. 1B shows the fluorescence of a dilution series of BT474 cell lysate when BT-Tyr was used at a low concentration (1: 1000). FIG. 1 shows the results obtained from an exemplary embodiment of the present invention. FIG. 1A shows the dilution series fluorescence of BT474 cell lysate when biotin-tyramide (BT-Tyr) was used at high concentration (1: 500). FIG. 1B shows the fluorescence of a dilution series of BT474 cell lysate when BT-Tyr was used at a low concentration (1: 1000). FIG. 2 shows a microscopic image of the result shown in FIG. 1B. FIG. 2A shows an image of a microcarrier incubated with a low concentration (1: 1000) of biotin-tyramide with 75 cells of the sample. FIG. 2B shows an image of a microcarrier incubated with a low concentration (1: 1000) of biotin-tyramide with 7.5 cells of the sample. FIG. 2 shows a microscopic image of the result shown in FIG. 1B. FIG. 2A shows an image of a microcarrier incubated with a low concentration (1: 1000) of biotin-tyramide with 75 cells of the sample. FIG. 2B shows an image of a microcarrier incubated with a low concentration (1: 1000) of biotin-tyramide with 7.5 cells of the sample.

I. Introduction
[0038] The present invention provides methods, devices, methods of use, and kits for rapidly identifying and quantifying analyte components such as protein biomarkers. In the multiplexed immunoassay of the present invention, a plurality of capture antibodies are bound to an encoded microcarrier to retain, for example, a biomarker derived from a cell lysate. The encoded microcarrier can be used to identify a specific capture antibody and, in turn, to identify a biomarker, ie, a captured analyte component. In certain instances, the invention may identify a patient suffering from a disease, identify whether a patient responds to treatment, identify an appropriate drug dosage for administration to a patient, etc. Provide in vitro use of multiplexed immunoassays to identify analyte components (eg, biomarkers) for diagnostic purposes. These objectives can be achieved by measuring the expression or activity level of the identified analyte component.

  [0039] Utilization of the present invention enables rapid and accurate diagnosis of medical conditions such as metabolic diseases, brain and cognitive diseases, gastrointestinal diseases, or cancer, identification of appropriate treatments, and optimization of therapeutic doses As such, patients and medical professionals have significant benefits.

  [0040] Advantageously, the methods, devices, uses, and kits of the present invention can perform diagnostic tests in real time or near real time, so that the tests that can be performed are similar as not utilizing the system of the present invention. Promotes diagnostic tests that are more efficient than point-of-care tests and can be provided at the point of care. A point-of-care diagnostic test is a test at or near a location where a patient is treated, such as a clinic or hospital or where medical care is provided. Due to the short time required to obtain assay results, there are many such as real-time evidence-based judgment, patient emergency treatment, minimizing unnecessary testing, minimizing side effects, and significant cost-effectiveness Benefits are provided.

  [0041] In certain instances, the methods and apparatus of the present invention allow clinical tests to be performed outside the central laboratory, in close proximity to the patient. In certain aspects, the method comprises a sample, ie, whole blood, plasma, serum, fine needle aspirate (FNA), or cerebrospinal fluid (CSF), and a ready-to-use reagent. And a disposable assay cartridge. In certain embodiments, the device of the present invention is portable and utilizes the sample content and quantity of each individual.

II. Definition
[0042] As used herein, the following terms have the meanings ascribed to them unless stated otherwise.

  [0043] The term "analyte component" encompasses any molecule of interest, typically including a macromolecule such as a polypeptide whose presence, amount, and / or identity is specified. In certain instances, the analyte component is a circulating cell of a solid tumor or a cellular component of cerebrospinal fluid, peripheral blood, or serum. Preferably, the analyte component or biomarker is a signaling molecule, pathological molecule, or autoantibody.

  [0044] The term "signaling molecule" or "signaling factor" refers to a response by which a cell transduces extracellular or intracellular signals, ie typically involved in a series of biochemical reactions inside the cell. Includes proteins and other molecules that perform the process by induction. Examples of signal transduction molecules include receptor tyrosine kinases, such as EGFR (eg, EGFR / HER-1 / ErbB1, HER-2 / Neu / ErbB2, HER-3 / ErbB3, HER-4 / ErbB4), respectively. VEGFR-1 / FLT-1, VEGFR-2 / FLK-1 / KDR, VEGFR-3 / FLT-4, FLT-3 / FLK-2, PDGFR (eg, PDGFRA, PDGFRB), c-KIT / SCFR, INSR (Insulin receptor), IGF-IR, IGF-IIR, IRR (insulin receptor related receptor), 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, V-cadherin, LTK (leukocyte tyrosine kinase), ALK (undifferentiated lymphoma kinase), ROR 1-2, MUSK, AATYK 1-3, RTK 106, and cleavage Receptor tyrosine kinases such as p95ErbB2; non-receptor tyrosine kinases such as BCR-ABL, Src, Frk, Btk, Csk, Abl, Zap70, Fes / Fps, Fak, Jak, Ack, and LIMK; tyrosine kinase Signaling cascade components such as AKT, MAPK / ERK, MEK, RAF, PLA2, MEKK, JNKK, JNK, p38, Shc (p66), PI3K, Ras (eg, K-Ras, N-Ras, H-Ras ), Rho, Rac1, Cdc42 , PLC, PKC, p70 S6 kinase, p53, cyclin D1, STAT1, STAT3, PIP2, PIP3, PDK, mTOR, BAD, p21, p27, ROCK, IP3, TSP-1, NOS, PTEN, RSK 1-3, JNK C-Jun, Rb, CREB, Ki67, and paxillin; nuclear hormone receptors such as estrogen receptor (ER), progesterone receptor (PR), androgen receptor, glucocorticoid receptor, mineralocorticoid receptor, Vitamin A receptor, vitamin D receptor, retinoid receptor, thyroid hormone receptor, and orphan receptor; nuclear receptor coactivators and repressors, such as amplified in breast cancer-1 (AIB1) and nuclear Acceptance Corepressor 1 (NCOR), and combinations thereof without limitation. Additional examples of signaling molecules include AMP-activated protein kinase (AMPK), AMPK kinase (AMPKK), calmodulin-dependent protein kinase kinase (CAMKK), LKB1, transforming growth factor-β-activated kinase 1 (TAK1). ), AKT, adiponectin, leptin, glucose, glycogen, AMP, and ATP, but are not limited thereto.

  [0045] The term "pathological molecule" refers to Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, spinal muscular atrophy, and similar diseases Proteins and other molecules involved in the pathology of diseases or disorders such as neurodegenerative diseases.

  [0046] The term "capture antibody" is specific to (ie, binds to, binds to, or forms a complex with) one or more analyte components of interest in a sample, such as a cell extract. It is intended to encompass immobilized antibodies. In preferred embodiments, the capture antibody is immobilized on a microcarrier (eg, a solid support) in an array device. Suitable capture antibodies for immobilizing any of a variety of signaling molecules on a solid support are Upstate (Temecula, Calif.), Biosource (Camarillo, Calif.), Cell Signaling Technologies (Danvers, Mass.), R & D Systems. (Minneapolis, Minn.), Lab Vision (Fremont, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), Sigma (St. Louis, Mo.), and BD Biosciences (Can. It is.

  [0047] As used herein, the term "detection antibody" is specific to (ie, binds to, binds to, or forms a complex with) one or more analyte components of interest in a sample. Includes antibodies that contain a detectable label). The term also encompasses antibodies specific for one or more analyte components of interest, which can be bound by another type of antibody that contains a detectable label. Examples of detectable labels include, but are not limited to, biotin / streptavidin labels, nucleic acid (eg, oligonucleotide) labels, chemically reactive labels, fluorescent labels, enzyme labels, radioactive labels, and combinations thereof. . Suitable detection antibodies for detecting the activation state and / or total amount of any of the various signaling molecules include Upstate (Temecula, Calif.), Biosource (Camarillo, Calif.), Cell Signaling Technologies (Danvers). Mass.), R & D Systems (Minneapolis, Minn.), Lab Vision (Fremont, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), Sigma (St. Louis, B. MoS. , Calif.).

  [0048] The term "activation state dependent antibody" is specific to (ie, binds to, binds to or together with) a particular activation state of one or more analyte components of interest in a sample. A detection antibody that forms a complex). In preferred embodiments, the activation state dependent antibody detects phosphorylation, ubiquitination, and / or complex state of one or more analyte components, such as one or more signaling molecules. In some embodiments, antibodies that depend on the activation state are used to detect phosphorylation of members of the EGFR family of receptor tyrosine kinases and / or detection of heterodimer formation between EGFR family members. Non-limiting examples of activation states (shown in parentheses) suitable for detection by antibodies that depend on activation states include: EGFR (EGFRvIII, phosphorylated (p-) EGFR, EGFR: Shc, ubiquitination (u-)) EGFR, p-EGFRvIII); ErbB2 (p95: truncated (Tr) -ErbB2, p-ErbB2, p95: Tr-p-ErbB2, HER-2: Shc, ErbB2: PI3K, ErbB2: EGFR, ErbB2: Erb3: ErbB4 (p-ErbB3, ErbB3: PI3K, p-ErbB3: PI3K, ErbB3: Shc); ErbB4 (p-ErbB4, ErbB4: Shc); ER (p-ER (S118, S167); p-IGF-1R, IGF-1R: RS, IRS: PI3K, p-IRS, IGF-1R: PI3K); INSR (p-INSR); KIT (p-KIT); FLT3 (p-FLT3); HGFRI (p-HGFR1); HGFR2 (p-HGFR2) ); RET (p-RET); PDGFRa (p-PDGFRa); PDGFRP (p-PDGFRP); VEGFRI (p-VEGFRI, VEGFRI: PLCG, VEGFR1: Src); VEGFR2 (p-VEGFR2, VEGFR2: PLCy, VEGFR2: Src, VEGFR2: heparin sulfate, VEGFR2: VE-cadherin); VEGFR3 (p-VEGFR3); FGFR1 (p-FGFR1); FGFR2 (p-FGFR2); FGFR3 (p-FGFR3); FGFR4 (p-FGFR4); 1 (p-Tie1); Tie2 (p-Tie2); EphA (p-EphA); EphB (p-EphB); NFKB and / or IKB (p-IK (S32), p-NFKB (S536), p- P65: IKBa); Akt (p-Akt (T308, S473)); PTEN (p-PTEN); Bad (p-Bad (S112, S136), Bad: 14-3-3); mTor (p-mtor ( P70S6K (p-p70S6K (T229, T389)); Mek (p-Mek (S217, S221)); Erk (p-Erk (T202, Y204)); Rsk-1 (p-Rsk-1 ( T357, S363)); Jnk (p-Jnk (T183, Y185)); P38 (p-P38 (T180, Y182)); Stat3 ( p-Stat-3 (Y705, S727)); Fak (p-Fak (Y576)); Rb (p-Rb (S249, T252, S780)); Ki67; p53 (p-p53 (5392, S20)); CREB (p-CREB (S133)); c-Jun (pc-Jun (S63)); cSrc (p-cSrc (Y416)); Paxillin (p-paxillin (Y118)); AMPK (p-AMPK ( T172, T183); and tau (p-tau (Y18, Y29, S46, T181, S199, S202, T205, T212, S214, T217, T231, S235, S262, S356, S396, S400, S404, S412, S422, S470, S492, S498, S515, S516, S519, T534, T548, 552, S622, S641, S713, S721, S726, etc. T739) and the like.

  [0049] The term "activation state independent antibody" is specific to (ie, binds to, binds to or together with) one or more analyte components of interest in a sample, regardless of activation state. A detection antibody that forms a complex). For example, an antibody that does not depend on its activation state can detect both phosphorylated and non-phosphorylated forms of one or more analyte components, such as one or more signaling molecules.

  [0050] The term "nucleic acid" or "polynucleotide" encompasses deoxyribonucleotides or ribonucleotides and polymers thereof, such as DNA and RNA, in either single-stranded or double-stranded form. Nucleic acids contain known nucleotide analogs or modified backbone residues or bonds that are synthetic, naturally occurring, and non-naturally occurring and have similar binding properties as the reference nucleic acid sequence. Includes nucleic acids. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidic acids, methylphosphonic acids, chiral-methylphosphonic acids, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNA). Unless otherwise stated, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise stated, specific nucleic acid sequences also implicitly include their conservatively modified variants, as well as complementary sequences and explicitly indicated sequences.

  [0051] The term "oligonucleotide" refers to a single stranded oligomer or polymer of RNA, DNA, RNA / DNA hybrids, and / or mimetics thereof. In certain instances, oligonucleotides are composed of naturally occurring (ie, unmodified) nucleobases, sugars, and internucleoside (backbone) linkages. In certain other examples, the oligonucleotide comprises a modified nucleobase, sugar, and / or internucleoside linkage.

  [0052] The term "incubate" is synonymous with "contact" and "expose" and does not imply any specific time or temperature unless otherwise stated.

  [0053] As used herein, the term "dilution series" is intended to encompass a series of specific samples (eg, cell lysates) or reagents (eg, antibodies) with decreasing concentrations. . A dilution series typically involves the process of mixing a fixed amount of sample or reagent with an initial concentration with a diluent (eg, a dilution buffer) to produce a low concentration of sample or reagent in the desired number of serial dilutions. Is generated by repeating a number of times sufficient to obtain By diluting the sample or reagent at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, or 1000 times The concentration of the sample or reagent is at least 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/11, 1 / 12, 1/13, 1/14, 1/15, 1/16, 1/17, 1/18, 1/19, 1/20, 1/25, 1/30, 1/35, 1/40, A dilution series can be generated that is reduced to 1/45 or 1/50. For example, a dilution series that includes a 2-fold serial dilution of a capture antibody formulation at an initial concentration of 1 mg / mL, mixes an amount of the initial concentration of capture antibody with the same amount of dilution buffer to give a concentration of 0.5 mg / mL. A capture antibody can be generated and this process can be repeated to obtain capture antibodies at concentrations such as 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.0325 mg / mL.

  [0054] As used herein, the term "excellent dynamic range" refers to the ability of an assay to detect specific analyte components in as little as one cell or thousands of cells. For example, the immunoassays described herein use 1 to 10,000 (eg, about 1, 5, 10, 25, 50, 75, 100, 250, 500) using a concentration dilution series of capture antibodies. , 750, 1000, 2500, 5000, 7500, or 10,000 cells), a specific signal transduction molecule of interest is advantageously detected, and thus has an excellent dynamic range.

  [0055] As used herein, the term "microcarrier" is a microparticle or microscale solid support functionalized on at least one surface, from which analyte components in a sample Can be used to analyze and / or react with such analyte components. In the present specification, the term “functionalized microcarrier” is simultaneously used. “A set of microcarriers” refers to one or more microcarriers used to capture and / or detect one particular target analyte component. This set may be due to only one type of microcarrier or may be due to one or more types of microcarriers. A set of microcarriers may carry one or more capture molecules (eg, capture antibodies or capture ligands) for the purpose of capturing two or more molecules of the target analyte component, but this still Is also referred to as a function. Two different sets of microcarriers that can be distinguished from each other differ in the capture molecules bound to the surface.

  [0056] As used herein, the term "code" is any property or characteristic of a microcarrier that is identifiable upon observation or detection and is identified by a microcarrier or a specific population of microcarriers (eg, a predetermined population). To a group of microcarriers having the following functions. The code on the microcarrier can be measured independent of position and can be measured independently of the results of the assay, i.e. the target analyte component to be detected need not be present. Typically, the code is characterized by the optical or magnetic response of the microcarriers when viewed. This response can be defined for the entire microcarrier (eg, the color of the microcarrier) or patterned layout (eg, the color of the microcarrier by spatially varying in or on the microcarriers). A bar code obtained by the change of (1) may be obtained. Examples of codes include color, shape, size, imprinted or engraved pattern, hole arrangement, holographic pattern, magnetic code, chemical composition, change in light transmission or reflection properties, emission of quantum dots, specific detection Possible foreign molecules (eg, oligonucleotides or other polymers), including, but not limited to, the surface of the IC chip attached or embedded.

  [0057] The term "encoded microcarrier" refers to a microcarrier having a code. Each microcarrier is encoded, ie, each microcarrier can have multiple microcarriers (typically a set of microcarriers) carrying a code of the same value (ie, the microcarrier is the microcarrier itself) Even if the code cannot be distinguished from the other code alone, it carries its own code. Different sets of encoded microcarriers can be identified and / or identified independently of the position of the microcarriers in the microchannels and independent of the results of the assay.

  [0058] The term "reaction chamber" refers to a space that captures and / or detects a reaction that occurs between a target analyte component and a capture molecule (eg, capture antibody or capture ligand) and / or a detection antibody.

  [0059] As used herein, the term "circulating cells" includes extratumoral cells that have metastasized or micrometastasized from a solid tumor. Examples of circulating cells include circulating tumor cells, cancer stem cells, and / or cells that migrate into the tumor (eg, circulating endothelial precursor cells, circulating endothelial cells, angiogenic circulating bone marrow cells, circulating dendritic cells, etc.). However, it is not limited to these.

  [0060] As used herein, the term "sample" encompasses any biological specimen obtained from a patient. Samples include whole blood, plasma, serum, red blood cells, white blood cells (eg, peripheral blood mononuclear cells), cerebrospinal fluid, ductal lavage fluid, nipple aspirate, lymph (eg, lymphatic disseminated tumor cells) Bone marrow aspiration fluid, saliva, urine, feces (ie, feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (eg, collected by random fine needle aspiration around the areola), any other body fluid, Examples include, but are not limited to, tissue samples such as tumor cytology (eg, needle biopsy) (eg, tumor tissue), or lymphatic vessels (eg, sentinel lymph node biopsy), and cell extracts thereof. In some embodiments, the sample is whole blood or a fractional component such as plasma, serum, or cell pellet. In other embodiments, the sample is obtained by isolating solid tumor circulating cells from whole blood or their cell fraction by any technique known in the art. In other embodiments, the sample is, for example, a formalin-fixed paraffin-embedded (FFPE) tissue sample derived from a breast solid tumor.

  [0061] "Biopsy" refers to the process of excising a tissue sample for diagnosis or prognostic evaluation and the tissue specimen itself. Any biopsy method known in the art can be utilized for the methods and compositions of the present invention. The biopsy method utilized will generally vary depending on the tissue being evaluated and the size and type of tumor (ie, solid or suspension (ie, blood or ascites)), among other factors. Exemplary biopsy methods include excision biopsy, incision biopsy, needle biopsy (eg, needle biopsy, fine needle aspiration biopsy, etc.), surgical biopsy, and bone marrow biopsy. Biopsy methods are discussed, for example, by Harrison Internal Medicine, edited by Kasper et al., 16th edition, 20005, Chapter 70, Part V. One skilled in the art will recognize biopsy methods that can identify cells of interest in a given tissue sample.

  [0062] The term "subject" or "patient" or "individual" typically includes humans, but other animals such as other primates, rodents, dogs, cats, horses, sheep Pigs, and similar animals can also be included.

  [0063] The present invention is useful where a point-of-care is done or where a laboratory-based clinical chemistry test is performed. As used herein, point of care (POC) locations include hospital tests, bedside tests, emergency rooms, clinics, outpatient clinics, blood banks, operating rooms, homes, laboratories Or it means a place such as an inspection at a nursing home or a remote inspection. It is particularly useful when performed in an analytical system or device where the sample and results of a test or assay performed at the same location as the point-of-care test are collected. However, it should be understood that the present invention can be used in facilities other than clinical laboratories.

  [0064] As used herein, the terms "metabolic disease", "metabolic disorder", and "metabolic syndrome" are used interchangeably and are caused by a disorder in normal metabolism (eg, metabolic balance). Point to. Metabolism is the process of converting food to energy at the cellular level. Typically, metabolic disorders are characterized by elevated blood pressure, hyperglycemia, abdominal excess body fat, insulin resistance, impaired glucose tolerance, and / or abnormal cholesterol levels. Non-limiting examples of metabolic diseases / disorders include type 1 diabetes, type 2 diabetes, prediabetes, diabetes related diseases, hyperinsulinemia, lysosomal storage disease, Gaucher disease, impaired glucose tolerance, fasting hyperglycemia, insulin Resistance, and dyslipidemia.

  [0065] The term "brain and cognitive disorder" or "brain and cognitive impairment" refers primarily to neurological / disorders and / or disorders related to mental health that affect learning, memory, perception and problem solving. / Refers to obstacles. The terms include delirium, severe and mild cognitive neurological disorders, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, spinal muscular atrophy. And all known neurological diseases / disorders characterized by reduced human mental activity and adverse effects on daily life.

  [0066] The term "cancer" is intended to encompass members of a disease class characterized by uncontrolled growth of abnormal cells. The term encompasses all known cancers and neoplastic diseases characterized by being malignant, benign, soft tissue, or solid, as well as all stages and grades of cancer, including pre- and post-migration cancers. . Examples of different types of cancer include breast cancer; lung cancer (eg, non-small cell lung cancer); gastrointestinal and gastrointestinal cancers such as colorectal cancer, gastrointestinal stromal tumor, gastrointestinal carcinoid, colon cancer, rectal cancer, anus Cancer, bile duct cancer, small intestine cancer, and gastric cancer; esophageal cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; ovarian cancer; kidney cancer (eg, renal cell cancer); cancer of the central nervous system; Examples include, but are not limited to: choriocarcinoma; head and neck cancer; osteosarcoma; and hematological cancer. As used herein, a “tumor” includes one or more cancer cells.

III. BEST MODE FOR CARRYING OUT THE INVENTION
[0067] In one aspect, the invention provides cancer, metabolic disease, gastrointestinal disease or brain and cognition by performing a multiplexed assay with a proximity dual detection assay on a microcarrier in an assay device. Methods for diagnosing disease, brain disease, or cognitive disease are provided. The method comprises a) contacting a patient sample with a plurality of encoded microcarriers placed or immobilized in a microchannel of the assay device to produce a plurality of captured analyte components; b) Contacting a plurality of captured analyte components on a microcarrier with at least two detection antibodies; [at least one detection antibody having a member of a signal amplification pair and capable of detection; Generating a component]; c) contacting a plurality of detectable captured analyte components with a second member of the signal amplification pair to generate an amplified signal; and d) a signal amplification pair. Detecting an amplified signal generated from the first and second members of.

  [0068] Utilizing the presence or absence, expression (eg, total amount) level and / or activity (eg, phosphorylation) level of a target analyte component in a patient sample, Determine if you have cancer.

  [0069] In some examples, HER1, HER2, HER3. HER4, VEGFR-1, VEGFR-2, VEGFR-3, FLT-3, FLK-2, PDGR, c-KIT, INSR, IGF-IR, IGF-IIR, IRR, CSF-IR, cMET, and combinations thereof The presence or absence, expression (eg, total amount) level and / or activity (eg, phosphorylation) level of one or more of the signaling molecules such as is used to diagnose a patient's cancer.

  [0070] In other examples, AMP-activated protein kinase (AMPK), AMPK kinase (AMPKK), calmodulin-dependent protein kinase kinase (CAMKK), LKB1, transforming growth factor-β-activated kinase 1 (TAK1), Presence / absence, expression (eg, total amount) level and / or activity (eg, phosphorylation) of one or more of signaling molecules such as AKT, adiponectin, leptin, glucose, other AMP modifiers, and combinations thereof Use it to diagnose a patient's diabetes.

  [0071] In other examples, one or more of GAD65 autoantibodies, insulinoma antigen 2 (IA-2) autoantibodies, insulin autoantibodies (IAA), zinc transporter protein 8 (ZnT8) autoantibodies, and combinations thereof The presence or absence and / or expression (eg, total amount) level of autoantibodies is used to diagnose diabetes in the patient.

  [0072] In other examples, tau, amyloid beta (Aβ) protein, amyloid precursor protein (APP), amyloid beta (Aβ) peptide (eg, β-amyloid protein 28, β-amyloid protein 40, β-amyloid protein 42, β-amyloid protein 43, soluble amyloid precursor protein beta (sAPPβ), and similar proteins), any Aβ protein fragment, VILIP-1, and combinations thereof, and the presence or absence of one or more pathological molecules The expression (eg total amount) level and / or activity (eg phosphorylation) level is used to diagnose Alzheimer's disease in a patient.

  [0073] In yet another example, the presence or level of at least one cytokine or cytokines in a sample is particularly useful in the present invention. As used herein, the term “cytokine” includes various polypeptides or proteins that are secreted by immune cells and control the functional range of the immune system, and also include low molecular weight cytokines such as chemokines. . The term “cytokine” also encompasses adipocytokines that are secreted by adipocytes and include, for example, a group of cytokines that function in body weight control, hematopoiesis, angiogenesis, wound healing, insulin resistance, immune response, and inflammatory response. Cytokines can also be used as biomarkers for disease, dysregulation, or dysfunction.

[0074] In certain embodiments, TNF-α, a TNF-related weak inducer of apoptosis (TWEAK), osteoclastogenesis inhibitor (OPG), IFN-α, IFN-β, IFN- γ, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, and IL-27, including but not limited to at least The presence and level of one cytokine is evaluated. In certain other embodiments, for example, CXCL1 / GRO1 / GROα, CXCL2 / GRO2, CXCL3 / GRO3, CXCL4 / PF-4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP- 2, CXCL9 / MIG, CXCL10 / IP-10, CXCL11 / I-TAC, CXCL12 / SDF-1, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15, CXCL16, CXCL17 / DMC, CCL1, CCL2 / MCP-1, CCL3 / MIP-1α, CCL4 / MIP-1β, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / CCL10, CCL11 / eotaxin, CCL12 / MCP-5, CCL13 / MC -4, CCL14 / HCC-1, CCL15 / MIP-5, CCL16 / LEC, CCL17 / TARC, CCL18 / MIP-4, CCL19 / MIP-3β, CCL20 / MIP-3α, CCL21 / SLC, CCL22 / MDC, CCL23 The presence or level of at least one chemokine such as / MPIF1, CCL24 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CL1, CL2, and CX ₃ CL1 is evaluated .

  [0075] In certain further embodiments, leptin, adiponectin, resistin, activity or total amount of plasminogen activator inhibitor-1 (PAI-1), visfatin, and retinol binding protein 4 (RBP4) in a sample. The presence or level of at least one adipocytokine is evaluated, including but not limited to.

  [0076] In other examples, the methods and systems of the present invention can be utilized to assess the presence or level of one or more growth factors in a sample. As used herein, the term “growth factor” includes various peptides, polypeptides, or proteins that are capable of stimulating cell growth and / or cell differentiation.

  [0077] In certain embodiments, also known as epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), vascular endothelial growth factor (VEGF), pigment epithelial derived factor (PEDF; SERPINF1) in a sample ), Amphiregulin (AREG; also known as Schwannoma-derived growth factor (SDGF)), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α) ), Transforming growth factor-β (TGF-β), bone morphogenetic protein (eg, BMP1-BMP15), platelet-derived growth factor (PDGF), nerve growth factor (NGF), β-nerve growth factor (β-NGF) , Neurotrophic factors (eg, brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotroph 4 (NT4), etc.), growth differentiation factor-9 (GDF-9), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), myostatin (GDF-8), erythropoietin (EPO), and the presence or level of at least one growth factor, including but not limited to thrombopoietin (TPO), is assessed. In one aspect, the presence or level of EGF, VEGF, PEDF, amphiregulin (SDGF), and / or BDNF is assessed.

[0078] Various kinetic binding parameters can be evaluated. Such parameters include, for example, binding kinetics parameters (e.g., association _{rate, k on;} dissociation _{rate, koff;} and affinity constant, _{K a)} can be mentioned. Using these parameters, for example, direct intermolecular interactions such as receptor-ligand interactions, enzyme-substrate interactions, protein-nucleic acid interactions, protein-antibody interactions, and protein-protein interactions. The effect can be confirmed and measured.

  [0079] Detecting the amplified signal includes reading the fluorescence and correlating the fluorescence intensity with the level (eg, amount, presence, concentration) of the target analyte component in the patient sample. This step can also include identifying microcarriers. In some embodiments, fluorescence detection is performed utilizing an optical sensor such as, but not limited to, a CCD or C-MOS photosensor.

  [0080] One skilled in the art will recognize that (a)-(c) of the multiplexed assay process is performed in such a way as to detect one or more specific target analyte components. Let's go. For example, the multiplex assay of the present invention measures the level of at least two different target analyte components.

  [0081] In some embodiments, the methods of the invention are performed in a microfluidic device, such as a Dynamic Multi-Analyte Technology (DMAT) device (Biocartis, Mechelen, Belgium).

A. Specimen component detection assay
[0082] Details about single detection assays and proximity dual detection assays can be found, for example, in US Pat. No. 8,163,499, which is hereby incorporated by reference in its entirety for all purposes. Incorporated.

1. Single detection assay
[0083] In some embodiments, the assay comprises a metabolic component or a brain component of interest, a brain cognitive impairment, a neurological disease / disorder and / or a disorder related to mental health, or a subject component of interest related to cancer, etc. It is for detecting a specific analyte component (for example, a signal transduction molecule or a neurodegenerative molecule) in a cell extract in an activated state of interest. In certain examples, specimen components can be internal to tumor cells, such as circulating cells of solid tumors. Very advantageously, the present invention provides a multiplex high throughput two antibody assay with excellent dynamic range. By way of non-limiting example, the two antibodies used in the assay include (1) a capture antibody specific for the analyte component; and (2) a detection antibody specific for the analyte component in the activated form (ie, in an activated state). Dependent antibodies). An antibody that depends on the activation state can detect, for example, phosphorylation, ubiquitination, and / or complex state of the analyte component. Alternatively, the detection antibody includes an antibody independent of the activation state, which detects the total amount of analyte components in the cell extract. Antibodies that are independent of the activation state are generally capable of detecting both activated and non-activated forms of the analyte component.

  [0084] In a preferred embodiment, the two-antibody assay comprises: (i) incubating a cell extract with multiple dilution series of capture antibodies to produce a plurality of captured analyte components; Incubating a captured analyte component with an activation state dependent antibody specific for the corresponding analyte component to produce a plurality of captured captured analyte components; (iii) detection Incubating possible captured analyte components with the first and second members of the signal amplification pair to produce an amplified signal; (iv) first and second of the signal amplification pair; Detecting an amplified signal generated from the members of

  [0085] The two-antibody assays described herein are typically antibody-based arrays that include a plurality of different capture antibodies, with a range of concentrations of capture antibody bound to the microcarrier surface.

  [0086] Capture and detection antibodies are preferably selected such that competition in analyte component binding is minimized (ie, capture and detection antibodies are their corresponding signaling or neurodegenerative molecules). Can be combined at the same time).

[0087] Thus, in one embodiment, the present invention provides a method of performing a multiplexed immunoassay on a sample, the method comprising:
(A) contacting a cell lysate with a plurality of capture antibodies that are specific to at least one analyte component immobilized on an encoded microcarrier to produce a plurality of captured analyte components Generating,
(B) contacting a plurality of captured analyte components with at least one detection antibody specific for the corresponding analyte component to produce a plurality of captured analyte components that are detectable [here Wherein at least one detection antibody has a first member of a signal amplification pair];
(C) contacting a detectable plurality of captured analyte components with a second member of a signal amplification pair to produce an amplified signal;
(D) detecting the amplified signal generated from the first and second members of the signal amplification pair.

  [0088] In certain embodiments, the detection antibody comprises one detection antibody composed of a plurality of detection antibodies labeled with a first member of a signal amplification pair specific for at least one analyte component. The facilitating moiety produces an oxidant that reacts with the first member of the signal amplification pair. In certain embodiments, the facilitating moiety is glucose oxidase (GO). A substrate such as dextran can be bound to GO, but not an antibody. Multiple GO moieties can be attached to dextran.

  [0089] In one embodiment, the detection antibody comprises a first member of a binding pair (eg, biotin) and the first member of the signal amplification pair comprises a second member of the binding pair (eg, streptavidin). . The member of the binding pair can be coupled directly or indirectly to the detection antibody or the first member of the signal amplification pair using methods well known in the art. In certain examples, the first member of the signal amplification pair is a peroxidase (eg, horseradish peroxidase (HRP), catalase, chloroperoxidase, cytochrome c peroxidase, eosinophil peroxidase, glutathione peroxidase, lactoperoxidase, myeloperoxidase, Thyroid peroxidase, deiodinase, etc.), and the second member of the signal amplification pair is a tyramide formulation (eg, biotin-tyramide).

[0090] Alternatively, the first member of the signal amplification pair can be directly bound to the detection antibody. In certain instances, peroxidase oxidation of a tyramide agent produces an amplified signal that produces activated tyramide in the presence of hydrogen peroxide (H ₂ O ₂ ).

[0091] In one aspect, hydrogen peroxide (H ₂ O ₂ ) is generated in situ. For example, a facilitating moiety such as glucose oxidase (GO) can be introduced into an immunoassay that is not conjugated to an antibody. That is, GO can be bound to a substrate such as dextran. Multiple GO moieties can be attached to dextran. In the presence of glucose, glucose oxidase produces an oxidant such as hydrogen peroxide that reacts with the first member of the signal amplification pair (eg, HRP).

  [0092] Activated tyramide is detected directly or by addition of a signal detection agent such as, for example, a streptavidin-labeled fluorophore or a combination of streptavidin-labeled peroxidase and a color former. . Examples of suitable fluorophores for use in the present invention include Alexa Fluor® dye (eg, Alexa Fluor® 555, Alexa Fluor® 647), fluorescein, fluorescein isothiocyanate (FITC), Oregon Green (TM) rhodamine, Texas Red, tetrarhodamine in isothiocyanate (TRITC), CyDye (TM) fluor (e.g., Cy2, Cy3, Cy5) and the like. Using methods well known in the art, the streptavidin label can be coupled directly or indirectly to the fluorophore or peroxidase. Non-limiting examples of color formers suitable for use in the present invention include 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), 2,2 ′. -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 4-chloro-1-naphthol (4CN), and / or porphyrinogen.

  [0093] In another embodiment, the present invention provides a kit for performing the two-antibody assay described above, the kit comprising: (a) a dilution series of a plurality of capture antibodies immobilized on a microcarrier; and (B) A plurality of detection antibodies (for example, an antibody independent of the activation state and / or an antibody dependent on the activation state). In some examples, the kit may further comprise instructions for how to use the kit to detect the activation state of multiple signaling molecules in circulating cells of a solid tumor. The kit may contain additional reagents in addition to the above from the viewpoint of carrying out the specific method of the present invention, for example, the first and second members of the signal amplification pair, tyramide signal amplification An agent, GO dextran, washing buffer and the like may be included.

  [0094] In another embodiment of the two-antibody approach, the present invention provides a method for detecting the presence or absence of a truncated receptor, the method comprising (a) cell extract, extracellular domain (ECD) incubating with a plurality of beads specific for the binding region and [ECD binding region is specific for full length receptor]; (b) by removing the plurality of beads from the cell extract Removing the full length receptor to produce a cell extract free of the full length receptor; (c) incubating the cell extract without the full length receptor with a plurality of capture antibodies [The plurality of capture antibodies are specific for the intracellular domain (ICD) binding region of the cleaved receptor, and the plurality of capture antibodies are immobilized on a solid support, Captured (D) incubating a plurality of captured cleaved receptors with a detection antibody specific for the corresponding cleaved receptor to produce a plurality of detectable, captured receptors (E) incubating a plurality of detectable, captured cleaved receptors with the first and second members of the signal amplification pair and amplified Generating a signal; (f) detecting the amplified signal generated from the first and second members of the signal amplification pair.

  [0095] In certain embodiments, the cleaved receptor is p95 ErbB2, and the full-length receptor is ErbB2 (HER-2). In certain other embodiments, the plurality of beads specific for the extracellular domain (ECD) binding region comprises a streptavidin-biotin pair, the biotin is bound to the bead, and biotin is an antibody (eg, this The antibody binds to the ECD binding region of the full length receptor).

  [0096] The present invention, in another embodiment of the two-antibody approach, provides a method for detecting the presence or absence of a cleaved protein, the method comprising: (a) dividing a cell extract into a plurality of full-length proteins specific; (B) removing the plurality of beads from the cell extract to remove the full length protein to produce a cell extract free of the full length receptor; c) incubating the full-length protein-free cell extract with multiple capture antibodies [the multiple capture antibodies are specific for the cleaved protein, and the multiple capture antibodies are: Fixed to microcarriers, resulting in multiple captured cleaved proteins]; (d) multiple captured cleaved proteins correspondingly cleaved Incubating with a detection antibody specific for the protein to produce a plurality of detectable, captured, cleaved proteins; (e) a plurality of detectable, captured, cleaved proteins; Incubating with the first and second members of the signal amplification pair to generate an amplified signal; (f) the amplified signal generated from the first and second members of the signal amplification pair; Detecting.

2. Proximity dual detection assay
[0097] In some embodiments, certain analyte components (eg, signaling molecules, eg, AMPK modulators, or pathological conditions) of interest in a cellular extract of cells, such as circulating cells, etc. Assays for detecting molecules, such as tau, Aβ peptides, etc., are multiplex high throughput proximity (ie, 3 antibody) assays with excellent dynamic range. By way of non-limiting example, the three antibodies used in the proximity assay include (1) a capture antibody specific for the analyte component; (2) a detection antibody specific for the analyte component in the activated form (ie, activated (3) a detection antibody that detects the total amount of analyte components (ie, an antibody that does not depend on the activation state). An antibody that depends on the activation state can detect, for example, phosphorylation, ubiquitination, and / or complex state of the analyte component. Antibodies that depend on the activation state can generally detect both activated and non-activated forms of the analyte component.

[0098] In one embodiment, the proximity assay comprises
(I) for example, incubating a cell extract from cerebrospinal fluid with a plurality of dilution series of capture antibodies against tau protein to generate a plurality of captured tau proteins;
(Ii) multiple captured tau proteins, multiple antibodies against all tau proteins (eg, antibodies that are independent of activation state) and multiple antibodies against phosphorylated tau proteins (eg, depending on activation state) A plurality of detectable, captured, activated, tau analyte components, and [antibodies against all tau proteins are labeled with a facilitating moiety (eg, glucose oxidase). The antibody against the phosphorylated tau protein is labeled with a first member of a signal amplification pair (eg, horseradish peroxidase) and the facilitating moiety (eg, glucose oxidase) produces an oxidant, Channel and react to the first member of the signal amplification pair];
(Iii) incubating a plurality of detectable, captured, activated tau analyte components with a second member of a signal amplification pair (eg, biotin-tyramide) to produce an amplified signal;
(Iv) using streptavidin-Alexa Fluor® dye as a signal detection agent to detect the amplified signal generated from horseradish peroxidase and biotin-tyramide.

  [0099] In a preferred embodiment, the proximity assay comprises (i) incubating a cell extract with a plurality of dilution series of capture antibodies to produce a plurality of captured analyte components; Captured analyte components can be detected by incubating the captured analyte component with a detection antibody that is specific to the corresponding analyte component and includes multiple activation state independent antibodies and multiple activation state dependent antibodies. A plurality of analyte components produced and [an antibody independent of activation state is labeled with a facilitating moiety, an antibody dependent on activation state is labeled with a first member of a signal amplification pair, and the facilitating moiety is oxidized And (iii) the oxidizing agent channels and reacts with the first member of the signal amplification pair]; (iii) a plurality of detectable captured analyte components Incubating with a second member of the amplification pair to produce an amplified signal; (iv) detecting the amplified signal produced from the first and second members of the signal amplification pair; ,including.

  [0100] Alternatively, antibodies that depend on the activation state can be labeled with a facilitating moiety, and antibodies that do not depend on the activation state can be labeled with the first member of the signal amplification pair.

  [0101] The capture antibody, the activation state independent antibody, and the activation state dependent antibody are preferably such that competition is minimized in binding to the analyte component (ie, all antibodies are It is preferred that they are selected (which can bind simultaneously to their corresponding signaling molecules).

  [0102] In some embodiments, the activation state independent antibody further comprises a detectable moiety. In such an example, the amount of detectable moiety corresponds to the amount of one or more analyte components in the cell extract. Examples of detectable moieties include, but are not limited to, fluorescent labels, chemically reactive labels, enzyme labels, radioactive labels and the like. Preferably, the detectable moiety is Alexa Fluor® dye (eg, Alexa Fluor® 647), fluorescein, fluorescein isothiocyanate (FITC), Oregon Green ™; rhodamine, Texas red, tetrarhodamine Fluorophores such as inisothiocyanate (TRITC), CyDye ™ fluor (eg, Cy2, Cy3, Cy5). Utilizing methods well known in the art, the detectable moiety can be coupled directly or indirectly to the antibody independent of the activation state.

[0103] In certain instances, antibodies that do not depend on activation status are directly labeled with a facilitating moiety. Utilizing methods well known in the art, the facilitating moiety can be attached to the antibody independent of the activation state. Suitable accelerating moieties for use in the present invention include any molecule capable of generating an oxidant, which oxidant is in close proximity to (ie, near or in space with) the accelerating moiety. ) Channeling (ie, directed) to another molecule and reacting (ie, bind, bind, or form a complex together). Examples of facilitating moieties include enzymes such as glucose oxidase or any other enzyme that catalyzes oxidation / reduction reactions involving oxygen molecules (O ₂ ) as electron acceptors, and photosensitization. Agents such as, but not limited to, methylene blue, rose bengal, porphyrin, squarate dye, phthalocyanine and the like. Non-limiting examples of oxidants include hydrogen peroxide (H ₂ O ₂ ), singlet oxygen, and any other compound that transfers oxygen atoms or gains electrons in oxidation / reduction reactions. . Preferably, in the presence of a suitable substrate (eg, glucose, light, etc.), the facilitating moiety (eg, glucose oxidase, photosensitizer, etc.) is oxidant (eg, hydrogen peroxide (H ₂ O ₂ ), Singlet oxygen, etc., and when the two parts are in close proximity to each other, the first member of the signal amplification pair (eg, horseradish peroxidase (HRP), a hapten protected by a protecting group, It reacts by channeling to an enzyme inactivated by thioether bonding to an enzyme inhibitor.

  [0104] In certain other examples, an activation state independent antibody comprises an oligonucleotide linker conjugated to an activation state independent antibody and a complementary oligonucleotide linker conjugated to a facilitating moiety. Labeled with a facilitating moiety indirectly via hybridization between the two. Methods that are well known in the art can be used to attach oligonucleotide linkers to antibodies that are independent of the facilitating moiety or activation state. In some embodiments, the oligonucleotide linker conjugated with the facilitating moiety has 100% complementarity with the oligonucleotide linker conjugated with the activation state independent antibody. In other embodiments, the oligonucleotide linker pair has, for example, at least 1, 2, 3, 4, 5, or 6 or more mismatch regions upon hybridization under stringent hybridization conditions. One skilled in the art will recognize that antibodies that are specific for different analyte components and do not depend on the activation state can be conjugated to either the same or different oligonucleotide linkers.

  [0105] The length of the oligonucleotide linker conjugated to the facilitating moiety or the antibody independent of activation state can be varied. Generally, the linker sequence can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in length. Typically, random nucleic acid sequences are generated for coupling. As a non-limiting example, a library of oligonucleotide linkers can be designed having three consecutive domains: a spacer domain; a signature domain; and a composite domain. Preferably, the oligonucleotide linker is designed to bind efficiently without compromising the function of the facilitating moiety or without compromising the function of the antibody that is conjugated to them and does not depend on the activation state.

  [0106] Oligonucleotide linker sequences can be designed to prevent or minimize the formation of any secondary structures under various assay conditions. Typically, their melting temperature is carefully monitored so that each fragment within the linker is involved in the overall assay. Generally, the dissolution temperature of the linker sequence fragment is 1-10 ° C. Analyze each of the three different domains within each linker using a computer algorithm (eg, OLIGO 6.0) to measure dissolution temperature, secondary structure, and hairpin structure under a defined ion concentration be able to. The binding sequence can also be analyzed overall for structural properties, as well as similarities to other composite oligonucleotide linker sequences, eg, whether it hybridizes to a complementary oligonucleotide linker under stringent conditions.

  [0107] The spacer region of the oligonucleotide linker maintains a sufficient distance between the oligonucleotide bridging moiety and the composite domain. The complex domain functions to bind molecules that are labeled with complementary oligonucleotide linker sequences to the complex domain by nucleic acid hybridization. Nucleic acid mediated hybridization can be performed either before or after antibody-analyte component (ie, antigen) complex formation, providing a more flexible assay format. Unlike many direct antibody conjugation methods, binding a relatively small oligonucleotide to an antibody or other molecule can affect the specific affinity of the antibody for target analyte components, or The impact on functionality is minimized.

  [0108] In some embodiments, signature sequence domains of oligonucleotide linkers can be used in complex multiplexed protein assays. Multiple antibodies can be conjugated with oligonucleotide linkers having different signature sequences. In multiplex immunoassays, reporter oligonucleotide sequences labeled with appropriate probes can be utilized to detect cross-reactivity of antibodies and their antigens in a multiplex assay format.

  [0109] Several different methods can be utilized to conjugate the oligonucleotide linker to the antibody or other molecule. For example, an oligonucleotide linker with a thiol group at either the 5 'or 3' end can be synthesized. The thiol group can be deprotected with a reducing agent (eg, TCEP-HCl), and the resulting linker can be purified using a desalting spin column. The resulting deprotected oligonucleotide linker can be conjugated to a primary amine of an antibody or other type of protein utilizing a heterobifunctional cross-linking linker such as SMCC. Alternatively, the 5 'phosphate group on the oligonucleotide can be treated with a water-soluble carbodiimide EDC to form a phosphate ester and subsequently coupled to an amine-containing molecule. In certain instances, the diol on the 3 'ribose residue can be oxidized to an aldehyde group and then conjugated to the amino group of an antibody or other type of protein by reductive amination. In certain other examples, oligonucleotide linkers can be synthesized by biotin modification at either the 3 'or 5' end and conjugation to a streptavidin labeled molecule.

  [0110] Oligonucleotide linkers are described in Usman et al. , J .; Am. Chem. Soc. 109: 7845 (1987); Scaringe et al. , Nucl. Acids Res. 18: 5433 (1990); Wincott et al. , Nucl. Acids Res. , 23: 2677-2684 (1995); and Wincott et al. , Methods Mol. Bio. 74:59 (1997), such as those described in the art. In general, the synthesis of oligonucleotides utilizes common nucleic acid protecting groups and coupling groups, such as 5'-terminal dimethoxytrityl and 3'-terminal phosphoramidites. Suitable reagents for oligonucleotide synthesis, nucleic acid deprotection methods, and nucleic acid purification methods are known in the art.

  [0111] In certain instances, antibodies that depend on the activation state are directly labeled with the first member of the signal amplification pair. Methods well known in the art can be used to bind signal amplification pair members to antibodies that depend on their activation state. In certain other examples, the activation state dependent antibody comprises a first member of a binding pair complexed to an activation state dependent antibody and a binding pair complexed to a first member of a signal amplification pair. Via indirect binding to the second member of the first of the signal amplification pair. Using methods well known in the art, members of a binding pair (eg, biotin / streptavidin) can be conjugated to a member of a signal amplification pair or an antibody that depends on the activation state. Examples of members of signal amplification pairs include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), catalase, chloroperoxidase, cytochrome c peroxidase, eosinophil peroxidase, glutathione peroxidase, lactoperoxidase, myeloperoxidase Thyroid peroxidase, deiodinase and the like. Other examples of signal amplification pair members include haptens protected by protecting groups, enzymes inactivated by thioether linkages to enzyme inhibitors, and the like.

[0112] In one example of proximity channeling, the facilitating moiety is glucose oxidase (GO) and the first member of the signal amplification pair is horseradish peroxidase (HRP). When GO is contacted with a substrate such as glucose, an oxidizing agent (ie, hydrogen peroxide (H ₂ O ₂ )) is generated. When HRP is present in the adjacent channeling of GO, the H ₂ O ₂ generated by GO channels to HRP to form the HRP-H ₂ O ₂ complex, which is a signal amplification pair. Amplified in the presence of a second member (eg, a chemiluminescent substrate such as luminol or isoluminol, or a fluorogenic substrate such as tyramide (eg, biotin-tyramide), homovanillic acid, or 4-hydroxyphenylacetic acid) Generate a signal. Methods utilizing GO and HRP in proximity assays are described, for example, in Langry et al. , U. S. Dept. of Energy Report No. UCRL-ID-136797 (1999). When biotin-tyramide is utilized as the second member of a signal amplification pair, the HRP-H ₂ O ₂ complex oxidizes tyramide to produce a reactive tyramide radical, which becomes a nearby nucleophilic residue. Covalently join. Activated tyramide is detected directly or detected by the addition of a signal detection agent, such as a streptavidin-labeled fluorophore or a combination of streptavidin-labeled peroxidase and a color former. Examples of suitable fluorophores for use in the present invention include Alexa Fluor® dye (eg, Alexa Fluor® 555, Alexa Fluor® 647, etc.), fluorescein, fluorescein isothiocyanate ( FITC), Oregon Green ™; rhodamine, Texas Red, tetrarhodamine in isothiocyanate (TRITC), CyDye ™ fluor (eg, Cy2, Cy3, Cy5) and the like. Using methods well known in the art, the streptavidin label can be coupled directly or indirectly to the fluorophore or peroxidase. Non-limiting examples of color formers suitable for use in the present invention include 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), 2,2 ′. -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 4-chloro-1-naphthol (4CN), and / or porphyrinogen.

  [0113] In another example of proximity channeling, the facilitating moiety is a photosensitizer and the first member of the signal amplification pair is a large molecule labeled with multiple haptens, wherein the multiple haptens are It is protected by a protecting group that prevents binding of the hapten to a specific binding partner (eg, ligand, antibody, etc.). For example, dextran molecules labeled with protected biotin, coumarin, and / or fluorescein molecules can be signal amplification pair members. Suitable protecting groups include, but are not limited to, phenoxy-, analino-, olefin-, thioether-, and selenoether-protecting groups. Other photosensitizers and protected hapten molecules suitable for the proximity assay of the present invention are described in US Pat. No. 5,807,675. When the photosensitizer is excited by light, an oxidant (ie, singlet oxygen) is generated. If the hapten molecule is present in channeling close to the photosensitizer, the singlet oxygen generated by the photosensitizer is channeled to and reacts with the thioether on the hapten protecting group, A carbonyl group (ketone or aldehyde) and sulfinic acid are generated, and the protecting group is released from the hapten. The unprotected hapten can then specifically bind to the second member of the signal amplification pair (eg, a specific binding partner that can produce a detectable signal). For example, when the hapten is biotin, the specific binding partner can be labeled streptavidin. Examples of the enzyme include alkaline phosphatase, β-galactosidase, HRP and the like. After washing away unbound reagents, a detectable signal is generated by adding a substrate that is detectable by an enzyme (eg, fluorescence, chemiluminescence, color development, etc.). It can be detected by a known measuring means. Alternatively, a detectable signal can be amplified by amplification of the tyramide signal, and the activated tyramide can be detected directly, or can be detected by the addition of the above signal detection agent.

  [0114] In yet another example of proximity channeling, the facilitating moiety is a photosensitizer and the first member of the signal amplification pair is an enzyme-inhibitor complex. The enzyme and inhibitor (eg, phosphonic acid labeled dextran) are linked together by a cleavable linker (eg, thioether). When the photosensitizer is excited by light, an oxidant (ie, singlet oxygen) is generated. When the enzyme-inhibitor complex is present in a channel adjacent to the photosensitizer, the singlet oxygen generated by the photosensitizer is channeled under the cleavable linker and reacts with it. The inhibitor is released from the enzyme by activating the enzyme. An enzyme substrate is added to produce a detectable signal, or an amplification reagent is added to produce an amplified signal.

[0115] In a further example of proximity channeling, the facilitating moiety is HRP, the first member of the signal amplification pair is a protected hapten or enzyme-inhibitor complex as described above, and the protecting group is p- Contains alkoxyphenol. The addition of phenylenediamine and H ₂ O ₂ produces a reactive phenylene imine, this molecule is protected hapten or an enzyme - it is channeled to the original inhibitor complex and reacts with p- alkoxy phenol protecting group To produce exposed haptens or reactive enzymes. Amplified signals are generated and detected as described above (eg, US Pat. No. 5,532,138 and US Pat. No. 5,445,944).

  [0116] In another embodiment, the present invention provides a kit for performing the proximity assay described above, the kit comprising: (a) a dilution series of a plurality of capture antibodies immobilized on a microcarrier; b) a plurality of detection antibodies (for example, an antibody independent of the activation state and an antibody dependent on the activation state). In some examples, the kit may further include instructions for using the kit to detect the activation status of multiple signaling molecules associated with circulating cells of the solid tumor. it can. The kit includes, for example, the first and second members of a signal amplification pair, a tyramide signal amplifying agent, a substrate for a facilitating moiety, a washing buffer, etc. Any additional reagents may be included.

B. Micro carrier
[0117] In the analyte component capture and detection assays of the present invention described above, in particular the capture antibody is bound to a microcarrier. Details about the microcarriers useful in the present invention are described, for example, in International Publication No. WO 2012/106827 and U.S. Patent No. 2011/0306506 and U.S. Patent No. 2013/0095574. The entire contents for purposes are incorporated herein by reference.

  [0118] In certain embodiments, provided microcarriers are solid supports in the shape of spheres, wafers, squares, disks, rectangles, circles, triangles, hexagons, or polygons. In some embodiments, the height of the microcarrier is less than both the width and height of the embodiment. In some embodiments, the microcarrier has an essentially parallel and essentially flat surface on each of the top and bottom.

  [0119] In some embodiments, the microcarriers have a disk-like shape (eg, a wafer shape) with a circular shaped sensing surface. In some examples, the microcarrier body ensures that there is a gap between the plane and the detection surface when the encoded microcarrier detection surface is placed on the plane facing the plane. And at least a spacing member protruding from the main body.

  [0120] In some embodiments, the diameter of the disk-like shape is about 1 to about 300 [mu] m, for example 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150. 175, 200, 225, 250, 275, or 300 μm.

  [0121] Microcarriers can be identified by a code, such as a digital code, that can constitute a hole across the microcarrier. A set (eg, a group) of microcarriers is composed of individual microcarriers having the same code and having the same function. Two different sets of microcarriers may have different functions and different codes.

  [0122] The code may be any pattern of holes through and across the entire thickness of the microcarrier including the detection surface. In some examples, the code also includes asymmetric directional marks, such as triangular or L-shaped signs.

  [0123] In some embodiments, the code is measured by reading (imaging). The code can be identified using common imaging and decoding methods. The code is preferably used to specify the function of the microcarrier.

  [0124] In some embodiments, a set of microcarriers has the same function, such as capturing and / or detecting a particular analyte component. For example, a set of microcarriers is composed of a plurality of coded individual microcarriers associated with a particular analyte component of interest. In certain embodiments, the function of the microcarrier corresponds to the identification of a capture antibody or capture ligand bound to the surface.

[0125] Microcarriers can be made from any material commonly used in high-throughput screening and diagnostic methods. Non-limiting examples of these materials include latex, polystyrene, crosslinked dextran, polymethylstyrene, polycarbonate, polypropylene, cellulose, polyacrylamide, polydimethylacrylamide, fluorinated ethylene-propylene, and microfabrication or micromilling. mentioned general materials utilized, for example, glass, SiO _2, silicon, PMMA (polymethylmethacrylate), gold, silver, aluminum, steel, or the like other metals, or epoxy-based photosensitive agent, for example, and SU-8 It is done. The microcarrier may be any shape and size. In some embodiments, the microcarrier is made of silicon.

  [0126] Microcarriers can be biofunctionalized with one or more capture antibodies of the present invention. Any known method can be utilized to bind the capture antibody to the microcarrier.

C. Diagnostic system
[0127] In the diagnostic systems provided herein, encoded microcarriers are placed in a microchannel of an assay device of a microfluidic device. In some embodiments, the microchannel is provided as a reaction chamber that includes at least one set of microcarriers. Typically, at least one side of the microchannel is made transparent so that the fluorescence emitted by the microcarriers can be detected. In some embodiments, the microfluidic channel has a transparent viewing wall.

  [0128] The term "microfluidic channel" or "microchannel" refers to about 1 to about 500 [mu] m, preferably about 10 to about 300 [mu] m, such as 1, 10, 20, 30, 40, 50, 60, 70, 80, Refers to a closed channel (eg, an elongate path for fluid) with a cross-section such as 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 μm. The microfluidic channel is not necessarily straight and has a longitudinal direction that coincides with the fluid direction induced in the microfluidic channel. It is preferred if the direction corresponds to the average velocity vector of the fluid and is thus considered to be in a laminar basin.

  [0129] While the microcarriers form a monolayer over the entire length of the microchannel, to limit the volume around the microcarriers to ensure that the sample passes through the maximum number of microcarriers, and while the sample and reagent fluids flow The microchannel is designed to limit the movement of the microcarriers along the length of the microchannel.

  [0130] Microchannels are silicon, SU-8 (epoxy-based photoresist), polyimide (PI), polydimethylsiloxane (PDMS), silicone, or other thermoplastic elastomer (TPE), polymethyl methacrylate (PMMA), Teflon (PTFE), thermoplastic elastomer (TPE), Victrex PEEK (trademark), polycarbonate, polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), polystyrene (PS), fluorinated ethylene-propylene (FEP) , Cyclic olefin (co) polymers (COP or COC), or other thermoplastic polymers, quartz, glass, or metals that can be plateable, such as nickel, silver, or gold, most preferably Transparent Polymers and the like (incluyding) is not limited thereto.

  [0131] The microcarrier of the assay device is such that at least two optional microcarriers do not contact each other and do not contact the outer periphery of the microchannel over the entire length of the microchannel, relative to the cross-sectional shape of the microchannel. It has a shape that can be placed side by side.

D. Sample components
[0132] Non-limiting examples of analyte components such as signaling molecules that can be investigated for expression (eg, total amount) levels and / or activity (eg, phosphorylation) levels in cell extracts include receptor tyrosine kinases, Non-receptor tyrosine kinase, tyrosine kinase signaling cascade component, nuclear hormone receptor, nuclear receptor coactivator, nuclear receptor repressor, ligand, receptor, signaling cascade component, and combinations thereof Is mentioned. Additional examples of analyte components include, but are not limited to, autoantibodies, neurodegenerative molecules, and pathological molecules.

  [0133] In one embodiment, the method of the invention comprises the following analyte components in a cell extract: HER1, HER2, HER3. HER4, VEGFR-1, VEGFR-2, VEGFR-3, FLT-3, FLK-2, PDGR, c-KIT, INSR, IGF-IR, IGF-IIR, IRR, CSF-IR, cMET, and combinations thereof Measuring the presence or absence, expression level (eg, total amount) and / or activity level (eg, phosphorylation level or complex formation) of at least one of the above.

  [0134] In another embodiment, the method of the invention comprises the following analyte components in a cell extract: AMP activated protein kinase (AMPK), AMPK kinase (AMPKK), calmodulin-dependent protein kinase kinase (CAMKK). , LKB1, transforming growth factor-β-activated kinase 1 (TAK1), AKT, adiponectin, leptin, glucose, other AMP regulatory factors, and the presence or absence of at least one of these combinations, expression level (for example, , Total amount) and / or activity level (eg, phosphorylation level or complex formation).

  [0135] In yet another embodiment, the method of the invention comprises an autoantibody, eg, GAD65 autoantibody, insulinoma antigen 2 (IA-2) autoantibody, insulin autoantibody (IAA), zinc transporter protein 8 (ZnT8 ) Measuring the presence and / or expression level (eg, total amount) of at least one or more of autoantibodies and combinations thereof.

  [0136] Glutamate decarboxylase 65 (GAD65) is a neuronal enzyme and is involved in the synthesis of γ-aminobutyric acid (GABA). To date, it has been shown that GAD65 autoantibodies can be used as biomarkers for autoimmune diseases associated with type 1 diabetes (Atkinson et al., Lancet, 335: 1357-60 (1990)). Furthermore, GAD65, IA-2, IAA, ZnT8 autoantibodies have been proposed for the prediction of type 1 diabetes (Mueller et al., Clinical Laboratory News, 36 (10), October 2010; Bingley, PJ., J Clin. Endocrinol. Metab., 95 (1): 25-33 (2010)).

  [0137] In another embodiment, the method of the invention comprises the following analyte components in a cell extract: tau, amyloid beta (Aβ) peptide (eg, β-amyloid protein 42), soluble amyloid precursor protein β. Measuring the presence, expression level (eg, total amount) and / or activity level (eg, phosphorylation level or complex formation) of at least one or more of (sAPPβ), VILIP-1, and combinations thereof . The concentrations of several analyte components such as β-amyloid protein 42 (Aβ42), sAPPβ, total tau protein, and phosphorylated tau have been associated with Alzheimer's disease.

  [0138] In certain embodiments, the invention relates to the presence or absence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more analyte components, expression levels (eg, Measuring the total amount) and / or activity level (eg phosphorylation level or complex formation).

  [0139] In certain embodiments, the present invention employs one or more (eg, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or more) It further comprises measuring the expression level (eg, total amount) and / or activity level (eg, phosphorylation level or complex formation) of the analyte component. In some embodiments, one or more (eg, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50) or more), the receptor tyrosine kinase, non-receptor type Tyrosine kinase, tyrosine kinase signaling cascade component, nuclear hormone receptor, nuclear receptor coactivator, nuclear receptor repressor, ligand, receptor, signaling cascade component; one or more neurodegenerative proteins One or more pathological protein aggregates; and one or more signaling molecules selected from the group consisting of combinations thereof.

IV. Example
[0140] The following examples are provided for purposes of illustration and are not intended to limit the scope of the claims of the present invention.

Example 1. Microfluidic CEER for measuring phosphorylated HER2 levels in patient samples
[0141] This example illustrates a method for detecting activated HER2 levels in a cell lysed sample utilizing a microcarrier to which a proximity dual detection assay (eg, CEER assay) is coupled and a microfluidic device. .

  [0142] This study aimed to determine the optimal conditions for measuring activated analyte components in cell-derived samples. Test parameters included the amount of capture antibody (eg, concentration), the amount of sample, and the amount of the second member of the signal amplification pair (eg, dilution).

  [0143] HER2 capture antibodies at concentrations of 0.5 mg / ml and 1.0 mg / ml were bound to the barcoded microcarriers. IgG prepared as a negative control for the assay was also bound to another set of microcarriers. The bound, encoded microcarriers were then blocked with 5% BSA-PBST for 1 hour and then loaded into an assay cartridge (assay device).

  [0144] Cell solubilized samples from HER2 overexpressing cells (ie, BT474 cells) were then loaded into cartridges and incubated for 80 minutes with the previously bound microcarriers in the reaction chamber. Test samples include solubilization corresponding to 75 cells (eg, 100 in 80 μl volume), 7.5 (eg, 10 in 80 μl volume) and 0.75 (eg, 1 in 80 μl volume). The product was included. After the incubation time, the sample was discharged from the discharge port by the microfluidic device.

  [0145] Bound microcarriers and detection antibodies (eg, 4G10-HRP and HER2-AB4-GO) were incubated for 20 minutes and then drained by the microfluidic device. Antibody 4G10-HRP specifically binds to phosphotryosine and antibody HER2-AB4-GO recognizes the HER2 polypeptide.

  [0111] Next, biotin-tyramide (BT-tyr), the second member of the signal amplification pair, was added to the reaction chamber and incubated for 30 minutes and removed from the reaction chamber. Two dilutions of biotin-tyramide (1: 500 and 1: 1000) were tested. It has been found that the higher the concentration (eg, level) of biotin-tyramide, the more fluorescent molecule aggregates are produced that interfere with fluorescence readings. The signal detection reagent streptavidin-Alexa647 was added at a concentration of 0.5 μg / ml and incubated for 5 minutes.

  [0146] Microcarrier fluorescence was detected and quantified using standard microscopic imaging methods and fluorescence analysis software.

  [0112] The results show that activated HER2 levels were detected and quantified even in samples containing only 0.75 cells (FIGS. 1A and 1B). FIG. 1A shows the fluorescence intensity of a high BT-Tyr dilution series. From left to right, anti-HER2 (0.5 mg / ml; 114), anti-HER2 (1.0 mg / ml; 133), and IgG (negative control) (1.0 mg / ml; 41.2), respectively. FIG. 1B shows the fluorescence intensity of a low BT-Tyr dilution series. From left to right, anti-HER2 (0.5 mg / ml; 104.2), anti-HER2 (1.0 mg / ml; 131.5), and IgG (negative control) (1.0 mg / ml; 38.5, respectively) ). Furthermore, HER2 levels were detectable at any capture antibody and biotin-tyramide concentrations. This data shows that the sensitivity of the microcarrier-based assay was comparable to a conventional CEER assay that utilizes a nitrocellulose-coated and capture antibody-conjugated slide.

  [0112] FIG. 2 shows that the fewer the cells loaded into the reaction chamber, the lower the signal background ratio. In particular, FIG. 2A shows fluorescent microcarriers in an assay device incubated with 75 BT474 cells of a sample. FIG. 2B shows a similar fluorescent microcarrier incubated with 7.5 cells of the sample.

  [0147] This example illustrates a method for detecting activated HER2 levels in a cell lysis sample utilizing a microcarrier and a microfluidic device coupled to a proximity dual detection assay (eg, CEER assay). .

Example 2 Microfluidic CEER for measuring phosphorylated tau levels in patient samples
[0148] This example describes a method for measuring phosphorylated tau levels in a patient-derived sample utilizing the microfluidic CEER assay described herein. The level of activated tau can be used to assess whether a patient has Alzheimer's disease (AD) or has an increased risk of developing AD.

  [0149] Lumbar puncture is performed according to standard outpatient practice protocols, and cerebrospinal fluid samples are collected from the patient. Standard solubilization buffer is used to solubilize the sample cells to produce a cell extract.

  [0150] A set of encoded microcarriers is coupled to a capture antibody that recognizes non-phosphorylated tau using standard binding procedures, such as the Biocartis capture molecule binding protocol. The bound microcarriers were blocked with 5% BSA-PBST solution for 1 hour. The bound microcarrier was loaded into the reaction chamber of the assay cartridge using a microfluidic instrument.

  [0151] The cell extract was loaded into the reaction chamber and contacted with the bound microcarriers for about 80 minutes. The cell extract was removed by laminar flow of fluid and the detection antibody was loaded into the reaction chamber. The detection antibody contains a first antibody that is against non-phosphorylated tau and bound to glucose oxidase, and a second antibody that is against phosphorylated tau and bound to horseradish peroxidase. The detection antibody is incubated for 20 minutes with the captured tau protein bound to the microcarrier and then removed. The reaction chamber containing the captured tau protein to be detected, bound to the microcarrier, is then filled with biotin-tyramide. The signal amplification reaction is performed for 30 minutes. The signal detection reagent streptavidin-Alexa Fluor® 647 is then incubated with the captured analyte component to be detected for 5 minutes. Finally, a fluorescent signal from the microcarrier is detected using a CCD camera and quantified using standard imaging software. Furthermore, the code on the microcarrier is read to decipher the function of the microcarrier.

  [0152] The measured fluorescence corresponds to the level of target analyte component (eg, activated tau) present in the sample. If the level of phosphorylated tau in a patient sample is higher than that of a non-AD subject, the patient is assessed as having AD or an increased risk of developing AD.

[0153] While the invention has been described in some detail for purposes of illustration and illustration for purposes of clarity of understanding, those skilled in the art will perceive certain changes and modifications within the scope of the appended claims. It will be appreciated that it can be implemented. Further, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was incorporated by reference.

Claims (58)

A method for performing a multiplexed immunoassay on a sample, comprising:
(A) contacting a cell lysate with a plurality of capture antibodies that are specific for at least one analyte component and immobilized on an encoded microcarrier to produce a plurality of captured analyte components Generating,
(B) contacting the plurality of captured analyte components with at least two detection antibodies specific for the corresponding analyte components to produce a plurality of captured analyte components that are detectable ( Wherein at least one detection antibody has a first member of a signal amplification pair);
(C) contacting the detectable plurality of captured analyte components with a second member of the signal amplification pair to produce an amplified signal;
(D) detecting the amplified signal generated from the first and second members of the signal amplification pair. The encoded microcarriers, latex, polystyrene, cross-linked dextran, polymethyl styrene, polycarbonate, polypropylene, cellulose, polyacrylamide, dimethylacrylamide, fluorinated ethylene - propylene, glass, SiO _2, silicon, PMMA ( The method of claim 1, made of a material selected from the group consisting of (polymethyl methacrylate), gold, silver, aluminum, steel, and SU-8.   The method according to claim 1, wherein the encoded microcarrier is made of silicon.   The method according to claim 1, wherein the encoded microcarrier has a function determined by reading the code.   The encoded microcarrier is formed into a shape selected from the group consisting of a sphere, a wafer, a square, a disk, a rectangle, a circle, a triangle, and a hexagon. The method according to item.   The method according to claim 1, wherein the encoded microcarrier is formed into a wafer shape.   The method according to claim 1, wherein the encoded microcarrier comprises a plurality of microcarriers.   The method of claim 7, wherein the plurality of microcarriers has at least two groups.   9. The method of claim 8, wherein each of the at least two groups of encoded microcarriers has a different function.   The method of claim 9, wherein the specific function is identifiable by the code.   The method according to any one of claims 1 to 10, wherein the cord is configured to form a hole that traverses the microcarrier.   The method according to any one of claims 9 to 11, wherein the specific function is identification of a plurality of capture antibodies.   The sample is selected from the group consisting of whole blood, serum, plasma, urine, sputum, bronchial wash, tears, nipple aspirate, lymph, saliva, fine needle aspirate (FNA), cerebrospinal fluid, and combinations thereof. The method according to any one of claims 1 to 12.   14. The method according to any one of claims 1 to 13, wherein the at least one analyte component comprises at least one signaling molecule, pathological molecule, or autoantibody. The detection antibody is
(I) a plurality of antibodies labeled with a facilitating moiety and independent of activation state;
The method according to any one of claims 1 to 14, comprising (ii) a plurality of antibodies that are labeled with a first member of a signal amplification pair and depend on an activation state.   16. The method of claim 15, wherein the facilitating moiety produces an oxidizing agent that channels and reacts to the first member of the signal amplification pair.   17. A method according to any one of claims 15 or 16, wherein the facilitating moiety is glucose oxidase. The method of claim 16, wherein the oxidant is hydrogen peroxide (H ₂ O ₂ ).   The method according to any one of claims 1 to 18, wherein the first member of the signal amplification pair is a peroxidase.   20. The method of claim 19, wherein the peroxidase is horseradish peroxidase (HRP).   21. The method according to any one of claims 1 to 20, wherein the second member of the signal amplification pair is a tyramide formulation.   24. The method of claim 21, wherein the tyramide agent is biotin-tyramide.   23. A method according to any one of claims 1 to 22, wherein the amplified signal is produced by biotin-tyramide being oxidized by peroxidase to produce activated tyramide.   24. The method of claim 23, wherein the activated tyramide is detected directly.   24. The method of claim 23, wherein the activated tyramide is detected by the addition of a signal detection agent.   26. The method of claim 25, wherein the signal detection agent is a streptavidin labeled fluorophore.   The method according to claim 25, wherein the signal detection agent is a combination of a streptavidin-labeled peroxidase and a color former.   28. The method of claim 27, wherein the color former is 3,3 ', 5,5'-tetramethylbenzidine (TMB).   29. A method according to any one of claims 1 to 28, wherein the method is used as a diagnostic test for human disease.   30. The method of claim 29, wherein the disease is a metabolic disease, brain and cognitive disease, gastrointestinal disease, or cancer.   The cancer is selected from the group consisting of breast cancer, colorectal cancer, gastrointestinal stromal tumor, gastrointestinal carcinoid, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, prostate cancer, and gastric cancer. The method of claim 30.   32. The method of claim 30, wherein the metabolic disease is diabetes.   32. The method of claim 30, wherein the brain and cognitive disorder is Alzheimer's disease.   The at least one signaling molecule is AMP-activated protein kinase (AMPK), AMPK kinase (AMPKK), calmodulin-dependent protein kinase kinase (CAMKK), LKB1, transforming growth factor-β-activated kinase 1 (TAK1). ), AKT, adiponectin, leptin, glucose, and a combination thereof, according to any one of claims 1 to 30 and 32.   The at least one autoantibody is from the group consisting of GAD65 autoantibodies, insulinoma antigen 2 (IA-2) autoantibodies, insulin autoantibodies (IAA), zinc transporter protein 8 (ZnT8) autoantibodies, and combinations thereof. 33. A method according to any one of claims 1 to 30 and 32, which is selected.   The at least one pathological molecule is selected from the group consisting of tau, amyloid beta (Aβ) peptide, soluble amyloid precursor protein β (sAPPβ), VILIP-1, and combinations thereof. 34. A method according to any one of 30 and 33.   32. The method of any one of claims 1-31, wherein the at least one signaling molecule is a receptor tyrosine kinase.   The receptor tyrosine kinase is HER1, HER2, HER3, HER4, VEGFR-1, VEGFR-2, VEGFR-3, FLT-3, FLK-2, PDGFR, c-KIT, INSR, IGF-IR, IGF-IIR 38. The method of claim 37, selected from the group consisting of, IRR, CSF-1R, cMET, and combinations thereof.   39. The method of claim 37 or 38, wherein the at least one receptor tyrosine kinase further comprises a non-receptor tyrosine kinase.   40. The method of any one of claims 1-39, wherein the sample is distributed in a microchannel that contains encoded microcarriers.   41. The method of claim 40, wherein at least one side of the microchannel is transparent.   42. The method of any one of claims 1-41, wherein the amplified signal is associated with the identity of the encoded microcarrier.   43. The method of any one of claims 1-42, wherein the amplified signal is related to a kinetic binding parameter.   44. The detection of the amplified signal is performed using an optical sensor array coupled to optical means, wherein the sensor is a CCD or C-MOS photosensor. The method according to item.   45. The method of any one of claims 1-44, wherein the method is performed as an in vitro method. A point-of-care diagnostic system,
(A) an assay device comprising a reaction chamber, wherein the reaction chamber comprises a microchannel;
(B) i) a first group of individually encoded microcarriers having a function; and ii) a kit comprising at least two detection antibodies;
The first group of individually encoded microcarriers has a first plurality of capture antibodies bound to the microcarriers;
The at least two detection antibodies are:
a) a plurality of antibodies labeled with a facilitating moiety and independent of activation state;
b) labeled with a first member of a signal amplification pair and comprising a plurality of antibodies depending on the activation state,
Point-of-care diagnostic system.   47. The point-of-care diagnostic system according to claim 46, wherein the device includes means for allowing fluid to flow through the microchannel while restricting movement of the microcarriers in the longitudinal direction of the microchannel.   48. The kit further comprises a second group of individually encoded microcarriers, wherein the second group of individually encoded microcarriers has a second plurality of capture antibodies. The point-of-care diagnostic system according to any one of the above.   48. The method of any one of claims 46 to 47, wherein the first group of individually encoded microcarriers has the function and the second group of individually encoded microcarriers has a different function. The point-of-care diagnostic system described.   50. The point-of-care diagnostic system of claim 49, wherein the function is identifiable by the code.   50. The point-of-care diagnostic system of claim 49, wherein the function is identification of the plurality of capture antibodies.   52. The point-of-care diagnostic system according to any one of claims 46 to 51, wherein the code is configured to form a hole that traverses the microcarrier. The encoded microcarriers, latex, polystyrene, cross-linked dextran, polymethyl styrene, polycarbonate, polypropylene, cellulose, polyacrylamide, dimethylacrylamide, fluorinated ethylene - propylene, glass, SiO _2, silicon, PMMA ( 53. Point-of-care diagnostic system according to any one of claims 46 to 52, made of a material selected from the group consisting of (polymethylmethacrylate), gold, silver, aluminum, steel, and SU-8. .   54. The point-of-care diagnostic system of claim 53, wherein the encoded microcarrier is made of silicon.   55. The point-of-care diagnostic system according to any one of claims 46 to 54, wherein the function of the encoded microcarrier is determined by reading the code.   56. The encoded microcarrier is formed into a shape selected from the group consisting of a sphere, a wafer, a square, a disk, a rectangle, a circle, a triangle, and a hexagon. Point-of-care diagnostic system as described in the section.   57. The point-of-care diagnostic system according to any one of claims 46 to 56, wherein the coded microcarrier is molded into a wafer.   Multiplexed, microfluidic coordinated enzyme-enhanced reaction (CEER) immunoassays for diagnostic purposes to identify patients with disease, whether the patient is responsive to treatment, or drug dosages suitable for administration to patients In vitro use to perform and identify analyte components.

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