Detection of biomarkers on vesicles for diagnosis and prognosis of diseases and disorders

Abstract

To provide a method for detecting and quantizing biomarkers on a vesicle without dissolution or transmission processing, a composition and a kit, and to use a biomarker identified on the vesicle in a method for diagnosis and prognosis of a variety of diseases and disorders.SOLUTION: The present invention is a method which includes: a) a step providing a biosample including a vesicle; b) a step which brings a solid support body containing scavenger coupled to a solid support body into contact with the biological sample under the condition that the scavenger is selectively coupled to a first biomarker, thereby capturing a vesicle having the first biomarker on the solid support body; c) a step separating the solid support body from the biosample; and d) a step which detects a second biomarker on the vesicle captured on the solid support body by using a detection agent selectively coupled to the second biomarker, the step in which the vesicle is not dissolved or transmission processed.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 510,726, filed May 24, 2017, and U.S. Provisional Patent Application No. 62 / 547,024, filed August 17, 2017, both of which are incorporated herein by reference in their entireties.

[0002] Technical Field The present invention relates to methods, compositions, and kits for detecting and quantifying biomarkers on vesicles without lysis or permeabilization of the vesicles, and the use of biomarkers identified on vesicles in methods of diagnosis and prognosis of various diseases and disorders, including neurological disorders, immunological disorders, placental disorders, and cancer. [Background technology]

[0003] 2. Background of the Invention Since exosomes harbor physiological and pathological materials (proteins, metabolites, RNA, small molecules, etc.) of the mother cells from which they originate and the microenvironment nearby the mother cells, exosomes in biological fluids are potentially useful diagnostically for various diseases. However, even though exosomes are recognized as a useful diagnostic resource, current analytical methods for analyzing exosomes are too complex and expensive to be routinely used in diagnostic tests.

[0004] More than 5.4 million Americans and 35 million worldwide have Alzheimer's disease, the most common form of dementia. Currently, the only definitive way to diagnose Alzheimer's disease is by direct examination of brain tissue after the patient's death. Physicians use brain imaging, behavioral assessments, psychiatric tests, and other procedures to diagnose the disease in patients suspected of having Alzheimer's disease, but none are highly accurate and many are costly or impractical.

[0005] In 2017, an estimated 1,688,780 new cancer cases will be diagnosed in the United States, and 600,920 cancer deaths are expected. The most common cancers are breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, bladder cancer, melanoma of the skin, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, leukemia, endometrial cancer, and pancreatic cancer. Similarly, millions of Americans suffer from immune disorders and diseases caused by immune system dysfunction.

[0006] Thus, there is a need in the art for biomarkers and methods for diagnosing diseases and other disorders, such as, for example, neurological disorders, cancer, immunological disorders, and placental disorders. Additionally, there is a need in the art for compositions for detecting biomarkers, as well as compositions and methods useful for treating diseases and other disorders. The present invention fulfills this need by providing accurate, non-invasive methods for diagnosing diseases and other disorders. The present invention further provides novel methods, assays, biomarkers, kits, and compositions for diagnosing, prognosing, predicting, and treating various diseases and disorders. Summary of the Invention

[0007] Summary of the Invention The present invention relates to methods, compositions, and kits for detecting and quantifying biomarkers on vesicles. In particular, the present invention provides methods for selectively capturing vesicles bearing a surface marker of interest on a solid support, and detecting membrane-bound and adsorbed biomarkers on the captured vesicles without the need for lysis or permeabilization of the vesicles. Measurement of biomarkers on vesicles is useful in diagnostic and prognostic methods for various diseases. In particular, the present invention provides methods for diagnosing or prognosticating a disease or disorder in a subject, identifying a subject at risk for a disease or disorder, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with a disease or disorder. In certain embodiments, the present invention provides methods for diagnosing or prognosticating a neurological disorder in a subject, identifying a subject at risk for a neurological disorder, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with a neurological disorder. In other embodiments, the present invention provides methods for diagnosing or prognosticating an immunological disorder in a subject, identifying a subject at risk for an immunological disorder, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with an immunological disorder. In other embodiments, the invention provides methods of diagnosing or prognosing cancer in a subject, identifying a subject at risk for cancer, or prescribing a therapeutic regimen or predicting the benefit of treatment in a subject with cancer.In other embodiments, the invention provides methods of diagnosing or prognosing placental disease in a subject, identifying a subject at risk for placental disease, or prescribing a therapeutic regimen or predicting the benefit of treatment in a subject with placental disease.

[0008] In one aspect, the invention provides a method comprising: a) providing a biological sample comprising vesicles; b) contacting a solid support comprising a capture agent bound to the solid support with the biological sample under conditions in which the capture agent selectively binds to the first biomarker, if present on the vesicles, thereby capturing vesicles having the first biomarker on the solid support; c) separating the solid support from the biological sample; and d) detecting a second biomarker on the vesicles captured on the solid support using a detection agent that selectively binds to the second biomarker, where the vesicles have not been lysed or permeabilized.

[0009] In some embodiments, the method of the present invention further comprises detecting a secreted protein on the vesicles captured on the solid support. In some aspects, the secreted protein is selected from the group consisting of a cytokine, a growth factor, a chemokine, and an interleukin. In other aspects, the cytokine is selected from the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B, nociceptin, GnRH, FasL, and TNFSF13.

[0010] In certain embodiments, the vesicle is selected from the group consisting of exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, ectosomes, and apoptotic bodies.

[0011] In certain embodiments, the first and second biomarkers are membrane-bound or adsorbed proteins of the vesicles. For example, the first or second biomarkers are exosome surface markers (e.g., CD81, CD63, CD171), neuron-specific proteins (e.g., synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), tau (Tau), phosphorylated tau, αβ-42, and synaptophysin), astrocyte-specific proteins (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1)), microglia-specific proteins (CD11b), dopamine transporter proteins (e.g., DAT), oligodendrocyte-specific proteins (e.g., myelin basic protein (MBP), oligodendrocyte myelin glycoprotein (OMG), or chemokines (CX3CL1) or cytokines (IL1b, IL34, FasL). , or IL12B). In another embodiment, the first or second biomarker is a metabolite. In another embodiment, the method further comprises detecting a cytosolic protein (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA), tyrosine hydroxygenase (TH), cathepsin D (CTSD), AchE, LAMP1, REST, SYT, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL) on the vesicles captured on the solid support. In another embodiment, the method further comprises detecting one or more additional biomarkers on the vesicles captured on the solid support.In some embodiments, the biomarkers are CD81, acetylcholinesterase (AchE), lysosomal associated membrane protein 1 (LAMP1), CTSD, RE1 silencing transcription factor (REST), synaptotagmin (SYT), synaptophysin (SYP), synaptopodin (SYNPO), postsynaptic density protein 95 (PSD95), synaptic vesicle glycoprotein 2A (SV2A), NGRN, monocyte chemoattractant protein-1 (CCL2), IL34, glycogen synthase (GYS), (OR), death receptor 6 (DR6), heat shock protein (HSP), IL12 The biomarkers are selected from the group consisting of beta, alpha-beta (Aβ), beta secretase (BACE), dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptors, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), neuropeptide receptors (orexin receptors, opioid receptors KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments, the first biomarker is SNAP25 and the second biomarker is selected from the group consisting of CD81, tau, NEFL, TNFa, and IL8. In some embodiments, the first biomarker is EAAT1 and the second biomarker is CD81 or GFAP. In some embodiments, the first biomarker is OMG and the second biomarker is CD81 or MBP. In some embodiments, the first biomarker is CD11b and the second biomarker is SYP. In some embodiments, the first biomarker is DAT and the second biomarker is CD81, SNCA, or TH. In some embodiments, the first biomarker is CD11b and the second biomarker is SYP.

[0012] In another embodiment, the vesicles captured on the solid support are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes.

[0013] In certain embodiments, the solid support is a plate, non-magnetic bead, magnetic bead, filter, slide, wafer, rod, particle, strand, disk, membrane, or tube surface, channel, column, flow cell apparatus, or microfluidic device. The solid support can include, for example, glass, quartz, silicon, metal, ceramic, plastic, nylon, polyacrylamide, hydrogel, or resin.

[0014] In certain embodiments, the solid support comprises one or more capture agents bound to the solid support, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more different capture agents that selectively bind different biomarkers on the vesicles.

[0015] In certain embodiments, detection of biomarkers on vesicles captured on a solid support includes using one or more detection agents, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more different detection agents that selectively bind to different biomarkers on the vesicles.

[0016] In certain embodiments, the capture agent comprises an antibody, antibody fragment, antibody mimetic, or aptamer that specifically binds to a first biomarker on the vesicles. In other embodiments, the detection agent comprises an antibody, antibody fragment, antibody mimetic, or aptamer that specifically binds to a second biomarker on the vesicles. The capture agent and detection agent may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, a Fab fragment, a Fab' fragment, a F(ab') fragment, or a Fab' fragment. 2 Fragment, F v Fragment, or scF v It may include fragments.

[0017] In certain embodiments, the detection agent further comprises a detectable label, eg, a fluorescent, chemiluminescent, electrochemiluminescent, bioluminescent, isotopic, or radioactive label.

[0018] In certain embodiments, detecting the biomarkers on the vesicles includes performing an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), an immunopolymerase chain reaction assay, an electrochemiluminescence immunoassay (ECLIA), and a radioimmunoassay (RIA).

[0019] The biological sample containing the vesicles may be from a subject or a cell culture supernatant. In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, ascites, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0020] In certain embodiments, the biological sample is obtained from a subject diagnosed with or suspected of having a neurological disorder, such as Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, neurofibrillary tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Parkinsonism-dementia complex of Guam, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), stroke, depression, bipolar disorder, epilepsy, autism, schizophrenia, brain tumor, white matter disease, cerebral atrophy, mental retardation, cerebellar ataxia, multiple system atrophy, concussion, subconcussive impact, or Parkinson's disease.

[0021] In some embodiments, the biological sample is obtained from a subject diagnosed with or suspected of having cancer, such as breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penic cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous or intraocular melanoma, anal region cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, brain stem glioma, or spinal cord tumor.

[0022] In some embodiments, the biological sample is obtained from a subject diagnosed or suspected of having an immunological disorder, hi other embodiments, the biological sample is obtained from a subject diagnosed or suspected of having a placental disorder.

[0023] In one aspect, the present invention provides a method for diagnosing and treating a disease or disorder in a subject, comprising: a) obtaining a biological sample comprising exosomes from a subject; b) contacting a solid support comprising a capture agent bound to the solid support with the biological sample under conditions in which the capture agent selectively binds to a first biomarker of the exosomes, where the exosomes are captured on the solid support; c) separating the solid support from the biological sample; d) measuring the level of a second biomarker of the exosomes captured on the solid support, where the exosomes are not lysed or permeabilized; e) diagnosing the subject having the disease or disorder by comparing the level of the second biomarker to a control level of the second biomarker; and f) treating the subject for the disease or disorder if the subject is diagnosed with the disease or disorder. In some embodiments, the first biomarker is a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, or an oligodendrocyte-specific protein. In other embodiments, the second biomarker is a cytosolic protein, a secreted protein, a receptor protein, a transporter protein, or a membrane protein. In some embodiments, the cytosolic protein is selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, SNCA, alpha beta-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL. In other embodiments, the secreted protein is selected from the group consisting of cytokines, growth factors, chemokines, and interleukins. In some embodiments, the cytokine is selected from the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B, nociceptin, GnRH, FasL, and TNFSF13.In other embodiments, the neurotransmitter receptor is selected from the group consisting of dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptors, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), and neuropeptide receptors (orexin receptors, opioid receptors KOR). In other embodiments, the membrane protein is selected from the group consisting of EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments, the transporter protein is a neurotransmitter transporter, a dopamine transporter (e.g., DAT), a serotonin transporter, a GABA transporter, a glutamate transporter, an insulin transporter, a tumor necrosis factor transporter, or a neuropeptide transporter. In some embodiments, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or cancer.

[0024] In another aspect, the present invention provides a method for producing a biomarker comprising: a) obtaining a biological sample comprising exosomes from a subject; b) contacting the biological sample with a solid support comprising a capture agent bound to the solid support under conditions in which the capture agent selectively binds to a first biomarker of exosomes selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, and an oligodendrocyte-specific protein, wherein neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes are captured on the solid support; and c) extracting exosomes from the biological sample. d) separating the solid support from the exosomes captured on the solid support; d) measuring a level of a second biomarker selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein of the exosomes captured on the solid support, where the exosomes have not been lysed or permeabilized; e) diagnosing the subject having a neuropathy by comparing the level of the second biomarker to a control level of the second biomarker; and f) treating the subject for the neuropathy if the subject is diagnosed with a neuropathy.

[0025] In other embodiments, the present invention provides a method for prognosing or monitoring the progression of a disease or disorder in a subject, comprising: a) obtaining a biological sample comprising exosomes from a subject; b) contacting a solid support comprising a capture agent bound to the solid support with the biological sample under conditions in which the capture agent selectively binds to a first biomarker of the exosomes, where the exosomes are captured on the solid support; c) separating the solid support from the biological sample; d) measuring the level of a second biomarker protein of the exosomes captured on the solid support, where the exosomes are not lysed or permeabilized, thereby prognosing or monitoring the progression of the disease or disorder in the subject. In some embodiments, the method further comprises obtaining a second biological sample comprising exosomes from the subject and measuring the level of a biomarker of the present invention and comparing the level of the biomarker in the second biological sample with the level of the biomarker in the first biological sample. In some embodiments, the first biomarker is a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, or an oligodendrocyte-specific protein. In other embodiments, the second biomarker is a cytosolic protein, a secreted protein, a receptor protein, a transporter protein, or a membrane protein. In some embodiments, the cytosolic protein is selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, SNCA, αβ-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD-95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL. In other embodiments, the secreted protein is selected from the group consisting of cytokines, growth factors, chemokines, and interleukins.In some embodiments, the cytokine is selected from the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B, nociceptin, GnRH, FasL, and TNFSF13. In other embodiments, the neurotransmitter receptor is selected from the group consisting of dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptors, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), and neuropeptide receptors (orexin receptors, opioid receptor KOR). In other embodiments, the membrane protein is selected from the group consisting of EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments, the transporter protein is a neurotransmitter transporter, a dopamine transporter, a serotonin transporter, a GABA transporter, a glutamate transporter, an insulin transporter, a tumor necrosis factor transporter, or a neuropeptide transporter. In some embodiments, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or a cancer.

[0026] In other embodiments, the present invention provides a method of prognosing or monitoring the progression of a neurological disorder in a subject, comprising: a) obtaining a biological sample comprising exosomes from a subject; b) contacting the biological sample with a solid support comprising a capture agent bound to the solid support under conditions where the capture agent selectively binds to a first biomarker of the exosomes selected from the group consisting of neuron-specific proteins, astrocyte-specific proteins, microglia-specific proteins, and oligodendrocyte-specific proteins, where neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes are captured on the solid support; c) separating the solid support from the biological sample; d) measuring a level of a second biomarker selected from the group consisting of neuron-specific proteins, astrocyte-specific proteins, microglia-specific proteins of the exosomes captured on the solid support, where the exosomes are not lysed or permeabilized, thereby prognosing or monitoring the progression of a neurological disease in the subject. In some embodiments, the method further comprises obtaining a second biological sample comprising exosomes from the subject and measuring the level of a biomarker of the invention and comparing the level of the biomarker in the second biological sample with the level of the biomarker in the first biological sample.

[0027] In yet another aspect, the present invention provides a method of diagnosing and treating a disease or disorder in a subject, comprising: a) obtaining a biological sample comprising exosomes from a subject; b) contacting a solid support comprising a capture agent bound to the solid support with the biological sample under conditions in which the capture agent selectively binds to a first biomarker of the exosomes, where the exosomes are captured on the solid support; c) separating the solid support from the biological sample; d) detecting a second biomarker of the exosomes captured on the solid support, where the exosomes are not lysed or permeabilized; e) diagnosing the subject having the disease or disorder by comparing the level of the second biomarker to a control level of the second biomarker; and f) treating the subject for the disease or disorder if the subject is diagnosed with the disease or disorder. In some embodiments, the first biomarker is a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, or an oligodendrocyte-specific protein. In other embodiments, the second biomarker is a cytosolic protein, a secreted protein, a receptor protein, a transporter protein, or a membrane protein. In some embodiments, the cytosolic protein is selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, SNCA, αβ-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL. In other embodiments, the secreted protein is selected from the group consisting of cytokines, growth factors, chemokines, and interleukins. In some embodiments, the cytokine is selected from the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B, nociceptin, GnRH, FasL, and TNFSF13.In other embodiments, the neurotransmitter receptor is selected from the group consisting of dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptors, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), and neuropeptide receptors (orexin receptors, opioid receptors KOR). In other embodiments, the membrane protein is selected from the group consisting of EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments, the transporter protein is a neurotransmitter transporter, a dopamine transporter, a serotonin transporter, a GABA transporter, a glutamate transporter, an insulin transporter, a tumor necrosis factor transporter, or a neuropeptide transporter. In some embodiments, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or cancer.

[0028] In yet another aspect, the present invention provides a method for producing a biomarker for exosomes comprising the steps of: a) obtaining a biological sample comprising exosomes from a subject; b) contacting the biological sample with a solid support comprising a capture agent bound to the solid support under conditions in which the capture agent selectively binds to a first biomarker of exosomes selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, and an oligodendrocyte-specific protein, wherein neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes are captured on the solid support; and c) contacting the biological sample with a solid support comprising a capture agent bound to the solid support under conditions in which the capture agent selectively binds to a first biomarker of exosomes selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, and an oligodendrocyte-specific protein, wherein neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes are captured on the solid support. The present invention provides a method for diagnosing and treating a neuropathy in a subject, comprising the steps of: separating the solid support from the body sample; d) detecting a second biomarker selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, and a microglia-specific protein in the exosomes captured on the solid support, where the exosomes have not been lysed or permeabilized; e) diagnosing the subject having a neuropathy by comparing the level of the second biomarker to a control level of the second biomarker; and f) treating the subject for the neuropathy if the subject is diagnosed with a neuropathy.

[0029] In certain embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, neurofibrillary tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Parkinsonism-dementia complex of Guam, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), stroke, depression, bipolar disorder, epilepsy, autism, schizophrenia, brain tumors, white matter disease, cerebral atrophy, mental retardation, cerebellar ataxia, multiple system atrophy, concussion, subconcussive impact, and Parkinson's disease.

[0030] In certain embodiments, the first and second biomarkers are vesicular membrane-bound or adsorbed proteins, including, but not limited to, synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), neuronal specific proteins selected from the group consisting of tau, phosphorylated tau, and synaptophysin, astrocyte specific proteins selected from the group consisting of glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1), microglia specific protein (CD11b), cytokines selected from the group consisting of IL1b, IL34, FasL, or IL12B, chemokines (CX3CL1), CD81, CD171, CTSD, CD63, αβ-42, myelin basic protein (MBP), and oligodendrocyte specific proteins selected from the group consisting of oligodendrocyte myelin glycoprotein (OMG). In some embodiments, the biomarkers are CD81, acetylcholinesterase (AchE), lysosomal associated membrane protein 1 (LAMP1), CTSD, RE1 silencing transcription factor (REST), synaptotagmin (SYT), NGRN, monocyte chemoattractant protein-1 (CCL2), IL34, glycogen synthase (GYS), olfactory receptor (OR), death receptor 6 (DR6), heat shock proteins (HSPs), IL12beta, alpha-beta (Aβ), beta secretase (BACE), dopamine In some embodiments, the first biomarker is selected from the group consisting of serotonin receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5, B1, B2), glutamate receptors (1 and 2), insulin receptor, tumor necrosis factor receptor superfamily (TRAL, TNF receptor, death receptors 5 and 6), neuropeptide receptors (orexin receptor, opioid receptor KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments, the first biomarker is SNAP25 and the second biomarker is selected from the group consisting of CD81, tau, NEFL, TNFa, and IL8.In some embodiments, the first biomarker is EAAT1 and the second biomarker is CD81 or GFAP. In some embodiments, the first biomarker is OMG and the second biomarker is CD81 or MBP. In some embodiments, the first biomarker is CD11b and the second biomarker is SYP.

[0031] In some embodiments, the level of one or more biomarkers of the exosomes in the biological sample is compared to the level of one or more biomarkers in a control sample, wherein the level of the one or more biomarkers in the biological sample is elevated compared to the control sample. In some embodiments, the level of one or more biomarkers in the biological sample is compared to the level of one or more biomarkers in a control sample, wherein the level of the one or more biomarkers in the biological sample is decreased compared to the control sample. In some embodiments, the biomarker level determines the disease or disorder status of the subject with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% specificity. In some embodiments, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or a cancer. In some embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, neurofibrillary tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Parkinsonism-dementia complex of Guam, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), stroke, depression, bipolar disorder, epilepsy, autism, schizophrenia, brain tumors, white matter disease, cerebral atrophy, mental retardation, cerebellar ataxia, multiple system atrophy, concussion, subconcussive impact, and Parkinson's disease. In other embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.In some embodiments, the cancer is breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penic cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous or intraocular melanoma, anal region cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, brain stem glioma, or spinal cord tumor.

[0032] In another embodiment, the present invention provides a method for the preparation of a biological sample comprising the steps of: a) obtaining a biological sample comprising exosomes from a subject; b) contacting the biological sample with a solid support comprising a capture agent bound to the solid support under conditions in which the capture agent selectively binds to a membrane marker of the exosomes, wherein the membrane marker is selected from the group consisting of SNAP25, OMG, CD11b, EAAT1 and CD171, and wherein the exosomes have the membrane marker captured on the solid support; c) separating the solid support from the biological sample; and d) isolating the exosomes captured on the solid support from the group consisting of CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau, CD63, αβ-42, CX3CL1, IL1b, and IL34. The present invention also includes methods of diagnosing and treating a disease or disorder in a subject, comprising the steps of: measuring a level of one or more biomarkers, where the exosomes have not been lysed or permeabilized; e) diagnosing the subject having the disease or disorder by comparing the level of the one or more biomarkers to a control level of the biomarkers in a control sample, where an increased level of the one or more biomarkers and / or a decreased level of the one or more biomarkers compared to the control level of the one or more biomarkers in the control sample indicates that the subject has the disease or disorder; and f) treating the subject for the disease or disorder if the subject is diagnosed with the disease or disorder.In some embodiments, the biomarkers are CD81, acetylcholinesterase (AchE), lysosomal associated membrane protein 1 (LAMP1), CTSD, RE1 silencing transcription factor (REST), synaptotagmin (SYT), NGRN, monocyte chemoattractant protein-1 (CCL2), IL34, glycogen synthase (GYS), olfactory receptor (OR), death receptor 6 (DR6), heat shock proteins (HSPs), IL12beta, alpha-beta (Aβ), beta secretase (BACE), dopamine In some embodiments, the disease or disorder is selected from the group consisting of: cytotoxic T cell receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptor, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), neuropeptide receptors (orexin receptors, opioid receptor KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or a cancer.

[0033] In another aspect, the present invention provides a kit for detecting biomarkers on vesicles, comprising: a) a solid support comprising a capture agent bound to the solid support, wherein the capture agent selectively binds to a first biomarker on the surface of the vesicle; and b) at least one detection agent selectively binds to a second biomarker on the surface of the vesicle. In certain embodiments, the vesicle is selected from the group consisting of exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, and ectosomes. In yet another aspect, the present invention provides a kit for detecting biomarkers on vesicles, comprising: a) a solid support comprising a capture agent bound to the solid support, wherein at least one capture agent selectively binds to a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, or an oligodendrocyte-specific protein of the exosome; and b) one or more detection agents, wherein the one or more detection agents selectively bind to a cytosolic protein, a secreted protein, a neurotransmitter receptor, or a membrane protein on the surface of the exosome. In some embodiments, the cytosolic protein is selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, SNCA, αβ-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL. In other embodiments, the secreted protein is selected from the group consisting of cytokines, growth factors, chemokines, and interleukins. In some embodiments, the cytokine is selected from the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B, nociceptin, GnRH, FasL, and TNFSF13.In other embodiments, the neurotransmitter receptor is selected from the group consisting of dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptors, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), and neuropeptide receptors (orexin receptors, opioid receptors KOR). In other embodiments, the membrane protein is selected from the group consisting of EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM.

[0034] In certain embodiments, the capture and detection agents in the kit include an antibody, antibody fragment, antibody mimetic, or aptamer that specifically binds to a first biomarker on a vesicle. The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, a Fab fragment, a Fab' fragment, a F(ab') fragment, or a Fab' fragment. 2 Fragment, F v Fragments, and scF vIn another embodiment, the capture or detection agent comprises an antibody selected from the group consisting of an anti-CD171 antibody, an anti-synaptosomal associated protein 25 (SNAP25) antibody, an anti-neurogranin (NRGN) antibody, an anti-tau antibody, an anti-synaptophysin antibody, and an anti-CD63 antibody, an anti-αβ-42 antibody, an anti-CD81 antibody, an anti-CTD antibody, an anti-GAPDH antibody, an anti-IL1b antibody, an anti-IL34 antibody, an anti-CX3CL1 antibody, an anti-glial fibrillary acidic protein (GFAP) antibody, an anti-excitatory amino acid transporter 1 (EAAT1) antibody, an anti-myelin basic protein (MBP) antibody, an anti-SNCA antibody, an anti-TH antibody, an anti-CD11b antibody, and an anti-oligodendrocyte myelin glycoprotein (OMG) antibody. In some embodiments, at least one capture agent selectively binds to CD171, CD63, CD81, SNAP25, EAAT1, CD11b, or OMG of the exosome. In other embodiments, the one or more detection agents selectively bind to CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), CD63, αβ-42, CX3CL1, IL1b, or IL34 on the surface of the exosome. In some embodiments, at least one of the detection agents selectively binds to CD81, acetylcholinesterase (AchE), lysosomal-associated membrane protein 1 (LAMP1), CTSD, RE1 silencing transcription factor (REST), synaptotagmin (SYT), NGRN, monocyte chemotactic protein-1 (CCL2), IL34, glycogen synthase (GYS), olfactory receptor (OR), death receptor 6 (DR6), heat shock protein (HSP), IL12beta, alpha-beta (Aβ), beta secretase (BACE), Selectively binds to dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), glutamate receptors (1 and 2), insulin receptor, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), neuropeptide receptors (orexin receptors, opioid receptor KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, or NCAM.

[0035] In certain embodiments, the detection agent in the kit further comprises a detectable label, eg, a fluorescent, chemiluminescent, electrochemiluminescent, bioluminescent, isotopic, or radioactive label.

[0036] In another embodiment, the kit further comprises reagents for performing an immunoassay. Exemplary immunoassays include enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays (IFAs), immunopolymerase chain reaction assays, electrochemiluminescence immunoassays (ECLIAs), and radioimmunoassays (RIAs).

[0037] In another embodiment, the invention provides a kit for diagnosing or prognosing a disease or disorder in a subject, identifying a subject at risk for a disease or disorder, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with a disease or disorder, comprising: a) a solid support comprising a capture agent bound to the solid support, wherein at least one capture agent selectively binds to CD171 membrane marker of exosomes; and b) at least two detection agents, wherein at least one detection agent selectively binds to phosphorylated tau T181 and at least one detection agent selectively binds to neurogranin on the surface of exosomes. In certain embodiments, at least one capture agent or detection agent comprises an antibody, antibody fragment, antibody mimetic, or aptamer that specifically binds to CD171, phosphorylated tau T181, or neurogranin. In certain embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, a Fab fragment, a Fab' fragment, a F(ab') 2 Fragment, F v Fragments, and scF v In another embodiment, the kit comprises an anti-neurogranin antibody, an anti-phosphorylated tau T181 antibody, and an anti-CD171 antibody. In one embodiment, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or a cancer.

[0038] In some embodiments, the invention provides methods for prognosing or monitoring the progression of a disease or disorder in a subject, comprising: a) obtaining a biological sample comprising vesicles or exosomes from a subject; b) contacting the biological sample with a solid support comprising a capture agent bound to the solid support under conditions where the capture agent selectively binds to a first biomarker of the vesicles or exosomes, where the vesicles or exosomes are captured on the solid support; c) separating the solid support from the biological sample; d) measuring a level of a second biomarker protein of the vesicles or exosomes captured on the solid support, where the vesicles or exosomes have not been lysed or permeabilized, thereby prognosing or monitoring the progression of a disease or disorder in the subject. In other embodiments, the method further comprises repeating (a), (b), (c), and (d) with a second biological sample at a second time point, where an increase in the level of the second biomarker compared to its level at the first time point indicates that the disease or disorder is progressing, and a decrease in the level of the second biomarker compared to its level at the first time point indicates that the disease or disorder is regressing. In other embodiments, the method further comprises treating the subject for the disease or disorder if the disease or disorder is progressing. In yet other embodiments, the method further comprises reducing or ceasing treatment for the disease or disorder if the disease or disorder is regressing. In some embodiments, the disease or disorder is a neurological disorder, an immunological disorder, a placental disorder, or a cancer.In other embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, neurofibrillary tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Parkinsonism-dementia complex of Guam, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), stroke, depression, bipolar disorder, epilepsy, autism, schizophrenia, brain tumors, white matter disease, cerebral atrophy, mental retardation, cerebellar ataxia, multiple system atrophy, concussion, subconcussive impact, and Parkinson's disease.

[0039] These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in light of the disclosure herein, and all such embodiments are specifically contemplated.

[0040] Each limitation of the present invention may encompass various embodiments of the present invention. Thus, each limitation of the present invention involving any one element or combination of elements is considered to be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction and the arrangement of components detailed below. The present invention is capable of other embodiments and can be practiced or embodied in various ways. Additionally, the phraseology and terminology used herein are for the purpose of description and should not be considered as limiting. The use of "including," "comprising," "having," "containing," "involving," and conjugations thereof herein means the inclusion of the items listed thereafter and equivalents thereof as well as additional items. It should be noted that the singular forms "a," "an," and "the" used in this specification and the appended claims include plural references unless the context clearly dictates otherwise. [Brief description of the drawings]

[0041] [Figure 1-1] Figures 1A and 1B show methods for quantifying cell-specific biomarkers in exosomes. Figure 1A shows a schematic diagram illustrating the generation of exosomes containing proteins of the cell (mother cell) from which they are derived. Figure 1B shows methods for screening exosomes for membrane and adsorption markers. [Figure 1-2] Same as above.

[0042] [Figure 2-1] Figures 2A-2F show exemplary detection of cytosolic (non-membrane) proteins on the surface of exosomes. Neurospecific anti-SNAP25 (Figures 2A-2C) or control mouse IgG (Figures 2D-2F) were immobilized on white ELISA plates. Various volumes (10, 2.5, and 0 uL) of plasma were suspended in PBS (▲), 0.1% tween-20 PBS (○), or 0.1% Triton-X100 PBS (□) in a final volume of 40 uL and applied to the ELISA plate. After overnight incubation at 4°C, unbound material was removed. Then, biotin-labeled antibodies against the exosome surface marker CD81 (Figures 2A and 2D), and two different soluble cytosolic proteins GAPDH (Figures 2B and 2E) and CTSD (Figures 2C and 2F) were applied to the ELISA plate. For PBS samples (▲), the antibodies were suspended in PBS without tween-20. For tween-20 (○) and Triton-X100 (□) samples, the labeled antibodies were suspended in 0.1% tween-20 PBS. A conventional chemiluminescence ELISA procedure was performed to determine the relative light units (RLU). [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above. [Figure 2-6] Same as above.

[0043] [Figure 3-1]Figures 3A-3D show the specificity of the marker proteins of the present invention. Antibodies against (Figure 3A) exosome surface marker CD81, (Figure 3B) neuronal surface marker SNAP25, (Figure 3C) astrocyte surface marker (EAAT1), or (Figure 3D) oligodendrocyte surface marker (OMG) were immobilized on white ELISA plates. 10 μL of plasma suspended in 40 μL of PBS was applied to the ELISA plate. After overnight incubation at 4°C, unbound material was removed. Biotin-labeled antibodies against exosome surface marker CD81, general cytosolic marker GAPDH, cytosolic protein in neurons (NRGN), oligodendrocytes (MBP), and astrocytes (GFAP) were applied to the ELISA plate. A control without antibody (tPBS) was also included. Conventional chemiluminescence ELISA was performed and the relative light units (RLU) were determined. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Diagram 3-4] Same as above.

[0044] [Figure 4-1] Figures 4A-4D show the detection and quantification of exosome biomarkers of the present invention. Anti-SNAP25 (Figures 4A and 4B), anti-EAAT1 (Figure 4C), or anti-OMG (Figure 4D) were immobilized on an ELISA plate. Various volumes of normal plasma were suspended in PBS and applied to ELISA wells in a final volume of 40 mL. Antibodies against NRGN (Figure 4A), tau (Figure 4B), GFAP (Figure 4C), and MBP (Figure 4D) were used as detection antibodies. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above.

[0045] [Figure 5-1]Figures 5A-5C show the screening of exosome-adhering markers. Control mouse IgG (open columns), mouse anti-human CD81 (light grey columns), or mouse anti-human synaptosomal associated protein 25 (SNAP25) (dark grey columns) were immobilized on an ELISA plate, and pooled human plasma was applied to all ELISA wells. ELISAs were performed using various detection antibodies as listed. The detection antibodies (all biotinylated) were against exosomal membrane proteins (CD81, CD171, CD63, and SNAP25) as well as various non-membrane proteins (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cathepsin D (CTSD), neurogranin (NRGN), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), tau, microtubule-associated protein tau (tau) and phosphorylated tau T181, and amyloid b1-42 peptide (Abeta42). As a negative control, PBS without any biotinylated antibodies was used. Figure 5A shows the detection of membrane proteins, CD81 and CD171, and non-membrane proteins, GAPDH and CTSD. Figure 5B shows the detection of membrane protein, CD63, and SNAP25. Figure 5C shows the detection of non-membrane proteins, NRGN, MBP, GFAP, tau, T181, and Abeta42. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0046] [Figure 6-1] Figures 6A-6E show clinical applications for the diagnosis of Alzheimer's disease. Anti-CD171 antibodies were immobilized on ELISA plates as shown in the assay schematic in Figure 6A. Twelve EDTA plasma samples from subjects with Alzheimer's disease and age- and sex-matched controls were applied to the ELISA wells, and antibodies against (Figure 6B) CD81, (Figure 6C) CTSD, (Figure 6D) NRGN, and (Figure 6E) p-tau T181 were used as detection antibodies. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above.

[0047] [Figure 7-1] Figures 7A-7L show the stability of plasma levels of various exosome biomarkers of the invention. Anti-CD81, anti-CD171, anti-SNAP25, anti-EAAT1, and anti-OMG were immobilized on ELISA plates. EDTA plasma was obtained from 7 control subjects every week for 2-3 weeks and applied to ELISA wells. Figure 7A shows an assay schematic for detecting exosome membrane targets. Figures 7B-7F show detection with (Figure 7B) anti-CD81 probe on CD81 plate for total exosomes (TE), (Figure 7C) anti-CD171 probe on CD81 plate for CD171-based NDE (cNDE), (Figure 7D) anti-SNAP25 probe on CD81 plate for SNAP25-based NDE (sNDE), (Figure 7E) anti-CD81 probe on EAAT1 plate (ADE), and (Figure 7F) anti-CD81 probe on OMG plate for ODE. Figure 7G shows an assay schematic for exosome surface protein targets. Figures 7H-7L show detection with (Figure 7H) a GFAP probe on an EAAT1 plate, (Figure 7I) an MBP probe on an OMG plate, (Figure 7J) an NRGN probe on a SNAP25 plate, (Figure 7K) a tau probe on a SNAP25 plate, and (Figure 7L) an NRGN probe on a CD171 plate, respectively. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 7-7] Same as above. [Figure 7-8] Same as above. [Figure 7-9] Same as above. [Figure 7-10] Same as above. [Figure 7-11] Same as above. [Figure 7-12] Same as above.

[0048] [Figure 8-1] Figures 8A and 8B show detection and quantification of exosome biomarkers of the invention in plasma using anti-NRGN-immobilized ELISA plates. Figure 8A shows an exemplary assay schematic. Figure 8B shows the assay results. Anti-NRGN (medium and dark grey columns) or control mouse IgG (black and light grey columns) were immobilized on the ELISA plate. EDTA plasma (light and dark grey columns) or PBS alone (black and medium grey columns) were applied to the ELISA wells. Antibodies against NRGN, CD171, SNAP25, CD81, EAAT1, OMG and PBS control were used as detection antibodies. [Figure 8-2] Same as above.

[0049] [Figure 9-1] Figures 9A-9D show microglial targeting of exosome biomarkers of the invention. Figure 9A shows the assay schematic. Figures 9B-9D show the assay results. Anti-CD11b (medium and dark grey columns) or control mouse IgG (black and light grey columns) were immobilized on the ELISA plate. EDTA plasma from seven different donors (IR1-IR7) (light and dark grey columns) or PBS alone (black and medium grey columns) was applied to the ELISA wells. Antibodies against GFAP (Figure 9B), MBP (Figure 9C), or NRGN (Figure 9D) were used as detection antibodies. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above.

[0050] [Figure 10-1] Figures 10A-10F show data demonstrating the detection of secreted proteins, cytokines (IL1b and IL34) and chemokine (CX3CL1), on the surface of brain-derived exosomes. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above.

[0051] [Figure 11-1] FIG. 11 shows data showing dilutions of standard plasma as ELISA quantification standards. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above.

[0052] [Figure 12-1] FIG. 12 shows data demonstrating duplicate variation for plasma levels of 16 exosomal biomarkers of the present invention. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above.

[0053] [Figure 13-1] FIG. 13 shows data illustrating standard curves for plasma levels of 16 exosomal biomarkers of the present invention. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above.

[0054] [Figure 14-1] FIG. 14 presents data showing quantification of target exosome surface protein biomarkers in plasma. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above.

[0055] [Figure 15-1] FIG. 15 presents data showing plasma exosome biomarker levels normalized to CD81. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above.

[0056] [Figure 16-1] FIG. 16 presents data showing plasma exosome biomarker levels normalized to SYT. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0057] [Figure 17] Figure 17 shows data illustrating the principle of an exemplary assay of the present invention. NDE, ADE, and ODE are specifically captured on ELISA plates immobilized with anti-SNAP25 (neuron marker), anti-EAAT1 (astrocyte marker), and anti-OMG (oligodendrocyte marker). Captured exosomes were further probed with biotinylated anti-CD81 (a general marker for exosomes).

[0058] [Figure 18-1] Figures 18A-18C show data demonstrating the specificity of an exemplary assay of the invention. Anti-SNAP25 (500 ng / mL) (A●), anti-EAAT1 (830 ng / mL) (B●), anti-OMG (830 ng / mL) (C●), and control mouse IgG (500 ng / mL) (△) were immobilized on ELISA plates as described in the Methods section. Serial dilutions of single donor plasma samples were prepared, applied to the ELISA plate, and probed with biotinylated anti-CD81. ELISA results are expressed as Relative Light Units (RLU). [Figure 18-2] Same as above. [Figure 18-3] Same as above.

[0059] [Figure 19-1] Figures 19A-19C show data showing inter-assay precision of an exemplary assay of the present invention. NDE (A), ADE (B), and ODE (C) were listed in duplicate for 52 samples (15 each of control, PD, MSA, and PSP), and each duplicate was analyzed in an xy plot to show the inter-assay variation. The dotted line is the 45° line and the solid line is the regression line. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0060] [Figure 20] 20A-20C show data demonstrating inter-assay precision for an exemplary assay of the invention. NDE (A), ADE (B), and ODE (C) were enumerated nine separate times in three different control plasma samples. In each experiment, seven dilutions of standard plasma (arbitrarily assigned 100 units / mL) and a buffer control were analyzed simultaneously, and ELISA measurements (RLU) were converted to units / mL. The mean ± standard deviation (CV%) of control plasma 1 (△), 2 (□), and 3 (●) were 64.6 ± 9.2 units / mL (14.2%), 1.6 ± 0.8 units / mL (52.9%), and 44.4 ± 5.5 units / mL (12.4%) for NDE, 7.5 ± 1.3 units / mL (16.8%), 2.6 ± 0.5 units / mL (20.3%), and 0.2 ± 0.1 units / mL (63.33%) for ADE, and 12.6 ± 1.3 units / mL (10.1%), 2.6 ± 0.3 units / mL (11.45%), and 0.2 ± 0.1 units / mL (34.5%) for ODE, respectively.

[0061] [Figure 21]21A-21C show data showing dilution linearity for an exemplary assay of the present invention. After screening various control plasma samples, we found three plasma samples (□, ●, ▲) that exhibited a wide range of NDE (A), ADE (B), and ODE (C). Plasma dilution studies were then performed to analyze the linearity and slope between the three plasma samples.

[0062] [Figure 22] Figures 22A-22C show data showing the enumeration of brain-derived exosomes in human subjects with Parkinson's disease. NDE (A), ADE (B), and ODE (C) were enumerated in controls, 15 each of PD, MSA, and 7 PSP patients. Each dot represents one individual, and p-values ​​are relative to controls.

[0063] [Diagram 23] Figures 23A-23C show data showing the receiver operating characteristics (ROC) of NDE and ODE for the differential diagnosis of Parkinson's disease and controls. The ROC was calculated using the NDE (A) and ODE (B) data of Parkinson's disease and controls. The area under the curve (AUC) was 88.9% and 88% for NDE and ODE, respectively.

[0064] [Figure 24] Figures 24A-24F show data showing the correlation between plasma BDE levels and disease severity in plasma samples from subjects with Parkinson's disease. NDE (A, D), ADE (B, E), and ODE (C, F) of PD patients are classified by mRS (AC) and Hoehn-Yahr (DF) severity scores and shown with the scores of controls. Each dot represents one individual, and statistical p-values ​​were between each severity group and controls.

[0065] [Diagram 25]Figures 25A-25D show data showing the correlation of brain-derived exosomes and Parkinson's disease severity. A: ADE / NDE ratio and disease duration. B: ADE / NDE ratio and mRS score. C: ADE / NDE ratio and UPDRS part 3 (motor). D: ODE / NDE ratio and UPDRS part 3 (motor).

[0066] [Figure 26] Figures 26A-26D show data showing the correlation between plasma BDE levels and disease severity in MSA. A: ODE of MSA patients (A) classified by mRS severity score and shown with the scores of controls. Each dot represents one individual. B: Same data as shown in Figure 3C. C-E: ODE of each MSA patient was compared with disease duration in months (C) and International Carotid Arthritis Rating Scale (ICARS) scores (D) and Unified Parkinson's Disease Rating Scale (UPDRS)3 (Motor) (E). r2 and p-values ​​are shown in E.

[0067] [Figure 27] Figures 27A-27F show data demonstrating the detection of pathological forms of α-synuclein on the surface of neuron-, astrocyte-, and oligodendrocyte-derived exosomes.

[0068] [Figure 28] Figures 28A-28B show data demonstrating detection of dopamine receptor D2 (DRD2) on the surface of neuron-derived exosomes (NDE).

[0069] [Figure 29] Figures 29A-29C present data showing the results of plasma dilution studies for DRD2.

[0070] [Diagram 30] Figures 30A-30G show data showing DRD2 detection in neuron-derived exosomes in plasma samples from humans with neurological diseases.

[0071] [Diagram 31] FIG. 31 shows data illustrating the ROC curve and area under the curve (AUC) for DRD2 levels among PD and control samples.

[0072] [Diagram 32] Figures 32A-32D present data showing detection of CD81, SNCA, SNCA oligomer F1, and TH on the surface of DAT+ vesicles.

[0073] [Diagram 33] FIG. 33 presents data showing detection of SNAP25, EAAT1, and OMG on the surface of vesicles in plasma.

[0074] [Diagram 34] FIG. 34 presents data showing particle size for captured DAT+CD81+ vesicles in plasma.

[0075] [Diagram 35] FIG. 35 shows data demonstrating detection of various biomarkers on the surface of vesicles in plasma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] Detailed Description of the Invention It is to be understood that the invention is not limited to the particular methodology, protocols, cell lines, assays, and agents described herein, as these may vary, and that the terminology used herein is intended to describe particular embodiments of the invention, and is not intended to limit the scope of the invention as set forth in the appended claims.

[0077] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "fragment" includes a plurality of such fragments, a reference to an "antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, preferred methods, devices, and materials are described below. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodology, agents, and tools reported in the publications that may be used in connection with the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate the above disclosure by prior invention.

[0079] Unless otherwise indicated, the practice of the present invention will employ conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art, such techniques being fully explained in the literature. For example, Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods in Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al.,eds. (1989)Molecular Cloning: A Laboratory Manual,2nd edition,Vols. I-III,Cold Spring Harbor Laboratory Press;Ausubel,FM et al.,eds. (1999)Short Protocols in Molecular Biology,4th edition,John Wiley & Sons;Ream et al.,eds. (1998)Molecular Biology Techniques: An Intensive Laboratory Course,Academic Press); PCR (Introduction to Biotechniques) Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag).

[0080] The present invention relates, in part, to the development of efficient methods for detecting and quantifying biomarkers on vesicles. In particular, vesicles are selectively captured on a solid support, and membrane-bound and adsorbed biomarkers of the captured vesicles are detected without lysis or permeabilization of the vesicles. The present invention further relates to the use of such biomarkers identified on vesicles in methods of diagnosis and prognosis for various diseases.

[0081] Moreover, the present invention provides compositions for use in the methods described herein, which may include a solid support, a capture agent that specifically binds to a surface marker of the vesicle (i.e., a membrane protein or an adsorbed protein) to capture the vesicle on the solid support, a detection agent that specifically binds to a biomarker on the vesicle, reagents for performing an immunoassay, and other reagents for performing the methods described herein.

[0082] The present invention further provides kits for detecting biomarkers on vesicles, which may include a solid support, one or more capture agents that specifically bind and capture vesicles on the solid support, one or more detection agents that specifically bind to biomarkers on the vesicles, and optionally, immunoassay reagents and other reagents for carrying out the methods described herein, one or more containers for collecting and / or holding a biological sample, and instructions for use of the kit.

[0083] The present invention further relates to the discovery that exosome biomarkers can be assayed to identify subjects having or likely to develop neurological disorders including, for example, Alzheimer's disease (AD), multiple sclerosis (MS), and frontotemporal dementia (FTD).

[0084] The present invention is based in part on the discovery of an unexpected decrease or increase in certain biomarkers in neuron-derived exosomes present in the blood circulation of subjects with neurological disease (e.g., Alzheimer's disease).The present invention demonstrates that exosomal levels of these biomarkers can be assayed to diagnose neurological disorders in subjects with neurological disease.The present invention further demonstrates that measurement of certain biomarkers in neuron-derived exosomes from a subject can be used to predict the subsequent development of a neurological disease (e.g., to identify subjects at risk of developing a neurological disorder).

[0085] The section headings are used herein for organizational purposes only and should not be construed as limiting the subject matter described herein in any way.

[0086] Biological samples A biological sample containing vesicles (e.g., exosomes) may be obtained from a subject. The biological sample obtained from a subject is typically blood, but may be any sample from bodily fluids, tissues, or cells that contain vesicles to be analyzed. Biological samples may include, but are not limited to, whole blood, serum, plasma, urine, interstitial fluid, ascites, cerebrospinal fluid, cervical swabs, tears, saliva, buccal swabs, skin, organs, and biopsies. Alternatively, exosomes may be obtained from cultured cells by collecting secreted exosomes from the surrounding medium.

[0087] In some embodiments, the biological sample of the present invention is obtained from blood. In some embodiments, about 1-10 ml of blood is drawn from a subject. In other embodiments, about 10-50 ml of blood is drawn from a subject. Blood can be drawn from any suitable area of ​​the body, including the arm, leg, or blood accessible via a central venous catheter. In some embodiments, blood is collected after a procedure or activity. For example, blood can be collected after a medical checkup. Also, the timing of collection can be adjusted to increase the number and / or composition of vesicles (e.g., exosomes) present in the sample. For example, blood can be collected after exercise or a procedure that induces vasodilation.

[0088] Blood can be combined with various components after collection to preserve or prepare the sample for subsequent techniques. For example, in some embodiments, blood is treated with anticoagulants, cell fixatives, protease inhibitors, phosphatase inhibitors, protein, DNA, or RNA preservatives after collection. In some embodiments, blood is collected by venipuncture using vacuum collection tubes containing anticoagulants such as EDTA or heparin. Blood can also be collected using heparin-coated syringes and hypodermic needles. Blood can also be combined with components that would be useful for cell culture. For example, in some embodiments, blood is combined with cell culture medium or supplemented cell culture medium (e.g., cytokines).

[0089] Enrichment or isolation of vesicles (exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, and ectosomes) The sample may be enriched for vesicles through positive selection, negative selection, or a combination of positive and negative selection. In some embodiments, the vesicles are captured directly. In other embodiments, blood cells are captured and vesicles are collected from the remaining biological sample. In some embodiments, the vesicles enriched in the biological sample are exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, or ectosomes. In some embodiments, the vesicles enriched in the biological sample are neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes.

[0090] A sample can also be enriched for vesicles based on differences in biochemical properties of the vesicles. For example, a sample can be enriched for vesicles based on antigen, nucleic acid, metabolic, gene expression, or epigenetic differences. In some embodiments based on antigen differences, antibody-bound magnetic or paramagnetic beads or fluorescently labeled antibodies in a magnetic field gradient are used with flow cytometry. In some embodiments based on nucleic acid differences, flow cytometry is used. In some embodiments based on metabolic differences, dye uptake / excretion measured by flow cytometry or other sorting techniques is used. In some embodiments based on gene expression, cell culture with cytokines is used. A sample can also be enriched for vesicles based on other biochemical properties known in the art. For example, a sample can be enriched for vesicles based on pH and motility. Additionally, in some embodiments, more than one method is used for enrichment for vesicles. In other embodiments, a sample is enriched for vesicles using antibodies, ligands, or soluble receptors.

[0091] In other embodiments, surface markers are used to positively enrich for vesicles in a sample. In some embodiments, the vesicles are exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, or ectosomes. In other embodiments, NCAM, CD171, CD9, CD63, CD81, SNAP25, EAAT1, OMG, neuron-specific enolase, various neuronal or astrocyte adhesion proteins, microglial CD18 / 11, or CD3T cell membrane cell surface markers are used to enrich for exosomes. In some embodiments, cell surface markers that are not found in the vesicle population are used to negatively enrich for vesicles by depleting the cell population. Flow cytometry sorting may be used to further enrich for exosomes using cell surface markers or intracellular or extracellular markers conjugated to fluorescent labels. Intracellular and extracellular markers may include nuclear stains or antibodies against intracellular or extracellular proteins preferentially expressed in vesicles. The cell surface marker may include an antibody against a cell surface antigen (e.g., NCAM) preferentially expressed on exosomes. In some embodiments, the cell surface marker is a neuron-derived exosome surface marker, including, for example, NCAM or CD171. In some embodiments, a monoclonal NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is used to enrich or isolate exosomes from a sample. In some aspects, the NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is biotinylated. In this embodiment, the biotinylated NCAM or CD171 antibody can form an antibody-exosome complex that can be subsequently isolated using streptavidin agarose resin or beads. In other embodiments, the NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is a monoclonal anti-human NCAM, CD9, CD63, CD81, neuron-specific enolase or CD171 antibody.In other embodiments, the cell surface markers include neuronal specific proteins (e.g., synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tau, αβ-42, and synaptophysin), astrocyte specific proteins (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1)), microglia specific proteins (CD11b), oligodendrocyte specific proteins (e.g., myelin basic protein (MBP), oligodendrocyte myelin glycoprotein (MGP), and EGFR-associated protein (EGFR-associated protein 1 (EGFR-associated protein 2 ... The proteins may be cytosolic proteins (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspases, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL), or chemokines (CX3CL1) or cytokines (IL1b, IL34, FasL, or IL12B).

[0092] In some embodiments, the vesicles enriched from the biological sample are then enriched for a particular type of vesicle. For example, the biological sample is enriched for exosomes, and then the enriched exosomes are then enriched for neurally derived exosomes. In some embodiments, the biological sample is enriched for a particular neural source of vesicles. In some aspects, the neural source of vesicles is microglia, neurons, or astrocytes.

[0093] In other embodiments, vesicles are isolated or enriched from a biological sample by a method comprising contacting the biological sample with an agent under conditions in which vesicles present in the biological sample bind to the agent to form a vesicle-drug complex, and isolating the vesicles from the vesicle-drug complex to obtain a sample containing the vesicles, wherein the purity of the vesicles present in the sample is greater than the purity of the vesicles present in the biological sample. In certain embodiments, the agent is an antibody or a lectin. Lectins useful for forming vesicle-lectin complexes are described in US Patent Publication No. 2012 / 0077263. In some embodiments, the vesicles are exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, or ectosomes. In some embodiments, the exosomes are neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes. In some embodiments, multiple isolation or enrichment steps are performed. In certain aspects of the present embodiments, a first isolation step is performed to isolate exosomes from a blood sample and a second isolation step is performed to isolate neurally derived exosomes from other exosomes.

[0094] In yet another embodiment, the method further comprises releasing the vesicles from the vesicle-drug complex. In another embodiment, the vesicles are released by exposing the vesicle-drug complex to a low pH between 3.5-1.5. In another embodiment, the vesicles are released using a competitor peptide that competes for binding of the selected antibody used in the method of the present invention. In yet another embodiment, the released vesicles are neutralized by adding a high pH solution. In another embodiment, the released vesicles are lysed by incubating the released vesicles with a lysis solution. In yet another embodiment, the lysis solution contains inhibitors for proteases and phosphatases.

[0095] Capture and detection of vesicles on solid supports In other embodiments, a subset of vesicles is separated from other vesicles in a biological sample using a capture agent immobilized on a solid support. Such capture agents selectively bind to surface markers (e.g., membrane proteins or adsorption proteins) of the vesicles, so that the capture agent can "capture" vesicles that have the surface marker. By "capture", we mean that the capture agent can bind to the surface marker of the vesicle, thereby separating the target vesicle from other vesicles in the sample.

[0096] The specificity of the capture agent determines the subset of vesicles from the biological sample captured on the solid support. One or more capture agents can be used in combination to capture vesicles with different surface markers. For example, the solid support can include one or more capture agents bound to the solid support, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more different capture agents that selectively bind to different biomarkers on the vesicles. In some embodiments, the vesicles targeted by the capture agent are exosomes, microparticles, microvesicles, nanosomes, extracellular vesicles, or ectosomes.

[0097] In some embodiments, the capture agent selectively binds to neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes. For example, the capture agent may selectively bind to exosome surface markers (e.g., CD81) for capturing exosomes, neuron-specific proteins (e.g., synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tau, αβ-42, and synaptophysin) for capturing neuron-derived exosomes, astrocyte-specific proteins (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1) for capturing astrocyte-derived exosomes, microglia-specific proteins (CD11b) for capturing microglia-derived exosomes, and exosome surface markers (e.g., CD82) for capturing oligodendrocyte-derived exosomes. The capture agent may be selected to selectively bind to an oligodendrocyte-specific protein (e.g., myelin basic protein (MBP) or oligodendrocyte myelin glycoprotein (OMG)) for capturing exosomes, a cytosolic protein (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA) or cathepsin D (CTSD)) for capturing brain-derived exosomes, or a chemokine (CX3CL1) or cytokine (IL1b, IL34, FasL, or IL12B) protein for capturing brain-derived exosomes. In some embodiments, the capture agent may be selected to selectively bind to an extracellular vesicle (EV) surface marker (e.g., DAT) for capturing extracellular vesicles in general.

[0098] Typically, the capture agent is directly or indirectly bound to a solid support. The capture agent may be immobilized on the surface of a solid support, such as, but not limited to, a plate, slide, wafer, non-magnetic beads, magnetic beads, rods, particles, strands, disks, membranes, films, or the inner surface of a tube, channel, column, flow cell apparatus, or microfluidic device. The solid support may comprise a variety of materials, including, but not limited to, glass, quartz, silicon, metal, ceramic, plastic, nylon, polyacrylamide, agarose, resin, porous polymer monolith, hydrogel, and composites thereof. Additionally, a substrate may be added to the surface of the solid support to facilitate attachment of the capture agent.

[0099] Once captured on a solid support, the vesicles can be screened for one or more membrane-bound and adsorbed biomarkers without the need for lysis or permeabilization of the vesicles using detection agents that selectively bind to the membrane-bound or adsorbed biomarkers of the vesicles. In certain embodiments, the detection agent is a neuron-specific protein (e.g., synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), αβ-42, tau, phosphorylated tau, and synaptophysin), an astrocyte-specific protein (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1)), a microglia-specific protein (CD11b), an oligodendrocyte-specific protein (e.g., myelin basic protein (MBP), oligodendrocyte myelin glycoprotein (OMG), a cytosolic protein (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, H, or a combination thereof). VESICLES SPECIFICALLY ARE CLAIMED BY THE INVENTORY OF A VESICLE ...

[0100] The capture and detection agents may include, for example, antibodies, antibody fragments, antibody mimetics, or aptamers that specifically bind to a surface marker of a vesicle (e.g., a membrane-bound or adsorbed protein). "Specifically (or selectively) bind" refers to a binding reaction that can determine the presence of a vesicle surface marker in a heterogeneous population of proteins and other biologics. Thus, under specified assay conditions, a particular capture or detection agent will bind to a particular surface marker of a vesicle at least twice the background and will not bind in a substantially significant amount to other proteins present in the sample.

[0101] In certain embodiments, the capture or detection agent comprises an antibody that specifically binds to a surface marker of the vesicle (e.g., a membrane protein or an adsorption protein). Any type of antibody may be used, including polyclonal and monoclonal antibodies, hybrid antibodies, modified antibodies, chimeric antibodies, and humanized antibodies, as well as hybrid (chimeric) antibody molecules (see, e.g., Winter et al. (1991) Nature 349:293-299; and U.S. Patent No. 4,816,567); F(ab') 2 and F(ab) fragment; F vmolecules (non-covalent heterodimers, see, e.g., Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single domain antibodies (sdAbs) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and British Patent Publication GB 2,276,169, published September 21, 1994); and any functional fragments derived from such molecules, where such fragments retain the specific binding properties of the parent antibody molecule (i.e., specifically bind to a target surface marker of a vesicle).

[0102] In other embodiments, the capture or detection agent comprises an aptamer that specifically binds to the target surface marker of the vesicle. Any type of aptamer may be used, including DNA, RNA, xenonucleic acid (XNA), or peptide aptamers that specifically bind to the target antibody isotype. Such aptamers can be identified, for example, by screening a combinatorial library. Nucleic acid aptamers (e.g., DNA or RNA aptamers) that selectively bind to the target antibody isotype can be produced by in vitro selection or repeated systematic evolution of ligands by exponential enrichment (SELEX). Peptide aptamers that bind to the target antibody isotype can be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection.For a description of how to produce aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (RN Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Nucleic Acid Aptamers: Selection, Characterization, and Application (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2016), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang eds., Springer, 2015), Cox et al. (2001) Bioorg. Med. Chem. 9(10):2525-2531; Cox et al. (2002) Nucleic Acids Res. 30(20): e108, Kenan et al. (1999) Methods Mol Biol. 118:217-231; Platella et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304-4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9):1440-1452.

[0103] In yet other embodiments, the capture or detection agent comprises an antibody mimetic. Any type of antibody mimetic may be used, including, but not limited to, affibody molecules, (Nygren (2008) FEBS J. 275(11):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372(1):172-185), affimers (Johnson et al. (2012) Anal. Chem. 84(15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383(5):1058-1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275(11):2677-2683), avimers (Silverman et al. (2005) Nat. Biotechnol. 23(12):1556-1561), darpins (Stumpp et al. (2008) Drug Discov. Today 13(15-16):695-701), fynomers (Grabulovski et al. al. (2007) J. Biol. Chem. 282(5):3196-3204), and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109).

[0104] The detection agent may further comprise a detectable label that facilitates detection and / or quantification of the biomarker on the vesicle. Detectable labels include fluorescent, chemiluminescent, electrochemiluminescent, or bioluminescent tags, metals, dyes, radionuclides, etc., attached to a specific binding agent (e.g., an antibody, antibody fragment, antibody mimetic, or aptamer that specifically binds to a membrane-bound or adsorbed biomarker of the vesicle).

[0105] Neuropathy The present invention provides methods for diagnosing or prognosing a neuropathy in a subject, identifying a subject at risk for a neuropathy, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with a neuropathy. In some embodiments, the present invention provides methods for the differential diagnosis of a neuropathy in a subject.

[0106] In some embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, neurofibrillary tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Parkinsonism-dementia complex of Guam, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), stroke, depression, bipolar disorder, epilepsy, autism, schizophrenia, brain tumors, white matter disease, cerebral atrophy, mental retardation, cerebellar ataxia, concussion, subconcussive impact, and Parkinson's disease.

[0107] In some embodiments, the invention allows a medical practitioner to diagnose or prognose one or more neurological disorders in a subject. In other embodiments, the invention allows a medical practitioner to rule out or eliminate one or more neurological disorders as diagnostic possibilities. In other embodiments, the methods of the invention allow a medical practitioner to distinguish between some forms of FTD and Alzheimer's disease. In yet other embodiments, the invention allows a medical practitioner to identify subjects at risk of developing a neurological disorder. In other embodiments, the invention allows a medical practitioner to predict whether a subject will subsequently develop a neurological disorder. In further embodiments, the invention allows a medical practitioner to prescribe a treatment regimen or predict the benefit of treatment in a subject with a neurological disorder.

[0108] cancer The present invention provides a method for diagnosing or prognosing cancer in a subject, identifying a subject at risk of developing cancer, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with cancer. Generally, cancer is characterized by the uncontrolled growth of abnormal cells anywhere in the body. Abnormal cells may be called cancer cells, malignant cells, or tumor cells. Cancer is not limited to humans; animals and other organisms can become cancerous.

[0109] In some instances, the cancer may be malignant. Alternatively, the cancer may be benign. The cancer may be recurrent and / or refractory. Most cancers can be classified as carcinomas, sarcomas, leukemias, lymphomas, myelomas, or cancers of the central nervous system.

[0110] The cancer may be a sarcoma. Sarcomas are cancers of bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Sarcomas include, but are not limited to, bone cancer, fibrosarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, bilateral vestibular schwannoma, osteosarcoma, soft tissue sarcoma (e.g., alveolar soft part sarcoma, angiosarcoma, phyllodes cystosarcoma, dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).

[0111] Alternatively, the cancer may be a carcinoma. Carcinomas are cells that originate from epithelial cells, cover the surface of the body, produce hormones, and make up glands. Non-limiting examples of carcinomas include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penic cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, skin or intraocular melanoma, anal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, brain stem glioma, and spinal cord tumors. In some examples, the cancer is a skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic (solar) keratosis. Preferably, the cancer is prostate cancer. Alternatively, the cancer may be thyroid cancer, bladder cancer, or pancreatic cancer.

[0112] In some instances, the cancer is lung cancer. Lung cancer begins in the airways that branch off the trachea and supply the lungs (bronchi) or small air sacs (alveoli) of the lungs. Lung cancer includes non-small cell lung cancer (NSCLC), small cell lung cancer, and mesothelioma. Examples of NSCLC include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Mesothelioma may be a cancerous tumor of the lining of the lungs and chest cavity (pleura) or the lining of the abdomen (peritoneum). Mesothelioma may result from asbestos exposure. The cancer may be a brain cancer, such as glioblastoma.

[0113] Alternatively, the cancer may be a central nervous system (CNS) tumor. CNS tumors may be classified as gliomas or non-gliomas. Gliomas may be malignant gliomas, high-grade gliomas, diffuse intrinsic pontine gliomas. Examples of gliomas include astrocytomas, oligodendrogliomas (or a mixture of oligodendrogliomas and astrocytoma elements), and ependymomas. Astrocytomas include, but are not limited to, low-grade astrocytomas, anaplastic astrocytomas, glioblastoma multiforme, pilocytic astrocytomas, xanthoastrocytomas multiforme, and subependymal giant cell astrocytomas. Oligodendrogliomas include low-grade oligodendrogliomas (or oligoastrocytomas) and anaplastic oligodendrogliomas. Non-gliomas include meningiomas, pituitary adenomas, primary CNS lymphomas, and medulloblastomas. In some instances, the cancer is a meningioma.

[0114] The cancer may be leukemia. The leukemia may be acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, or chronic myelogenous leukemia. Additional types of leukemia include hairy cell leukemia, chronic myelomonocytic leukemia, and juvenile myelomonocytic leukemia.

[0115] In some instances, the cancer is lymphoma, which is a cancer of the lymphocytes and may arise from either B or T lymphocytes. Lymphoma The two main types are Hodgkin lymphoma, formerly known as Hodgkin's disease, and Non-Hodgkin lymphomaHodgkin's lymphoma is characterized by the presence of Reed-Sternberg cells. Non-Hodgkin's lymphoma is any lymphoma that is not Hodgkin's lymphoma. Non-Hodgkin's lymphoma can be low-grade lymphoma and intermediate-grade lymphoma. Non-Hodgkin's lymphoma includes, but is not limited to, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), extranodal marginal zone B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis.

[0116] In some embodiments, the invention allows a medical practitioner to diagnose or prognose one or more cancers in a subject. In other embodiments, the invention allows a medical practitioner to rule out or eliminate one or more cancers as a diagnostic possibility. In other embodiments, the methods of the invention allow a medical practitioner to identify the origin of a cancer. In yet other embodiments, the invention allows a medical practitioner to identify a subject at risk of developing cancer. In other embodiments, the invention allows a medical practitioner to predict whether a subject will subsequently develop cancer. In further embodiments, the invention allows a medical practitioner to prescribe a treatment regimen or predict the benefit of treatment in a subject with cancer. Exemplary biomarkers of the invention that are useful in cancer diagnosis and prognosis include, but are not limited to, EpCAM, PD-L1, ErbB2, CK19.

[0117] Immunologic disorders The present invention provides methods for diagnosing or prognosing an immunological disorder in a subject, identifying a subject at risk for developing an immunological disorder, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with an immunological disorder. Immunological disorders are diseases or conditions caused by dysfunction of the immune system, including allergies, asthma, autoimmune diseases, autoinflammatory syndromes, and immune deficiency syndromes.

[0118] In some embodiments, the present invention allows a medical practitioner to diagnose or prognose one or more immunological disorders in a subject. In other embodiments, the present invention allows a medical practitioner to rule out or eliminate one or more immunological disorders as a diagnostic possibility. In yet other embodiments, the present invention allows a medical practitioner to identify subjects at risk for developing an immunological disorder. In other embodiments, the present invention allows a medical practitioner to predict whether a subject will subsequently develop an immunological disorder. In further embodiments, the present invention allows a medical practitioner to prescribe a treatment regimen or predict the benefit of treatment in a subject with an immunological disorder. Exemplary biomarkers of the present invention that are useful in the diagnosis and prognosis of immunological disorders include, but are not limited to, TCR, CD16, CD28, CD32, CD79a, and TREM2.

[0119] Placental disorders and fetal evaluation The present invention provides methods for diagnosing or prognosing placental disease in a subject, identifying subjects at risk for developing placental disease, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with placental disease. Generally, placental disease is characterized by: placenta The biomarkers of the present invention may also be used in fetal evaluation or diagnosis of fetal disorders, such as fetal alcohol syndrome or fetal genetic abnormalities. Exemplary biomarkers of the present invention that are useful in the diagnosis and prognosis of placental disorders or fetal evaluation include, but are not limited to, PLAP, CSH1, and PSG1.

[0120] Biomarkers The biomarker levels on the vesicles are assayed in a biological sample obtained from a subject having or at risk of having a disease. In some embodiments, the biomarker levels on the vesicles are assayed in a biological sample obtained from a subject having or at risk of having a neurological disorder (e.g., Alzheimer's disease). In other embodiments, the biomarker levels on the vesicles are assayed in a biological sample obtained from a subject having or at risk of having a cancer. In yet other embodiments, the biomarker levels on the vesicles are assayed in a biological sample obtained from a subject having or at risk of having an immunological disorder. In yet other embodiments, the biomarker levels on the vesicles are assayed in a biological sample obtained from a subject having or at risk of having a placental disorder. In some embodiments, the one or more biomarkers are selected from the group consisting of neuron-specific proteins (e.g., synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), tau, and synaptophysin), astrocyte-specific proteins (e.g., glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1)), microglia-specific proteins (CD11b), oligodendrocyte-specific proteins (e.g., myelin basic protein (MBP), oligodendrocyte myelin glycoprotein (OMG)), and extracellular vesicle-specific proteins (dopamine transporter, DAT). In another embodiment, the biomarkers are CD171, phosphorylated tau T181, SNCA, and NRGN. In other embodiments, the biomarkers are acetylcholinesterase (AchE), lysosomal associated membrane protein 1 (LAMP1), CTSD, RE1 silencing transcription factor (REST), synaptotagmin (SYT), monocyte chemoattractant protein-1 (CCL2), IL34, glycogen synthase (GYS), (OR), death receptor 6 (DR6), heat shock proteins (HSPs), IL12 beta, alpha-beta (Aβ), and beta secretase (BACE).In some embodiments, the one or more biomarkers are selected from the group consisting of cytosolic proteins, secreted proteins, membrane proteins and receptors and their pathological forms including aggregates and mutants. Biomarkers of the present invention include neurotransmitter receptors such as, for example, dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5.B1, B2), and glutamate receptors (1 and 2). Other receptor biomarkers of the present invention include insulin receptors, tumor necrosis factor receptor superfamily (TRAL, TNF receptors, death receptors 5 and 6), and neuropeptide receptors (orexin receptors, opioid receptors KOR). Biomarkers of the present invention include, for example, membrane proteins such as EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. Other known neurological disorder biomarkers may be used in combination with the biomarkers of the present invention. Examples of such biomarkers are provided in U.S. Patent Application Publication No. 2015 / 0119278, which is incorporated by reference.

[0121] Those skilled in the art have several methods and devices available for the detection and analysis of the markers of the present invention. For polypeptides or proteins on vesicles in patient test samples, immunoassay devices and methods are often utilized. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats to generate signals related to the presence or amount of the analyte of interest. Furthermore, certain methods and devices, such as biosensors and optical immunoassays, can be used to determine the presence and amount of analytes without the need for labeled molecules.

[0122] Preferably, the markers are analyzed using immunoassays, although other methods (e.g., measuring marker RNA levels) are well known to those skilled in the art. The presence or amount of markers is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), immunopolymerase chain reaction assay, electrochemiluminescence immunoassay (ECLIA), radioimmunoassay (RIA), competitive binding assay planar waveguide technology, etc. The immunological binding of specific antibodies to markers can be determined directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, or radionuclides bound to the antibody, etc. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, etc.

[0123] The use of immobilized antibodies specific for the surface markers of vesicles is also contemplated by the present invention. Antibodies can be immobilized on a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay location (such as a microtiter well), a piece of solid substrate material (plastic, nylon, paper, etc.), etc. An assay strip can be prepared by coating an array of solid supports with an antibody or antibodies. The strip can be immersed in a test sample to capture vesicles through binding to the surface markers, and then rapidly processed through a washing and detection step with a detection reagent as described above to generate a measurable signal, such as a colored spot.

[0124] Analysis of multiple markers may be performed separately or simultaneously with one test sample. Markers on some vesicles may be captured and / or detected using a combination of multiple capture and / or detection agents in one test for efficient processing of multiple samples. Furthermore, those skilled in the art will recognize the value of testing multiple samples (e.g., consecutive time points) from the same individual. Testing such consecutive samples will allow for the identification of changes in marker levels over time. An increase or decrease in marker levels, as well as the absence of changes in marker levels, will provide useful information about the disease state, including but not limited to identifying the approximate time from the onset of an event, the presence and amount of salvageable tissue, the appropriateness of drug therapy, the effectiveness of various treatments, identifying the severity of an event, identifying the severity of the disease, and identifying the patient's treatment outcomes, including the risk of future events.

[0125] Assays consisting of combinations of markers referred to in the present invention may be constructed to provide relevant information regarding differential diagnosis. Such panels may be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more, or individual markers. Analysis of single markers or subsets of markers, including larger panels of markers, may be performed in the methods described within the present invention to optimize clinical sensitivity or specificity in various clinical situations.

[0126] The analysis of the markers can be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation can be used to facilitate the processing of multiple test samples. Alternatively, single sample formats can be developed to facilitate timely and rapid treatment and diagnosis, for example, in ambulatory transport or emergency room settings. Particularly useful physical formats include surfaces with multiple, discrete, addressable locations for the detection of multiple different analytes. Such formats include protein microarrays or "protein chips" and capillary devices.

[0127] The biomarkers of the present invention play an important role in the early detection and monitoring of neurological disorders (e.g., Alzheimer's disease). Such disorder markers are typically substances found in body samples that can be measured. The measured amount can be correlated to the pathophysiology of the underlying disorder or disease, the presence or absence of neurological disorders, or the likelihood of future neurological disorders. When a patient undergoes treatment for his / her condition, the measured amount will also correlate to the responsiveness of the treatment. In some embodiments, a decrease or increase in the level of one or more biomarkers of the present invention indicates neurological disorders. For example, an increase in phosphorylated tau T181 levels and / or a decrease in NRGN levels of exosomes with CD171 membrane marker indicates Alzheimer's disease. Thus, the method of the present invention is useful for the differential diagnosis of Alzheimer's disease.

[0128] In some embodiments, the biomarker is measured by a method selected from the group consisting of immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprecipitation, electrochemiluminescence immunoassay, radioimmunoassay, immunopolymerase chain reaction, Western blotting, and ELISA.

[0129] Clinical Assay Performance The method of the present invention may be used in clinical assays to diagnose or prognose neuropathy in a subject, identify subjects at risk of neuropathy, and / or prescribe a treatment regimen or predict the benefit of treatment in a subject with neuropathy.Clinical assay performance can be evaluated by determining the sensitivity, specificity, area under the ROC curve (AUC), accuracy, positive predictive value (PPV), and negative predictive value (NPV) of the assay.Disclosed herein are assays for diagnosing or prognosing neuropathy in a subject, identifying subjects at risk of neuropathy, or prescribe a treatment regimen or predict the benefit of treatment in a subject with neuropathy.

[0130] The clinical performance of the assay may be based on sensitivity. The sensitivity of the assay of the present invention may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. The clinical performance of the assay may be based on specificity. The specificity of the assay of the present invention may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. The clinical performance of the assay may be based on area under the ROC curve (AUC). The AUC of the assay of the present invention may be at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95. The clinical performance of the assay may be based on accuracy. The accuracy of the assays of the invention may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.

[0131] composition Compositions useful in the methods of the present invention include compositions that specifically recognize biomarkers associated with neurological disorders. Such compositions include, for example, neuron-specific protein biomarkers such as synaptosomal associated protein 25 (SNAP25), αβ-42, neurogranin (NRGN), tau, phosphorylated tau, and synaptophysin, astrocyte-specific protein biomarkers such as glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1), oligodendrocyte-specific protein biomarkers such as myelin basic protein (MBP) and oligodendrocyte myelin glycoprotein (OMG), microglia-specific protein (CD11), and mitochondrial protein biomarkers such as mitochondrial protein (MP) and mitochondrial protein (MP). b), capture and / or detection agents that recognize cytosolic proteins (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspases, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL), or chemokines (CX3CL1) or cytokines (IL1b, IL34, FasL, or IL12B).

[0132] In yet other embodiments, the composition is selected from the group consisting of a peptide, a nucleic acid, an antibody, and a small molecule.

[0133] In certain embodiments, the present invention relates to compositions that specifically detect biomarkers associated with neurological disorders.As detailed elsewhere herein, the present invention is based on the discovery that GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau, synaptophysin, αβ-42, CX3CL1, IL1b, IL34, CD81, CD63, CD171, SNAP25, EAAT1, SNCA, CD11b, OMG, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, and BACE can be used as biomarkers for AD and other neurological disorders.In some embodiments, the compositions of the present invention specifically bind and detect such biomarkers. For example, the composition may include a solid support comprising a capture agent bound to the solid support that selectively binds to CD81, CD63, CD171, SNAP25, EAAT1, CD11b, or OMG. In another example, the composition may include a detection agent that selectively binds to GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau, synaptophysin, αβ-42, SNCA, CX3CL1, IL1b, IL34, OMG, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, and / or BACE.

[0134] In some embodiments, the composition comprises an antibody, which specifically binds to a biomarker or vesicle of the invention. The term "antibody" as used herein and further described below is intended to include fragments thereof that also specifically react with a biomarker or vesicle (e.g., an exosome). Antibodies can be fragmented using conventional techniques, and the fragments can be screened for utility in the same manner as described above for whole antibodies. For example, F(ab) 2 Fragments can be generated by treating the antibody with pepsin. The resulting F(ab) 2The fragments can be treated to reduce disulfide bridges to produce Fab fragments. Antigen-binding portions may also be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab') fragments. 2 , Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, diabodies, and polypeptides that contain at least a sufficient portion of an immunoglobulin to confer specific antigen binding to the polypeptide. In certain embodiments, the antibody further comprises a label that binds to the antibody and can be detected (e.g., the label can be a radioisotope, a fluorescent substance, an enzyme, or a co-enzyme for an enzyme).

[0135] In certain embodiments, the antibodies of the present invention are monoclonal antibodies, and in certain embodiments, the present invention makes available methods for generating novel antibodies that specifically bind to the biomarkers or exosomes of the present invention. For example, a method for generating monoclonal antibodies that specifically bind to a biomarker or exosome may include administering to a mouse an amount of an immunogenic composition comprising a biomarker or exosome, or a fragment thereof, effective to stimulate a detectable immune response, obtaining antibody-producing cells from the mouse (e.g., cells from the spleen), fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify hybridomas that produce monoclonal antibodies that specifically bind to the biomarker or exosome. Once obtained, the hybridomas can be grown in cell culture, optionally in culture conditions where cells derived from the hybridomas produce monoclonal antibodies that specifically bind to the biomarker or exosome. The monoclonal antibodies may be purified from the cell culture.

[0136] The term "specifically reacts with" as used with respect to an antibody is intended to mean that the antibody is sufficiently selective between the antigen of interest (e.g., a biomarker or exosome) and other antigens that are not of interest, as is generally understood in the art. In certain methods using antibodies, such as therapeutic applications, a higher degree of specificity in binding may be desired. Monoclonal antibodies generally have a greater tendency (compared to polyclonal antibodies) to effectively discriminate between the desired antigen and cross-reacting polypeptides. One characteristic that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen. While the desired specificity may reach a range of different affinities, generally, preferred antibodies have a specificity of about 10 -6 , 10 -7 , 10 -8 , 10 -9 or lower affinity (dissociation constant).

[0137] Antibodies can be generated to specifically bind to epitopes of exosomes or biomarkers of the invention, including, for example, neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes, or neuron-specific proteins selected from the group consisting of synaptosomal associated protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tau (tar), and synaptophysin, astrocyte-specific proteins selected from the group consisting of glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1), and oligodendrocyte-specific proteins selected from the group consisting of myelin basic protein (MBP) and oligodendrocyte myelin glycoprotein (OMG), microglia-specific proteins (CD11b), and chemokines (CX3CL1) or cytokines (IL1b, IL34, FasL, or IL12B). In another embodiment, the antibodies produced are anti-CD171 antibody, anti-synaptosomal associated protein 25 (SNAP25) antibody, anti-neurogranin (NRGN) antibody, anti-tau antibody, anti-synaptophysin antibody, and anti-CD63 antibody, anti-αβ-42 antibody, anti-CD81 antibody, anti-CTD antibody, anti-GAPDH antibody, anti-IL1b antibody, anti-IL34 antibody, anti-CX3CL1 antibody, anti-glial fibrillary acidic protein (GFAP) antibody, anti-excitatory amino acid transporter 1 (EAAT1) antibody, anti-SNCA antibody, anti-TH antibody, and anti-CD11b antibody, anti-myelin basic ta protein (MBP) antibody, anti-oligodendrocyte myelin glycoprotein (OMG) antibody, anti-dopamine transporter (DAT) antibody, anti-AchE antibody AchE, anti-LAMP1 antibody LAMP1, anti-REST antibody REST, anti-SYT antibody SYT, anti-SYP antibody, anti-SYNP O antibody, anti-PSD95 antibody, anti-SV2A antibody, anti-CCL2 antibody CCL2, anti-IL34 antibody IL34, anti-GYS antibody GYS, anti-OR antibody OR, anti-DR6 antibody DR6, anti-HSP antibody HSP, anti-IL12b antibody IL12b, anti-Aβ antibody Aβ, or anti-BACE antibody BACE.

[0138] Also, the technique used to screen antibodies to identify a desired antibody may affect the properties of the antibody obtained. A variety of different techniques are available to test antibody-antigen interactions to identify particularly desirable antibodies. Such techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore binding assays, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., paramagnetic bead systems, IGEN International, Inc., Gaithersburg, Md.), Western blots, immunoprecipitation assays, immunocytochemistry, and immunohistochemistry.

[0139] In some embodiments, the present invention relates to compositions used to treat or prevent neurological disorders. As detailed elsewhere herein, the present invention is based on the discovery that levels of CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, CD63, αβ-42, SNCA, CX3CL1, IL1b, IL34, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, and / or BACE are associated with the pathology of various neurological disorders, such as, for example, Alzheimer's disease.

[0140] In some embodiments, in addition to surface biomarkers, biomarkers within vesicles are analyzed. In certain embodiments, the present invention relates to a composition for dissolving vesicles (e.g., exosomes) in biological samples obtained from subjects. Dissolving agents useful in the methods of the present invention include RIPA buffer; Tris-HCl (pH 6.8); glycerin; SDS; 2-mercaptoethanol; Triton-X 100; M-PER reagent; T-PER solution; and CHAPS. Dissolving agents may be incubated with biological samples to disrupt the membrane of vesicles of the present invention and release vesicle cargo (e.g., exosome proteins) for subsequent analysis.

[0141] Treatment method The present invention provides methods of treating a neurological disorder in a subject comprising administering to the subject an effective amount of a composition, wherein the composition increases, decreases, or maintains levels of CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, CD63, αβ-42, SNCA, CX3CL1, IL1b, or IL34 in the subject. In yet other embodiments, the composition prevents an increase or decrease in CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, CD63, αβ-42, SNCA, CX3CL1, IL1b, IL34, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, or BACE levels. In other embodiments, the invention provides a method of treating a neurological disorder in a subject comprising administering to the subject an effective amount of a composition, wherein the composition normalizes levels of CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), synaptophysin, CD63, αβ-42, SNCA, CX3CL1, IL1b, IL34, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, and / or BACE to a reference level.

[0142] kit The above-mentioned assay reagents, including the capture agent-bound solid support, the detection agent, and optionally the reagents for performing an immunoassay such as ELISA, IFA, immunopolymerase chain reaction assay, ECLIA, or RIA, can be provided in a kit with appropriate instructions and other necessary reagents for performing an assay to detect biomarkers on vesicles, as described above. The kit will typically contain the capture agent-bound solid support, the detection agent, the control preparation (positive and / or negative), and other reagents required by the assay format in separate containers. Instructions (e.g., written, CD-ROM, DVD, Blu-ray, flash drive, digital download, etc.) for performing the assay are typically included in the kit. The kit can also contain other packaged reagents and materials (i.e., wash buffers, etc.), depending on the particular assay used. Assays such as those described above can be performed using these kits.

[0143] In another embodiment, the present invention includes a kit for detecting or monitoring a neurological disorder in a subject. A variety of kits with different components are contemplated by the present invention. Generally speaking, the kit will include a means for quantifying one or more biomarkers in a subject. In another embodiment, the kit will include a means for collecting a biological sample, a means for quantifying one or more biomarkers in the biological sample, and instructions for using the contents of the kit. In a particular embodiment, the kit will include a means for enriching or isolating exosomes in a biological sample. In a further aspect, the kit includes a solid support with a binding capture agent for isolating exosomes from a biological sample. In some aspects, the kit includes a means for quantifying the amount of a biomarker. In a further aspect, the means for quantifying the amount of a biomarker includes the necessary reagents for detecting the amount of a biomarker.

[0144] In another embodiment, the invention provides a method for diagnosing or prognosing a neuropathy in a subject, identifying a subject at risk for a neuropathy, or prescribing a treatment regimen or predicting the benefit of treatment in a subject with a neuropathy, comprising: a) a solid support comprising a capture agent bound to the solid support, where at least one capture agent selectively binds to CD171, CD63, CD81, SNAP25, EAAT1, or OMG; and b) one or more detection agents. wherein the one or more detection agents selectively bind to CD81, GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau (e.g., T181), CD63, αβ-42, CX3CL1, IL1b, IL34, AchE, LAMP1, REST, SYT, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, or BACE on the surface of the exosome. In certain embodiments, at least one capture or detection agent comprises an antibody, antibody fragment, antibody mimetic, or aptamer that specifically binds to CD171, phosphorylated tau T181, or neurogranin. In certain embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a recombinant fragment of an antibody, a Fab fragment, a Fab' fragment, a F(ab') 2 Fragment, F v Fragments, and scF v In another embodiment, the kit comprises an anti-neurogranin antibody, an anti-phosphorylated tau T181 antibody, and an anti-CD171 antibody.

[0145] These and other embodiments of the present invention will readily suggest themselves to those of ordinary skill in the art in light of the disclosure herein. EXAMPLES

[0146] The present invention will be further understood by reference to the following examples, which are intended to be merely exemplary of the present invention. These examples are provided only to illustrate the present invention. The present invention is not limited in scope by the exemplary embodiments, which are intended only as illustrations of single aspects of the invention. Any methods that are functionally equivalent are within the scope of the present invention. Various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0147] Example 1: Methods for quantifying cell-specific biomarkers in exosomes Exosomes in body fluids are a new resource for the diagnosis of various diseases, since they carry physiological and pathological materials (proteins, metabolites, RNA, small molecules, etc.) of the mother cells from which they originated and the microenvironment close to the mother cells (Figure 1A). Therefore, quantification of such pathological materials in exosomes is the basis for next-generation diagnostics.

[0148] However, current methods for exosome analysis are complex and expensive, limiting their use in routine diagnostic testing. Quantification of exosomal material typically involves isolating target exosomes, lysing or permeabilizing the exosomes to release exosomal cargo, and transferring them to a suitable assay vessel for analysis. The multiple steps involved in current procedures for exosome analysis are labor-intensive and produce large sample-to-sample and assay-to-assay variability.

[0149] Here we describe a method to measure exosomal material derived from the original mother cell at the surface of the exosome, thus removing the need for a lysis or permeabilization step: exosome isolation and analysis are performed on the same solid support without transferring the sample to another assay vessel, preventing loss of material.

[0150] As shown in Figure 1B, the method is carried out using antibodies against at least two different markers on the surface of exosomes, which may include exosome membrane proteins and / or adsorption markers. For example, a primary antibody against an exosome marker, such as a membrane marker (CD81, SNAP25, CD171, EAAT1, OMG, etc.) or an adsorption marker (tau, NRGN, GFAP, MBP, etc.), is immobilized on an ELISA plate, allowing the capture of exosomes bearing that marker. A secondary antibody against another exosome marker of interest, which may also be a membrane marker (CD81, SNAP25, CD171, EAAT1, OMG, etc.) or an adsorption marker (tau, NRGN, GFAP, MBP, etc.), is then used to screen the exosomes captured by the primary antibody.

[0151] Example 2: Detection of cytosolic non-membrane proteins on the surface of exosomes Cytosolic non-membrane proteins were detected on the surface of exosomes as follows: Neural-specific anti-SNAP25 (Figures 2A-2C) or control mouse IgG (Figures 2D-2F) were immobilized on white ELISA plates. Various volumes (10, 2.5, and 0 μL) of plasma were suspended in PBS, 0.1% tween-20, or 0.1% Triton-X100 PBS in a final volume of 40 μL and applied to the ELISA plate. After overnight incubation at 4°C, unbound material was removed, and then biotin-labeled antibodies against the exosome surface marker CD81, and two different soluble cytosolic proteins (GAPDH and CTSD) were applied to the ELISA plate. For PBS samples, antibodies were suspended in PBS without tween-20. For tween-20 and Triton-X100 samples, labeled antibodies were suspended in 0.1% tween-20 PBS. A conventional chemiluminescence ELISA procedure was then performed to determine the relative light units (RLU).

[0152] Since GAPDH and CTSD are cytosolic proteins, we hypothesized that they would be detected only after permeabilization of exosomes with tween-20 or Triton-X100, whereas the membrane protein CD81 would appear in both cases. However, as shown in Figures 2A-2C, all three markers were detected in a plasma volume-dependent manner, whereas no signal was detected in the mouse IgG-immobilized control ELISA plate. These results clearly demonstrated that soluble cytosolic proteins such as GAPDH and CTSD may be detected on the surface of exosomes. These results further demonstrated that the methods and compositions of the present invention are useful for detecting biomarkers on vesicles captured on a solid support, where the vesicles have not been lysed or permeabilized, using detection agents that selectively bind to the biomarkers.

[0153] Example 3: Origin of cytosolic non-membrane proteins on the surface of exosomes The origin of cytosolic non-membrane proteins on the surface of exosomes was determined as follows. Antibodies against exosome surface marker CD81, neuronal surface marker SNAP25, astrocyte surface marker (EAAT1), or oligodendrocyte surface marker (OMG) were immobilized on white ELISA plates. 10 μL of plasma suspended in 40 μL of PBS was applied to the ELISA plate. After overnight incubation at 4 °C, unbound material was removed, and then biotin-labeled antibodies against exosome surface marker CD81, general cytosolic marker GAPDH, cytosolic protein in neurons (NRGN), oligodendrocytes (MBP), and astrocytes (GFAP) were applied to the ELISA plate. A control without antibody was also included (tPBS). Then. A conventional chemiluminescence ELISA procedure was performed and the relative light units (RLU) were determined (Figure 3A-3D).

[0154] Both CD81 and GAPDH were detected in all ELISA plates (left), indicating that each ELISA plate captured exosomes with GAPDH attached to the surface. In the CD81 plate (Figure 3A), which captured whole exosomes, MBP and GFAP were not detected. NRGN was detected, but the values ​​were very low. In the SNAP25 plate (Figure 3B), where neuron-derived exosomes were captured, neuronal-specific NRGN was detected, but MBP and GFAP were very low. In the EAAT1 plate (Figure 3C), where astrocyte-derived exosomes were captured, astrocyte-specific GFAP was detected, but NRGN and MBP were also detected. In the OMG plate (Figure 3D), where oligodendrocyte-derived exosomes were captured, oligodendrocyte-specific MBP was detected, but NRGN and GFAP were also detected.

[0155] These results indicated that the soluble markers on the surface of exosomes originate from the mother cell before they are released to the outside or from the microenvironment immediately after they are released from the mother cell. These results further indicated that the methods and compositions of the present invention are useful for detecting biomarkers on vesicles that have not been lysed or permeabilized.

[0156] Example 4: Quantification of Marker Proteins The marker proteins of the present invention were quantified as follows: Because the immobilized antibody and biotin-labeled detection antibody bind to two different target molecules (such as CD81 and SNAP25), the detectable target standard must contain both antigens in a single molecule. Therefore, purified or recombinant proteins cannot be applied as a quantification standard, unlike traditional ELISA. Therefore, we first screened various plasma samples to find a suitable sample that contains a large amount of target exosomes. By allocating 100 units / mL to this plasma, dilution studies were performed on SNAP25, EAAT1, and OMG plates (Figures 4A-4D).

[0157] As shown in Figures 4A and 4B, the ELISA measurements RLU (x-axis) of NRGN (Figure 4A) and Tau protein (Figure 4B) on anti-SNAP25 immobilized ELISA (neuron-derived exosome, NDE) plates showed linear changes. Similarly, GFAP (Figure 4C) and MBP (Figure 4D) on anti-EAAT1 (astrocyte-derived exosome, ADE) and anti-OMG (oligodendrocyte-derived exosome, ODE) immobilized plates, respectively, showed linear changes. Thus, RLU can be converted to units / mL. These results indicated that the methods and compositions of the present invention are useful for detecting and measuring biomarkers on vesicles that are not lysed or permeabilized.

[0158] Example 5: Screening of exosome-adsorbed biomarkers Exosome-adsorbed biomarkers were identified as follows. Control mouse IgG, mouse anti-human CD81, or mouse anti-human synaptosomal associated protein 25 (SNAP25) were immobilized on ELISA plates, and pooled human plasma was applied to all ELISA wells (Figure 5A-5C). ELISAs were performed using various detection antibodies (all biotinylated) to detect not only antibodies against exosomal membrane proteins (CD81, CD171, CD63, and SNAP25), but also various non-membrane proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cathepsin D (CTSD), neurogranin (NRGN), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), tau, microtubule-associated protein tau (tau) and phosphorylated tau T181, and amyloid b). 1-42 As a negative control, PBS without any biotinylated antibodies was used.

[0159] As shown in FIG. 5A, the ELISA signal (RLU) was clearly positive on both anti-CD81 and anti-SNAP25-immobilized plates, while almost no signal was detected on the control mouse IgG-immobilized plate, indicating that the system is CD81 or SNAP25 specific. As shown in PBS, the non-specific signal without detection antibody was very low, indicating that the assay is detection antibody specific. Detection of membrane proteins (CD81, CD171, CD63, and SNAP25) confirmed that the assay captured exosomes (FIGS. 5A and 5B). Surprisingly, non-membrane proteins such as GAPDH, CTSD, NRGN, MBP, and GFAP were also detected (FIG. 5C). These results indicated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers on vesicles that are not lysed or permeabilized.

[0160] Example 6: Diagnosis of Alzheimer's Disease in Humans The methods and compositions of the present invention were used to diagnose Alzheimer's disease in biological samples from humans as follows: anti-CD171 was immobilized on an ELISA plate. Twelve samples of EDTA plasma from subjects with Alzheimer's disease (AD) and age- and sex-matched controls were applied to the ELISA wells, and antibodies against CD81, CTSD, NRGN, and p-tau T181 were used as detection antibodies (FIGS. 6A-6E).

[0161] As shown in Table 1 below, NRGN values ​​were lower in AD samples than controls, and T181 values ​​were higher in AD samples than controls. Chi-square test showed that these two groups were significantly different (p=0.002 for NRGN, p=0.0001 for T181) as shown in Table 1. These results indicated that the methods and compositions of the present invention can be used to diagnose Alzheimer's disease. [Table 1]

[0162] Example 7: Stability of plasma exosomal biomarker levels The stability of plasma exosome biomarker levels was determined as follows: anti-CD81, anti-CD171, anti-SNAP25, anti-EAAT1, and anti-OMG were immobilized on separate ELISA plates. EDTA plasma was obtained from 7 control subjects every week for 2-3 weeks and applied to ELISA wells. For detection of exosome membrane targets (Figures 7A-7F), anti-CD81 probes were used on CD81 plates (total exosomes, TE); anti-CD171 probes were used on CD81 plates (CD171-based NDE, cNDE); anti-SNAP25 probes were used on CD81 plates (SNAP25-based NDE, sNDE); anti-CD81 probes were used on EAAT1 plates (ADE); and anti-CD81 probes were used on OMG plates (ODE). For the detection of exosome surface protein targets (Figures 7G-7L), GFAP probe on EAAT1 plates, MBP probe on OMG plates, NRGN probe on SNAP25 plates, Tau probe on SNAP25 plates, and NRGN probe on CD171 plates, respectively.

[0163] Each value was highly dispersed among the test subjects (except for tau on the SNAP25 plate), but the values ​​were highly consistent within the same individual, except for NRGN on the SNAP25 plate. These results indicated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers on vesicles that are not lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in subjects. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing diseases or disorders.

[0164] Example 8: Plasma on anti-NRGN immobilized ELISA plates Plasma exosome biomarkers were identified as follows: Anti-NRGN or control mouse IgG was immobilized on ELISA plates. EDTA plasma or PBS alone was applied to ELISA wells. Antibodies against NRGN, CD171, SNAP25, CD81, EAAT1, OMG were used for detection, and a PBS control was used for comparison.

[0165] As shown in FIG. 8B, the plasma applied to the anti-NRGN immobilized ELISA plate tested positive for NRGN, CD171, SNAP25, and CD81, but not EAAT1 and OMG, indicating that the NRGN of the NAP25 plate is specific to neurons, but not to astrocytes or oligodendrocytes (FIG. 8B). These results indicated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers on vesicles that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in subjects. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing diseases or disorders.

[0166] Example 9: Microglial targeting of exosomal biomarkers Microglial targeting of exosome biomarkers was performed as follows: anti-CD11b antibody or control mouse IgG was immobilized on ELISA plates. EDTA plasma from seven different donors (IR1-IR7) or PBS alone was applied to ELISA wells. Antibodies against GFAP (Figure 9B), MBP (Figure 9C), or NRGN (Figure 9D) were used as detection antibodies.

[0167] As shown in Figures 9B-9D, microglial analysis was limited to CD11b, although no microglia-specific antibodies were available and anti-CD11b binds to both microglia and peripheral macrophages. +This can be done by assessing brain-derived proteins on the surface of exosomes. Donors 4 (GFAP) to 5 (MBP) showed higher positive signals than the three controls (PBS on IgG plates, plasma on IgG plates, and PBS on CD1b plates), indicating an interaction of microglia with astrocytes and oligodendrocytes in these donors. However, none of the 7 subjects showed NRGN (Figure 9D).

[0168] These results indicated that the methods of the present invention are useful for detecting biomarkers (GFAP, MBP, NGRN) of microglia-derived exosomes. These results indicated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers on vesicles that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in subjects. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing diseases or disorders.

[0169] Example 10: Detection of secreted proteins (cytokines IL1b and IL34 and chemokine CX3CL1) on the surface of brain-derived exosomes Secreted proteins (cytokines and chemokines) on the surface of brain-derived exosomes were detected as follows: Various concentrations of standard plasma (FIGS. 10A, 10C, and 10E) and fixed dilutions of seven control plasma samples (1 / 8 dilution for IL1b and IL34 and 1 / 4 dilution for CX3CL1) and PBS controls were applied to ELISA plates containing immobilized antibodies (anti-SNAP25, anti-EAAT1, anti-OMG, and control mouse IgG) to capture NDE, ADE, and ODE, respectively. Control mouse IgG was used to assess nonspecific binding.

[0170] As shown in Figures 10A, 10C, and 10E, normal plasma showed a volume-dependent increase in IL1b, IL34, and CX3CL1 levels on SNAP25, EAAT1, and OMG ELISA plates compared to control IgG-immobilized plates. IL1b and IL34 on anti-OMG-immobilized plates were saturated at 2.5% plasma (see Figures 10A and 10C).

[0171] Plasma samples (IR1, IR4, IR6) were negative for IL1b, IL34, and CX3CL1, as was the negative control PBS alone. However, IR2 was positive for IL1b on the SNAP25 ELISA plate, and IR3 was positive for CX3CL1 on the EAAT1 and OMG ELISA plates. IR5 was positive for IL1b levels and IL34 on the SNAP25 plate, as well as detectable levels of CX3CL1 on the EAAT1 and OMG ELISA plates. IR7 was positive for IL1b on all ELISA plates, IL34 on the SNAP25 ELISA plate, and showed detectable levels of CX3CL1 on both the EAAT1 and OMG ELISA plates.

[0172] These results indicated that the method of the present invention is useful for detecting cytokines (IL1b and IL34) and chemokines (CX3CL1) on the surface of exosomes (SNAP25+NDE, EAAT1+ADE, and OMG+ODE). These results also indicated that the cytokine and chemokine profiles are unique to each subset of brain-derived exosomes (NDE, ADE, and ODE). These results indicated that the method and composition of the present invention is useful for identifying, detecting, and measuring biomarkers on vesicles that are not lysed or permeabilized. These results further suggested that the method and composition of the present invention may be useful for diagnosing neurological disorders in subjects. These results further suggested that the method and composition of the present invention may be useful for diagnosing diseases or disorders.

[0173] Example 11: Detection of membrane proteins and receptors on the surface of exosomes Multiple membrane proteins and receptors on the surface of exosomes were detected as follows. Various antibodies were suspended in coating buffer and applied to the ELISA plate. After 1 hour of incubation, each well was washed once, then blocking solution (0.5% BSA in blocker casein) was added, and incubation was continued for another hour. Each well was washed twice, then plasma or buffer control (phosphate-buffered saline, PBS) was added, and incubation was continued overnight in the refrigerator. Each well was washed twice, then biotinylated anti-CD81 or anti-SNAP25 was added, and incubation was continued for 1 hour. Each well was washed twice, then streptavidin-horseradish peroxidase was added, and incubation was continued for another 30 minutes. Each well was washed three times, then SuperSignal substrate was added, and the chemiluminescent signal was measured in a luminometer.

[0174] As shown in Table 2 below, multiple cytosolic, secreted, and membrane proteins and receptors were detected on the surface of exosomes. Table 2. Immobilized antibodies probed with CD81 / SNAP25. [Table 2-1] [Table 2-2] [Table 2-3]

[0175] To confirm the proteins detected in Table 2 above, two series of experiments were performed as follows: Positive antibodies were biotinylated and used in an ELISA where PBS or plasma was applied to anti-SNAP25- or control IgG-immobilized plates. All positive antibodies from Table 2 were also positive in this series of experiments, as shown in Table 3 below. Table 3. Anti-SNAP25 or control IgG-immobilized plates probed with various biotinylated antibodies. [Table 3-1] [Table 3-2] [Table 3-3]

[0176] These results indicated that the methods of the present invention are useful for detecting cytosolic, secreted, and membrane proteins and receptors on the surface of exosomes. These results indicated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers in exosomes that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in a subject. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing a disease or disorder.

[0177] Example 12: Quantitation standards for multi-target ELISA. ELISA assays require a quantitative standard to determine the level of a target molecule in a test sample. The exemplary assay of the present invention utilizes two antibodies against different target molecules. Thus, purified or recombinant proteins are not applicable as a standard for such ELISA assays. Therefore, various plasma samples were screened for having high values ​​of exosomes, including NDE, ADE, and ODE positive exosomes, and one particular plasma sample was selected. This plasma sample was then used as a quantitative standard. As shown in FIG. 11, this plasma sample performed well as a standard for multiple ELISAs of the present invention. A four parameter logistic regression analysis was used to generate standard curves for each target.

[0178] Example 13: Differential diagnosis of Alzheimer's disease and mild cognitive impairment in humans The method and composition of the present invention were used for the differential diagnosis of Alzheimer's disease and mild cognitive impairment in biological samples from humans as follows: Anti-SNAP25 antibodies were immobilized on ELISA plates. Eight samples of EDTA plasma from subjects with Alzheimer's disease (AD, n=4) or mild cognitive impairment (MCI, n=4) and eight age- and sex-matched controls were applied to ELISA wells, and antibodies against 16 cytosolic, secreted, and membrane proteins and receptors were used as detection antibodies ( FIG. 12 ).

[0179] As shown in Figure 12, the duplicate variation of all 16 biomarkers was small. Figure 13 shows the standard curves of all 16 biomarkers. Quantification of target surface protein biomarkers is shown in Figure 14. As shown in Figure 14, AchE, LAMP1, and CCL2 levels were significantly higher in MCI compared to control sample levels. These results indicated that the method and composition of the present invention can be used to diagnose MCI and for the differential diagnosis of Alzheimer's disease and MCI.

[0180] To account for the variation in CD81 levels in plasma samples, the remaining 15 exosome biomarker levels were normalized with CD81 levels. As shown in Figure 15, AchE and LAMP1 levels were significantly greater in plasma samples from subjects with MCI and AD+MCI compared to control samples. Furthermore, NGRN, GYS, and DR6, HSP, IL12b, and BACE levels were significantly different between AD, MCI, AD+MCI, and control samples. These results indicated that the methods and compositions of the present invention can be used to diagnose AD, MCI, and for the differential diagnosis of AD and MCI.

[0181] In another series of experiments, the target biomarker levels of the plasma samples assayed above were normalized to SYT levels. Because the neural derived exosomes were captured by SNAP25-immobilized plates, SYT normalization provided a secondary normalization of neuronal markers. As shown in FIG. 16, AchE / SYT, LAMP1 / SYT, REST / SYT, CCL2 / SYT, HSP / SYT, and IL12b / SYT levels were significantly different between AD, MCI, AD+MCI, and control samples. These results indicated that the methods and compositions of the present invention can be used to diagnose AD, MCI, and for the differential diagnosis of AD and MCI. These results further suggested that the methods and compositions of the present invention would be useful for diagnosing diseases or disorders.

[0182] Example 14: Enumeration of brain-derived exosomes in human subjects with Parkinsonism-plus syndrome The methods and compositions of the present invention were used to enumerate plasma levels of neuron-derived, astrocyte-derived, and oligodendrocyte-derived exosomes (NDE, ADE, and ODE, respectively) obtained from patients with Parkinson's disease (PD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and control subjects as follows: A total of 52 subjects were included in the study, including 15 patients with PD (age 46-79, mean ± SD 64.7 ± 10.8, 9 males and 6 females), 15 patients with MSA (age 44-74, mean ± SD 63.3 ± 8.16, 10 males and 5 females), 7 patients with PSP (age 58-81, mean ± SD 71.6 ± 9.8, 2 males and 5 females), and 15 disease controls (age 47-77, mean ± SD 64.7 ± 8.5, 10 males and 5 females), and these groups were age-matched.

[0183] Plasma samples were obtained by venipuncture and a total of 8 mL of blood was collected into EDTA-containing tubes. After collection, plasma was separated by centrifugation at 2,000 g for 15 min, placed into polypropylene vials, and stored at −80°C until analysis.

[0184] Various concentrations of antibodies were suspended in 1x coating buffer (BioLegend, San Diego, CA) and 50 μL was applied to each well of a white flat-bottom strip microplate (Sigma-Aldrich, St. Louise, MO). After 1 hour of incubation at room temperature, each well was washed with 1x washing buffer (BioLegend), followed by the addition of 75 μL of blocker casein (Thermo Fisher Scientific, Waltham, MA) supplemented with 1% blocker bovine serum albumin (BSA) (Thermo Fisher), and incubation was continued for another hour. After each well was washed, each plate was immediately used for enzyme-linked immunosorbent assay (ELISA).

[0185] Forty microliters of each standard, control, and plasma sample were applied to each well of the ELISA plate and incubated overnight in a refrigerator. The next day, each well was washed, followed by the addition of 40 μL of biotinylated detection antibody suspended in phosphate-buffered saline, pH 7.4 (PBS) supplemented with 0.1% Tween-20 (Sigma-Aldrich), 1% blocker BSA (Thermo Fisher), and 8 μg / mL mouse IgG (Equitech-Bio, Kerrville, TX) and incubation for 1 h at room temperature. After incubation, each well was washed, followed by the application of 40 μL of streptavidin-horseradish peroxidase (SA-HRP) (Thermo Fisher) suspended in PBS supplemented with 0.1% Tween-20, 1% blocker BSA (Thermo Fisher), and 5% blocker casein. After 30 min of incubation, each well was washed and then 50 μL of substrate (SuperSignal, Thermo) was applied. After 4 min of incubation, the chemiluminescent signal (Relative Light Unit, RLU) was determined with a luminometer (ANSH Labs, Webster, TX).

[0186] Our sandwich ELISA used a combination of two different target antibodies, such as anti-CD81 and anti-SNAP25 (synaptosomal associated protein 25), so purified or recombinant proteins could not be applied as quantification standards. Therefore, we first screened various plasma samples obtained from commercial sources (Innovative Research, Novi, MI, and EquiTech Enterprise, Kerrville, TX) to find plasma samples with high concentrations of all NDE, ADE, and ODE. By assigning this plasma 100 units / mL, we performed dilution tests with each ELISA and obtained RLU at each dilution. Then, we converted the RLU of each sample to units / mL using a four-parameter logistic analysis.

[0187] To find antibodies against NDE, ADE, and ODE in plasma, various dilutions of plasma were applied to ELISA plates immobilized with anti-CD81 monoclonal antibody (clone JS-81, BD Biosciences, Sparks, MD) to capture total exosomes as well as control mouse IgG (Santa Cruz Biotechnology, Dallas, Texas) used as a negative control. After extensive washing steps to remove unbound material from each well, biotinylated antibodies against various targets were applied and ELISA was performed as described above. For biotin labeling, we used EZ-Link Sulfo-NHS-LC-biotin (Thermo, Rockford, USA). From this, we found that monoclonal antibodies against SNAP25 (clone B-8, Santa Cruz Biotechnology), polyclonal antibodies against excitatory amino acid transporter 1 (EAAT1) (= glutamate aspartate transporter (GLAST)) and oligodendrocyte myelin glycoprotein (OMG) (Bioss Antibodies, Woburn, MA) showed very strong signals on CD81-immobilized plates but not on control mouse IgG-immobilized plates. After finding suitable antibodies, anti-SNAP25, anti-EAAT1, and anti-OMG were immobilized on ELISA plates and probed with biotinylated anti-CD81 (clone 1.3.3.22, LS Bio, Seattle, WA). We used SNAP25 and polyclonal antibodies against excitatory amino acid transporter 1 (EAAT1) (= glutamate aspartate transporter (GLAST)) and oligodendrocyte myelin glycoprotein (OMG) (Bioss Antibodies, Woburn, MA) to detect SNAP25 and OMG, respectively. + , CD81 + ELISA results for NDE and EAAT1 + , CD81 + ADE and OMG for ELISA results + , CD81 + The ELISA results were reported as ODE (FIG. 17).

[0188] Logarithmic transformation of BDE measurements was used appropriately for certain analyses to reduce the influence of undue skew and outliers in the analysis of group differences and regressions between parameters. Mean differences in BDE between diagnostic groups and regressions between BDE levels and clinical parameters were assessed by t-tests (Excel). Because of the limited number of samples, additional nonparametric Mann-Whitney U-tests were performed (Prism, GraphPad, La Jolla, CA). We also derived receiver operating characteristic (ROC) curves for the diagnosis of PD using the levels of BDE as predictors and estimated the area under the curve (AUC; AUC = 0.5 would indicate no difference and AUC = 1 would indicate a diagnostic test) to assess the diagnostic ability of each predictor (plasma levels of NDE, ADE, and ODE). The significance level was set at p < 0.05.

[0189] ELISA specificity was determined as follows. ELISA plates were immobilized with either target antibodies (anti-SNAP25, anti-EAAT1, and anti-OMG) or control IgG, and serial dilutions of plasma were applied. As shown in Figures 18A-18C, the ELISA measurements (RLU) increased proportionally with the volume of plasma on the target antibody-immobilized ELISA plates (filled circles; ●) but not on the control IgG-immobilized plates (open triangles; △). We also tested the ELISA without biotin antibody to evaluate nonspecific binding of SA-HRP, but no signal was detected. Nonspecific IgG binding was eliminated in the assay because the biotin antibody was mixed with a 10-fold volume of control mouse IgG.

[0190] Next, the performance of the ELISA assays was determined. Intra-assay precision was determined by ELISAs performed twice using 52 different subjects. As shown in Figures 19A-19C, duplicate variation was very small in all three ELISAs (r for NDE, ADE, and ODE, respectively). 2= 0.9644, 0.9541, and 0.9791 and slopes = 1.08, 0.99, and 1.01). Inter-assay precision was determined using three control plasma samples run nine times. As shown in Figures 20A-20C, the CV% for high and mid range plasmas was <= 20%, while the very low range plasmas (<10 units / mL for NDE, <1 unit / mL for ADE and ODE) were 35-63%. Since an ELISA plate was constructed for each experiment, the CV% was acceptable.

[0191] In this ELISA, the quantification of target plasma samples is based on the dilution curve of standard plasma. This calculation is applicable when the dilution curves between samples and standards are the same. To verify this hypothesis, dilution linearity was determined by high, medium, and low range plasma samples. As shown in Figures 21A-21C, although the amounts of these three plasmas vary widely by about 1,000-fold, all three plasmas showed NDE, ADE, and ODE linear dilutions with almost identical slopes. As shown in Figures 21A-21C, the linear detection ranges were 0.1-5uL plasma (NDE), 0.01-5uL plasma (ADE and ODE), respectively.

[0192] The limits of detection (LODs) for NDE, ADE, and ODE were 0.519, 0, and 0.062 units / mL, respectively.

[0193] The enumeration of plasma BDEs and comparison between patients and controls was determined as follows: Plasma levels of NDE, ADE and ODE were significantly higher in PD samples than in control samples, respectively (Figures 22A-22C; p=0.003 (t-test), p=0.003 (U-test) for NDE, p=0.028 (t-test), p=0.021 (U-test) for ADE, and p=0.016 (t-test), p=0.008 (U-test) for ODE). When comparing between PD and MSA groups, plasma levels of NDE were significantly higher in patients with PD than those with MSA (Figures 22A-22C; p=0.028 (t-test), p=0.023 (U-test)), whereas no significant differences were found in ADE and ODE. Patients with PSP also showed significantly higher levels of plasma NDEs and ADEs compared to those of controls (Figures 22A-22B; p=0.038 (t-test), p=0.041 (U-test) for NDEs, and p=0.012 (t-test), p=0.025 (U-test) for ADEs).

[0194] Figures 23A-23C show the ROC curves for classification of patients with PD and controls based on plasma NDE, ADE, and ODE levels. The AUCs of the ROC curves for plasma NDE, ADE, and ODE are 0.89, 0.83, and 0.88, respectively, indicating that plasma levels of NDE, ADE, and ODE may be applicable to diagnostic testing of PD with substantial accuracy.

[0195] The correlation between BDE and clinical severity in subjects with PD and MSA was determined as follows. We studied the correlation between plasma BDE levels and clinical severity scores in patients with PD. As shown in Figures 24A-24F, plasma levels of all BDEs (NDE, ADE and ODE) were significantly higher in patients with advanced PD, i.e., those with mRS grade 4 (n=4) (p=0.003 (t-test), p=0.008 (U-test) for NDE, p=0.001 (t-test), p=0.006 (U-test) for ADE, and p<0.001 (t-test), p=0.006 (U-test) for ODE, and those with Horn-Yarr (HY) stage 4 (n=5). ) (p=0.001 (t test), p=0.003 (U test) for NDE, p=0.001 (t test), p=0.003 (U test) for ADE, and p<0.001 (t test), p=0.003 (U test) for ODE. Furthermore, plasma levels in NDE were significantly higher than in controls in both mRS grade 1-2 (n=5) (p=0.001 (t test), p=0.002 (U test)) or HY stage 1-2 (n=4) (p=0.003 (t test), p=0.00 Plasma levels of ODE were also significantly higher, even in mild PD patients with mRS grade 1-2 (p=0.002 (t test), p=0.005 (U test)) or HY stage 1-2 (p=0.011 (U test)), compared to controls. Furthermore, plasma levels of ADE were also significantly higher, even in mild PD patients with mRS grade 1-2 (p=0.023 (U test)), compared to controls. These results indicate that plasma levels of NDE and ODE may be useful as diagnostic biomarkers for PD patients, especially in the early disease stages. Although the levels of ODE fluctuated during the early disease course in mRS and Yahr scores 1-3, the levels of ODE were significantly higher in mRS and Yahr scores 4 than in scores 1-3 (p=0.002 (t-test), p=0.011 (U-test) for mRS, and p=0.004 (t-test), p=0.0024 (U-test) for Yahr).This suggests that ODE may be a potential surrogate biomarker for monitoring disease progression in PD.

[0196] We further studied the correlation between the levels of BDE or the ratios between them (ADE / NDE, ODE / NDE, and ODE / ADE) and various clinical parameters in the PD group (see Table 4). 2 = 0.51, n = 14, p = 0.0041), as well as the ADE / NDE ratio, which was significantly associated with patient disease duration (r 2 = 0.51, n = 15, p = 0.0028), mRS grade (r 2 = 0.47, n = 15, p = 0.0048), and UPDRS Part III score (r 2 = 0.52, n = 14, p = 0.0036) 2 These results also suggest that plasma levels of BDE may be useful for monitoring the severity of patients with PD. Table 4. Correlation between BDE and PD severity. *=r 2 value. [Table 4]

[0197] Next, we investigated the correlation between the levels of BDE and various clinical parameters in the MSA group. As shown in Figures 26A-26D, the plasma levels of ODE did not correlate with the mRS grade or disease duration in patients with MSA as a whole, or with the ICARS score in MSA-C, but showed a significant correlation with the UPDRS part III score (r 2 =0.57, n=6, p=0.048).

[0198] These results showed that the plasma levels of both NDE and ODE were significant even in the early stages of PD compared to controls, and that the levels of ODE increased as disease severity progressed in PD. These results further indicated that the plasma levels of BDE may be diagnostic and severity level biomarkers for PD and related diseases. These results indicated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers of exosomes that are not lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological diseases in subjects. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing diseases or disorders.

[0199] Example 15: Detection of pathological forms of α-synuclein on the surface of neuron-, astrocyte-, and oligodendrocyte-derived exosomes. Detection of pathological forms of α-synuclein on the surface of neuron-, astrocyte-, and oligodendrocyte-derived exosomes was performed as follows. 40 uL of EDTA plasma samples (1 / 4 dilution in PBS) were added to anti-SNAP25-, EAAT1-, and OMG-immobilized ELISA plates capturing neuron- (NDE), astrocyte- (ADE), and oligodendrocyte- (ODE) derived exosomes. After washing each well, biotinylated antibodies against aggregated forms (pathological forms) of α-synuclein (Syn-F1, Syn-O2 from BioLegend) were applied and further incubation for 1 h was continued, after which the usual ELISA procedure was performed (HRP reaction, Supersignal, measurement of chemiluminescent signal). As shown in Figure 27A-27F, pathological α-synuclein was detected in patients with PD, MSA, and PSP. Statistical significance was based on the Mann-Whitney U test.

[0200] These results demonstrated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers in exosomes that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in a subject. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing a disease or disorder.

[0201] Example 16: Detection of dopamine receptor D2 (DRD2) on the surface of neuron-derived exosomes. Detection of dopamine receptor D2 (DRD2) on the surface of neuron-derived exosomes was performed as follows. Anti-SNAP25 (neuron marker), anti-DRD2, and control mouse IgG were immobilized on an ELISA plate. Human EDTA plasma or buffer control (phosphate buffered saline, PBS) was then applied to the ELISA plate, followed by reaction with biotinylated anti-SNAP25. As shown in Figure 28A, plasma was positive on both the anti-SNAP25 and anti-DRD2 antibody ELISA plates, whereas PBS was not.

[0202] In another set of experiments, anti-SNAP25 and control mouse IgG were immobilized on ELISA plates and applied to human EDTA plasma or PBS ELISA plates, then reacted with biotinylated antibodies against SNAP25, CD81 (a common marker for exosomes), DRD2, and PBS controls. As shown in Figure 28B, plasma was positive with anti-SNAP25, CD81, and DRD2 probes on the anti-SNAP25 antibody ELISA plate.

[0203] Plasma dilution studies were performed as follows: anti-SNAP25 was immobilized on an ELISA plate, and serial dilutions of human EDTA plasma samples (three different donors) were applied to the ELISA plate, and then reacted with biotinylated anti-DRD2 and anti-CD81. As shown in Figures 29A and 29B, the slopes of plasma dilution were almost the same among the three donors. Thus, the ratio of DRD2 / CD81 was constant (see Figure 29C).

[0204] In another series of experiments, DRD2 was detected in neuron-derived exosomes in plasma samples from humans with neurological diseases. Standard plasma was assigned 100 units / mL, and then the ELISA measurements of each sample (DRD2 probe on SNAP25 plate, and CD81 probe on SNAP25 plate (NDE)) were converted to units / mL (U / mL) using a dilution curve. Plasma samples from controls, 15 each of Parkinson's disease (PD), multiple system atrophy (MSA), and 7 patients with progressive supranuclear palsy (PSP) were analyzed. PD and MSA patients were further classified by disease severity (mRS score). As shown in Figures 30A-30G, the levels of DRD2 in PD were significantly lower than controls, while the levels of NDE were significantly higher in PD than controls. Thus, the ratio (DRD2 / NDE) showed a more significant difference between PD and control samples. Furthermore, a decrease in DRD2 was shown in mild cases of PD (mRS score 1+2), indicating that it may be used as a marker for early screening of PD patients as well as monitoring PD progression. The ROC curve and area under the curve (AUC) for DRD2 levels between PD and control samples are shown in Figure 31.

[0205] These results demonstrated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers on vesicles that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in subjects. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing diseases or disorders.

[0206] Example 17: Detection of DAT-positive extracellular vesicles DAT-positive extracellular vesicles were captured and analyzed on an ELISA plate as follows. Plasma samples obtained from three different human control donors were diluted in PBS. The samples were applied to a pair of enzyme-linked immunosorbent assay (ELISA) wells, one of which was immobilized with an anti-DAT mouse monoclonal antibody (Figure 32, ●▲■) and the other well was immobilized with control mouse IgG (Figure 32, ○△□). + After extracellular vesicles (EVs) were captured in ELISA wells, unbound material was removed by extensive washing and then probed with labeled anti-CD81 to identify DAT. + -CD81 + Double positive signal (=DAT + We quantified the DAT protein (EV) (Figure 32A), and analyzed the DAT protein with antibodies against monomeric (Figure 32B), oligomeric α-synuclein (SNCA) (Figure 32C), and tyrosine hydroxylase (TH) (Figure 32D). + SNCA and TH on the surface of EVs were quantified.

[0207] As shown in FIG. + All of the plasma samples in the ELISA wells showed dose-dependent signal changes, whereas those in the control IgG ELISA wells showed no significant changes in DAT + These were much lower than those in the ELISA wells, 10-100 times lower. More importantly, the slopes of the three plasma samples were very similar, indicating that the dilution curve of one particular plasma can be used as a universal quantification standard.

[0208] These results indicate that the method and composition of the present invention are useful for capturing and detecting DAT-positive extracellular vesicles.These results further indicate that the method and composition of the present invention are useful for identifying, detecting and measuring biomarkers on vesicles that are not lysed or permeabilized.These results further suggest that the method and composition of the present invention will be useful for diagnosing neurological diseases or disorders in subjects.

[0209] Example 18: Detection of SNAP25-positive, EAAT1-positive, and OMG-positive extracellular vesicles SNAP25-positive, EAAT1-positive, and OMG-positive extracellular vesicles were captured and analyzed on ELISA plates as follows: Plasma samples obtained from control donors were applied to ELISA plates on which antibodies against SNAP25, EAAT1, and OMG had been previously immobilized. After extensive washing of the captured extracellular vesicles (EVs), EVs were eluted by incubation with pH 12.5 elution buffer for 5 min and immediately neutralized. These samples were applied to nanoparticle tracking analysis (NanoSight).

[0210] As shown in Figure 33, EVs were detected in all samples with a size range of 100-400 nm. Furthermore, the EV size of SNAP25 was smaller than that of EAAT1 and OMG.

[0211] These results showed that the methods and compositions of the present invention are useful for capturing and detecting SNAP25-positive, EAAT1-positive, and OMG-positive extracellular vesicles. These results further showed that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers on vesicles that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological diseases or disorders in subjects.

[0212] Example 19: Detection of biomarkers on the surface of exosomes Multiple membrane biomarkers were detected on the surface of exosomes as follows: Various antibodies were suspended in coating buffer and applied to the ELISA plate. After 1 hour of incubation, each well was washed once, then blocking solution (0.5% BSA in blocker casein) was added, and incubation was continued for another hour. Each well was washed twice, then plasma or buffer control (phosphate-buffered saline, PBS) was added, and incubation was continued overnight in the refrigerator. Each well was washed twice, then biotinylated anti-EAAT1, anti-SNAP25, anti-OMG, or anti-CD11b was added, and incubation was continued for 1 hour. Each well was washed twice, then streptavidin-horseradish peroxidase was added, and incubation was continued for another 30 minutes. Each well was washed three times, then SuperSignal substrate was added, and the chemiluminescent signal was measured in a luminometer.

[0213] As shown in FIG. 35, multiple biomarkers were detected on the surface of SNAP25+, EAAT1+, OMG+, and CD11b+ exosomes.

[0214] As shown in Figure 34, all three control plasmas exhibited particles ranging in size from 50-400 nm. These results demonstrated that the particles captured by the method described in Example 17 above were extracellular vesicles.

[0215] These results demonstrated that the methods of the present invention are useful for detecting biomarkers on the surface of exosomes. These results demonstrated that the methods and compositions of the present invention are useful for identifying, detecting, and measuring biomarkers in exosomes that have not been lysed or permeabilized. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing neurological disorders in a subject. These results further suggested that the methods and compositions of the present invention may be useful for diagnosing a disease or disorder.

[0216] Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are intended to fall within the scope of the appended claims.

[0217] All references cited herein are hereby incorporated by reference in their entirety.

Claims

1. a) contacting a solid support containing a capture agent bound to the solid support with a collected biological sample containing vesicles under conditions such that the capture agent selectively binds to a first biomarker, if present on the vesicles, thereby capturing vesicles bearing the first biomarker on the solid support, wherein the first biomarker is selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein and an oligodendrocyte-specific protein; b) separating the solid support from the biological sample; and c) detecting a second biomarker on the vesicles captured on the solid support using a detection agent that selectively binds to the second biomarker, where the vesicles have not been lysed or permeabilized, and the second biomarker is selected from the group consisting of a neuron-specific protein, an astrocyte-specific protein, a microglia-specific protein, and an oligodendrocyte-specific protein; A method comprising:

2. The method of claim 1, wherein the first biomarker and the second biomarker are membrane-bound or adsorbed proteins of a vesicle.

3. The method of claim 1, wherein the first biomarker or the second biomarker is an exosome surface marker.

4. 4. The method of claim 3, wherein the exosome surface marker is CD81, CD63, CD171, SNAP25, EAAT1, DAT, CD11b or OMG.

5. 2. The method of claim 1, wherein the neuron-specific protein is selected from the group consisting of NRGN, tau, phosphorylated tau, synaptophysin, αβ-42, α-synuclein (SNCA), AchE, LAMP1, REST, SYT, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, Aβ, and BACE.

6. 2. The method of claim 1, wherein the astrocyte-specific protein is selected from the group consisting of glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1).

7. 2. The method of claim 1, wherein the oligodendrocyte-specific protein is selected from the group consisting of myelin basic protein (MBP) and oligodendrocyte myelin glycoprotein (OMG).

8. 2. The method of claim 1, wherein the vesicles captured on the solid support are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes.

9. The method of claim 2, wherein the membrane-bound or adsorbed protein of the vesicle is dopamine transporter (DAT) or tyrosine hydroxygenase (TH).

10. The method of claim 1, wherein the second biomarker is GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau, synaptophysin, SNCA, αβ-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, or PSG1.

11. The method of claim 1, wherein the second biomarker is a pathological form comprising aggregates and / or mutant forms of GAPDH, CTSD, NRGN, MBP, GFAP, tau, phosphorylated tau, synaptophysin, SNCA, αβ-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, or PSG1.

12. The method of claim 1, wherein the second biomarker is selected from the group consisting of cytokines, growth factors, chemokines, interleukins, nociceptin (an opioid peptide), and GnRH.

13. 13. The method of claim 12, wherein the cytokine is selected from the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, TNFSF13, IL12B, and FasL.

14. 10. The method of claim 1, further comprising detecting a receptor protein, a transporter protein, or a membrane protein on the vesicles captured on the solid support.

15. 15. The method of claim 14, wherein the receptor protein is a neurotransmitter receptor, a dopamine receptor, a serotonin receptor, a GABA receptor, a glutamate receptor, an insulin receptor, a tumor necrosis factor receptor, or a neuropeptide receptor.

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