Method and system for detecting central nervous system diseases using multiple biomarkers in peripheral body fluids

Abstract

The present invention provides a method, system, composition and kit for diagnosing central nervous system diseases, and the diagnostic system includes a first detection module that detects central nervous system-derived markers in the biological fluids of a subject, a second detection module that detects central nervous system disease-related markers in the biological fluids of the subject, and a diagnostic module that diagnoses whether the subject is suffering from a central nervous system disease based on the detection results of the central nervous system-derived markers and the central nervous system disease-related markers obtained by the first detection module and the second detection module, respectively.

Description

[Technical field]

[0001] The present invention relates to the technical field of biomedicine, in particular to the technical field of in vitro diagnosis and detection, in particular to a detection method and system using dual markers and / or multiple markers, preferably a simultaneous detection method and system using dual markers and / or multiple markers. [Background technology]

[0002] With the development of science and technology, the detection of biomarkers plays an increasingly important role in the diagnosis, differential diagnosis and monitoring of disease progression.

[0003] Currently, the detection of biomarkers for central nervous system diseases is mainly developed based on the detection of cerebrospinal fluid and direct detection of peripheral body fluids. Detection using cerebrospinal fluid showed good performance because cerebrospinal fluid directly contacts the brain and spinal cord and can better reflect changes in the central nervous system. However, the acquisition of cerebrospinal fluid is invasive and has poor patient acceptance, which has severely limited the further development, clinical translation and widespread application of cerebrospinal fluid biomarkers. Detection using peripheral body fluids has poor performance and mainly has the following two problems. One is that due to the presence of the blood-brain barrier, not all components derived from the central nervous system can be detected in peripheral body fluids, and even biomarkers derived from the central nervous system, especially proteins, that exist in peripheral body fluids have very low abundance; the other is that many central nervous disease-related markers are not specifically expressed in the central nervous system, but are also produced in other peripheral organs and / or systems, so signals derived from the central nervous system are easily influenced by peripheral components.

[0004] And central nervous system diseases have the characteristic of brain region specificity. For example, the main pathological changes in Alzheimer's disease are atrophy of the cerebral cortex, such as the parietal lobe, temporal lobe, and hippocampus, while Parkinson's disease is mainly caused by insufficient dopamine secretion due to lesions or damage to the dopamine circuit in the substantia nigra-striatum of the basal ganglia. Currently, only brain biopsy can specifically detect and analyze markers in different brain regions, and none of the current in vitro detection techniques can distinguish the origin, so there are differences in the diagnostic effects for different diseases, and there is also room for improvement in the accuracy of disease diagnosis.

[0005] Therefore, the focus of research in this field is how to efficiently concentrate and specifically label biomarkers that can reflect changes in central nervous system diseases from peripheral body fluids. Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, there are no systems, methods, or kits in the art that can effectively detect central nervous system diseases using peripheral body fluids. The present application is intended to achieve the effect of effectively recognizing various markers derived from the central nervous system in peripheral body fluids, detecting them in combination with disease-related markers, and simultaneously detecting multiple labels, thereby reflecting central nervous system changes using peripheral body fluid samples without obtaining cerebrospinal fluid.

[0007] That is, the present application proposes, for the first time, a concept and a detection method for diagnosing central nervous system diseases by recognizing central nervous system-derived biomarkers in peripheral body fluids. [Means for solving the problem]

[0008] Specifically, the present application relates to the following: 1. A method for diagnosing a central nervous system disease, comprising: detecting a central nervous system-derived marker in a biological fluid of the subject; detecting a central nervous system disease-related marker in a biological fluid of the subject; and diagnosing whether or not the subject is suffering from a central nervous system disease based on the detection results of the central nervous system-derived marker and the central nervous system disease-related marker. 2. The method according to item 1 above, wherein the detection of the central nervous system-derived marker in the subject's biological fluid and the detection of the central nervous system disease-related marker in the subject's biological fluid are carried out simultaneously. 3. obtaining and pre-treating a biological fluid from the subject to concentrate the biological sample in the biological fluid; Detecting the central nervous system-derived marker in a biological fluid of the subject comprises detecting the central nervous system-derived marker in an enriched biological sample of the subject; 3. The method according to item 1 or 2 above, wherein detecting a central nervous system disease-associated marker in a biological fluid of a subject is detecting a central nervous system disease-associated marker in an enriched biological sample of the subject. 4. Number of biological samples in which both CNS-derived markers and CNS disease-related markers were detected count and / or Measuring particle size of a biological sample in which both a central nervous system-derived marker and a central nervous system disease-related marker have been detected; The method according to any one of items 1 to 3, further comprising a step of diagnosing whether or not the subject is suffering from a central nervous system disease based on the number of the biological samples and / or the particle size of the biological samples. 5. The biological sample is an extracellular vesicle; Preferably, the extracellular vesicles are neuron-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles, or microglia-derived extracellular vesicles.

[0009] 6. The method according to any one of items 1 to 5, wherein the biological fluid is one or more selected from blood, serum, plasma, saliva, urine, lymph, semen, and milk. 7. The method according to any one of items 1 to 6, wherein the central nervous system disease is one or more selected from the group consisting of cerebrovascular disease, spinal cord lesion, central nervous system infection, genetic disease of the nervous system, developmental abnormality of the nervous system, autonomic nervous system disease, demyelinating lesion of the nervous system, epilepsy, and degenerative disease of the nervous system. 8. The cerebrovascular disorder includes cerebral infarction, cerebral hemorrhage, cerebral blood flow deficiency, and transient ischemic attack; The spinal cord pathology includes acute myelitis, polio, and syringomyelia; The central nervous system infection includes encephalitis and meningitis. The nervous system genetic disease includes neurocutaneous syndrome and spinocerebellar ataxia; The nervous system development disorder includes congenital hydrocephalus and cerebral palsy; the autonomic nervous system disorder is autonomic dysfunction; 8. The method according to item 7, wherein the nervous system degenerative disease includes degenerative dementia, movement disorder, and prion disease. 9. The degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB); The method according to claim 8, wherein the movement disorder disease comprises motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), Sydenham's disease, hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). 10. The method according to any one of items 1 to 9, wherein the central nervous system-derived marker is a biomarker derived from a neuron or cell selected from the group consisting of mature neurons, oligodendrocytes, astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate progenitor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons.

[0010] 11. The method according to any one of items 1 to 10, wherein the central nervous system-derived marker and / or the central nervous system disease-related marker are one, two or three types selected from proteins, nucleic acids and lipids. 12.The central nervous system-derived markers include G Protein-Coupled Receptor 162 (GPR162), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1 (HCN1), Gamma-Aminobutyric Acid Type A Receptor Subunit Delta (GABRD), Neuroligin 3 (NLGN3), SHISA9, contactin-1 (CNTN1), NeuN, MAP2, neurofilament-L (NF-L: neurofilament light), neurofilament-M (NF-M: neurofilament medium), and neurofilament-H (NF-H: neurofilament light). heavy), synaptophysin, syntaxin, PSD95, L1CAM, doublecortin, beta III tubulin, NeuroD1, TBR1, stathmin 1, MPZ, NCAM, GAP43, S100, CNPase, olig 1, olig 2, olig 3, MBP, OSP, MOG, SOX10, NG2, GFAP, GLAST (EAAT1), GLT1 (EAAT2), glutamine synthetase synthetase), S100-β, ALDH1L1, CD11b, CD45, Iba1, F4 / 80, CD68, CD40, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, TBR2, MASH1 (Ascl1), vGluT1, vGluT2, NMDAR, glutaminase, glutamine synthetase, GABA transporter 1 (GAT1), GABA.B Receptor 1 (GABA B receptor 1), GABA B Receptor 2 (GABA B receptor 2), GAD65, GAD67, tyrosine hydroxylase (TH), dopamine transporter (DAT), FOXA2, GIRK2, Nurr1, LMX1B, tryptophan hydroxylase (TPH), serotonin transporter, Pet1, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AchE), Preferably, the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system, More preferably, the method according to item 10 or 11, wherein the central nervous system-derived marker is one or more selected from the group consisting of L1CAM, SHISA9, CNP enzyme, and GLT1 (EAAT2). 13. The method according to item 11, wherein the central nervous system disease-related marker is a protein, and the protein is one or more selected from Aβ1-40 (Aβ40) protein, Aβ1-42 (Aβ42) protein, oligomeric Aβ (oligo-Aβ) protein, tau protein, phosphorylated tau (p-tau) protein, α-synuclein protein, α-synuclein phosphorylated at position 129 (pS129) protein, oligomeric α-synuclein (oligomeric α-synuclein, oligo-α-syn) protein, and prion protein. 14. The method according to claim 11, wherein the central nervous system disease-related marker is a nucleic acid, and the nucleic acid is one or more types selected from DNA or RNA that is a biomarker for Alzheimer's disease (AD), and DNA or RNA that is a biomarker for Parkinson's disease (PD) and parkinsonian syndrome (e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB)). 15. The DNA or RNA that is a biomarker for AD is selected from the group consisting of the genes UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3, RNF7, RPLP0, SPARC, WTAP, HNRNPU, LINC00265, INMT, SLC35E2, CT60, SYNCRIP, RGS2, SMC6, ARSA, SPDYE7P, SMIM17, TRAF3IP2-AS1, KCNC2, SLC24A1, HLCS, GOSR1, MN1, and M The method according to claim 14, comprising DNA or RNA associated with GAT5, NBPF14, FBXO31, WDR52, TBC1D2B, ZNF648, NBPF16, PAGR1, AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1, BPTF, FLG, POM121L10P, NXPH3, H3F3A, SH3TC2, GGCX, and GREB1.

[0011] 16. The method according to claim 14, wherein the DNA or RNA that is a biomarker for PD includes DNA or RNA associated with the genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP, HNRNPAB, NXN, SLC9A4, EIF2AK1, PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5. 17. The method described in paragraph 3, wherein the method for obtaining and pretreating the subject's biological fluid and concentrating the biological sample is one or more methods selected from centrifugation, ultracentrifugation, filtration using an ultrafiltration tube, polymerization precipitation, and specific antibody capture. 18. The method according to claim 11, wherein the central nervous system-derived marker is a protein, and the step of detecting the central nervous system-derived marker in the subject's biological fluid comprises reacting the subject's biological fluid, preferably the subject's extracellular vesicles, with a labeled antibody that specifically reacts with the central nervous system-derived marker, and detecting the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system-derived marker. 19. The method according to claim 11, wherein the central nervous system disease-associated marker is a protein, and the step of detecting the central nervous system disease-associated marker in the subject's biological fluid comprises reacting the subject's biological fluid, preferably the subject's extracellular vesicles, with a labeled antibody that specifically reacts with the central nervous system disease-associated marker, and detecting the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system disease-associated marker. 20. The method according to item 18 or 19, wherein the label is one or more selected from a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, and a gold colloid label. 21. The method according to claim 11, wherein the central nervous system-derived marker is a nucleic acid, and the step of detecting the central nervous system-derived marker in the subject's biological fluid comprises dissolving the subject's biological fluid, preferably the subject's extracellular vesicles, and detecting the presence and content of central nervous system-derived nucleic acid by DNA or RNA probe or sequencing technology. 22. The method according to claim 11, wherein the central nervous system disease-associated marker is a nucleic acid, and the step of detecting the central nervous system disease-associated marker in the subject's biological fluid comprises dissolving the subject's biological fluid, preferably the subject's extracellular vesicles, and detecting the presence and content of nucleic acid associated with the central nervous system disease by DNA or RNA probes or sequencing techniques.

[0012] 23. A system for diagnosing a central nervous system disease, comprising: a first detection module for detecting a central nervous system-derived marker in a biological fluid of the subject; a second detection module for detecting a central nervous system disease-related marker in the subject's biological fluid; A diagnostic module that diagnoses whether or not a subject is suffering from a central nervous system disease based on the detection results of central nervous system-derived markers and central nervous system disease-related markers obtained by the first detection module and the second detection module, respectively. 24. The first detection module and the second detection module simultaneously detect a central nervous system-derived marker in the subject's biological fluid and a central nervous system disease-related marker in the subject's biological fluid; or The system described in paragraph 23, wherein the first detection module and the second detection module are the same module and simultaneously detect a central nervous system-derived marker in the subject's biological fluid and a central nervous system disease-related marker in the subject's biological fluid. 25. The method further includes a concentration module for obtaining and pre-treating a biological fluid from a subject and concentrating a biological sample in the biological fluid; 25. The system according to claim 23 or 24, wherein the first detection module and the second detection module detect a central nervous system-derived marker and a central nervous system disease-related marker in an enriched biological sample from a subject. 26. Number of biological samples in which both CNS-derived markers and CNS disease-related markers were detected count Quantity to be count Modules, and / or a particle size measurement module for measuring particle size of a biological sample in which both a central nervous system-derived marker and a central nervous system disease-related marker have been detected; 26. The system according to any one of claims 23 to 25, further comprising a diagnostic module for diagnosing whether or not a subject is suffering from a central nervous system disease based on the number of biological samples and / or the particle size of the biological samples. 27. The biological sample is an extracellular vesicle; Preferably, the extracellular vesicles are neuron-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles, or microglia-derived extracellular vesicles. 28. The system described in any one of items 23 to 27, wherein the biological fluid is one or more selected from blood, serum, plasma, saliva, urine, lymph, semen, and milk. 29. The system described in any one of items 23 to 28, wherein the central nervous system disease is one or more selected from cerebrovascular disorders, spinal cord lesions, central nervous system infections, genetic diseases of the nervous system, developmental disorders of the nervous system, autonomic nervous system diseases, demyelinating lesions of the nervous system, epilepsy, and degenerative diseases of the nervous system. 30. The cerebrovascular disorder includes cerebral infarction, cerebral hemorrhage, cerebral blood flow deficiency, and transient ischemic attack; The spinal cord pathology includes acute myelitis, polio, and syringomyelia; The central nervous system infection includes encephalitis and meningitis. The nervous system genetic disease includes neurocutaneous syndrome and spinocerebellar ataxia; The nervous system development disorder includes congenital hydrocephalus and cerebral palsy; the autonomic nervous system disorder is autonomic dysfunction; 30. The system according to claim 29, wherein the nervous system degenerative disease includes degenerative dementia, movement disorder, and prion disease.

[0013] 31. The degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB); The system described in paragraph 30, wherein the movement disorder disease includes motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), Sydenham's disease, hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). 32. The system according to any one of items 23 to 31, wherein the central nervous system-derived marker is a biomarker derived from a neuron or cell selected from the group consisting of mature neurons, oligodendrocytes, astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate progenitor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons. 33. The system described in any one of items 23 to 32, wherein the central nervous system-derived marker and / or central nervous system disease-related marker is one, two or three types selected from proteins, nucleic acids and lipids. 34.The central nervous system-derived markers include G Protein-Coupled Receptor 162 (GPR162), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1 (HCN1), Gamma-Aminobutyric Acid Type A Receptor Subunit Delta (GABRD), Neuroligin 3 (NLGN3), SHISA9, contactin-1 (CNTN1), NeuN, MAP2, neurofilament-L (NF-L: neurofilament light), neurofilament-M (NF-M: neurofilament medium), and neurofilament-H (NF-H: neurofilament light). heavy), synaptophysin, syntaxin, PSD95, L1CAM, doublecortin, beta III tubulin, NeuroD1, TBR1, stathmin 1, MPZ, NCAM, GAP43, S100, CNPase, olig 1, olig 2, olig 3, MBP, OSP, MOG, SOX10, NG2, GFAP, GLAST (EAAT1), GLT1 (EAAT2), glutamine synthetase synthetase), S100-β, ALDH1L1, CD11b, CD45, Iba1, F4 / 80, CD68, CD40, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, TBR2, MASH1 (Ascl1), vGluT1, vGluT2, NMDAR, glutaminase, glutamine synthetase, GABA transporter 1 (GAT1), GABA.B Receptor 1 (GABA B receptor 1), GABA B Receptor 2 (GABA B receptor 2), GAD65, GAD67, tyrosine hydroxylase (TH), dopamine transporter (DAT), FOXA2, GIRK2, Nurr1, LMX1B, tryptophan hydroxylase (TPH), serotonin transporter, Pet1, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AchE), Preferably, the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system, More preferably, the system according to item 32 or 33, wherein the central nervous system-derived marker is one or more selected from the group consisting of L1CAM, SHISA9, CNP enzyme, and GLT1 (EAAT2).

[0014] 35. The system described in item 34, wherein the central nervous system disease-related marker is a protein, and the protein is one or more selected from Aβ1-40 (Aβ40) protein, Aβ1-42 (Aβ42) protein, oligomeric Aβ (oligo-Aβ) protein, tau protein, phosphorylated tau (p-tau) protein, α-synuclein protein, α-synuclein phosphorylated at position 129 (pS129) protein, oligomeric α-synuclein (oligomeric α-synuclein, oligo-α-syn) protein, and prion protein. 36. The system described in paragraph 34, wherein the central nervous system disease-related marker is a nucleic acid, and the nucleic acid is one or more types selected from DNA or RNA that is a biomarker for Alzheimer's disease (AD), and DNA or RNA that is a biomarker for Parkinson's disease (PD) and Parkinsonian syndrome (e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB)). 37. The DNA or RNA that is a biomarker for AD is selected from the group consisting of the genes UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3, RNF7, RPLP0, SPARC, WTAP, HNRNPU, LINC00265, INMT, SLC35E2, CT60, SYNCRIP, RGS2, SMC6, ARSA, SPDYE7P, SMIM17, TRAF3IP2-AS1, KCNC2, SLC24A1, HLCS, GOSR1, MN1, and MG 37. The system according to claim 36, comprising DNA or RNA associated with AT5, NBPF14, FBXO31, WDR52, TBC1D2B, ZNF648, NBPF16, PAGR1, AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1, BPTF, FLG, POM121L10P, NXPH3, H3F3A, SH3TC2, GGCX, and GREB1. 38. The system described in paragraph 36, wherein the DNA or RNA that is a biomarker for PD includes DNA or RNA associated with the genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP, HNRNPAB, NXN, SLC9A4, EIF2AK1, PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5.

[0015] 39. The system described in paragraph 25, wherein the concentration module has sub-modules that perform one or more steps of centrifugation, ultracentrifugation, filtration through an ultrafiltration tube, polymerization precipitation, and specific antibody capture. 40. The system described in paragraph 33, wherein the central nervous system-derived marker is a protein, and the first detection module reacts the subject's biological fluid, preferably the subject's extracellular vesicles, with a labeled antibody that specifically reacts with the central nervous system-derived marker, and detects the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system-derived marker. 41. The system described in paragraph 33, wherein the central nervous system disease-associated marker is a protein, and the second detection module reacts the subject's biological fluid, preferably the subject's extracellular vesicles, with a labeled antibody that specifically reacts with the central nervous system disease-associated marker, and detects the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system disease-associated marker. 42. The system described in item 40 or 41, wherein the label is one or more selected from a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, and a gold colloid label. 43. The system described in paragraph 33, wherein the central nervous system-derived marker is a nucleic acid, and the first detection module dissolves the subject's biological fluid, preferably the subject's extracellular vesicles, and detects the presence and content of the central nervous system-derived nucleic acid by DNA or RNA probe or sequencing technology. 44. The system described in paragraph 33, wherein the central nervous system disease-related marker is a nucleic acid, and the second detection module dissolves the subject's biological fluid, preferably the subject's extracellular vesicles, and detects the presence and content of nucleic acid related to the central nervous system disease by DNA or RNA probes or sequencing technology.

[0016] 45. A composition for detecting a central nervous system disease, comprising: A composition comprising an antibody that targets a central nervous system-derived marker and an antibody that targets a central nervous system disease-associated marker. 46. ​​The composition described in item 45, wherein the central nervous system disease is one or more selected from cerebrovascular disorders, spinal cord lesions, central nervous system infections, genetic diseases of the nervous system, developmental disorders of the nervous system, autonomic nervous system diseases, demyelinating lesions of the nervous system, epilepsy, and degenerative diseases of the nervous system. 47. The cerebrovascular disorder includes cerebral infarction, cerebral hemorrhage, cerebral blood flow deficiency, and transient ischemic attack; The spinal cord pathology includes acute myelitis, polio, and syringomyelia; The central nervous system infection includes encephalitis and meningitis. The nervous system genetic disease includes neurocutaneous syndrome and spinocerebellar ataxia; The nervous system development disorder includes congenital hydrocephalus and cerebral palsy; the autonomic nervous system disorder is autonomic dysfunction; Item 47. The composition according to item 46, wherein the nervous system degenerative disease includes degenerative dementia, movement disorder, and prion disease. 48. The degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB); The composition according to item 47, wherein the movement disorder disease includes motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), Sydenham's disease, hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). 49. The composition according to any one of items 45 to 48, wherein the central nervous system-derived marker is a biomarker derived from a neuron or cell selected from the following: mature neurons, oligodendrocytes, astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate progenitor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons.

[0017] 50.The central nervous system-derived markers include G Protein-Coupled Receptor 162 (GPR162), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1 (HCN1), Gamma-Aminobutyric Acid Type A Receptor Subunit Delta (GABRD), Neuroligin 3 (NLGN3), SHISA9, contactin-1 (CNTN1), NeuN, MAP2, neurofilament-L (NF-L: neurofilament light), neurofilament-M (NF-M: neurofilament medium), and neurofilament-H (NF-H: neurofilament light). heavy), synaptophysin, syntaxin, PSD95, L1CAM, doublecortin, beta III tubulin, NeuroD1, TBR1, stathmin 1, MPZ, NCAM, GAP43, S100, CNPase, olig 1, olig 2, olig 3, MBP, OSP, MOG, SOX10, NG2, GFAP, GLAST (EAAT1), GLT1 (EAAT2), glutamine synthetase synthetase), S100-β, ALDH1L1, CD11b, CD45, Iba1, F4 / 80, CD68, CD40, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, TBR2, MASH1 (Ascl1), vGluT1, vGluT2, NMDAR, glutaminase, glutamine synthetase, GABA transporter 1 (GAT1), GABA.B Receptor 1 (GABA B receptor 1), GABA B Receptor 2 (GABA B receptor 2), GAD65, GAD67, tyrosine hydroxylase (TH), dopamine transporter (DAT), FOXA2, GIRK2, Nurr1, LMX1B, tryptophan hydroxylase (TPH), serotonin transporter, Pet1, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AchE), Preferably, the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system, More preferably, the composition according to any one of items 45 to 49, wherein the central nervous system-derived marker is one or more selected from L1CAM, SHISA9, CNP enzyme, and GLT1 (EAAT2). 51. The composition according to any one of items 45 to 50, wherein the central nervous system disease-related marker is one or more selected from Aβ1-40 (Aβ40) protein, Aβ1-42 (Aβ42) protein, oligomeric Aβ (oligo-Aβ) protein, tau protein, phosphorylated tau (p-tau) protein, α-synuclein protein, α-synuclein phosphorylated at position 129 (pS129) protein, oligomeric α-synuclein (oligomeric α-synuclein, oligo-α-syn) protein, and prion protein. 52. The composition described in any one of items 45 to 51, wherein the antibody targeting the central nervous system-derived marker and the antibody targeting the central nervous system disease-related marker are labeled antibodies, and preferably the label is one or more selected from a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, and a gold colloid label.

[0018] 53. A kit for detecting a central nervous system disease in a subject, comprising: A kit comprising a reagent for detecting a central nervous system-derived marker in a biological fluid of a subject, and a reagent for detecting a central nervous system disease-related marker in a biological fluid of a subject. 54. The kit described in item 53, wherein the reagent for detecting a central nervous system-derived marker in the subject's biological fluid and the reagent for detecting a central nervous system disease-related marker in the subject's biological fluid are the compositions described in any one of items 45 to 52. 55. The reagent for detecting a central nervous system-derived marker in the subject's biological fluid is a primer or a probe targeting the central nervous system-derived marker; Item 54. The kit according to Item 53, wherein the reagent for detecting a central nervous system disease-associated marker in a biological fluid of the subject is a primer or a probe targeting a central nervous system disease-associated marker. 56. The kit described in paragraph 53, further comprising reagents and devices for obtaining a biological sample from a subject, preferably reagents and devices for obtaining extracellular vesicles from a subject. 57. The kit described in item 53, wherein the central nervous system disease is one or more selected from cerebrovascular disorders, spinal cord lesions, central nervous system infections, genetic diseases of the nervous system, developmental disorders of the nervous system, autonomic nervous system diseases, demyelinating lesions of the nervous system, epilepsy, and degenerative diseases of the nervous system. 58. The cerebrovascular disorder includes cerebral infarction, cerebral hemorrhage, cerebral blood flow deficiency, and transient ischemic attack; The spinal cord pathology includes acute myelitis, polio, and syringomyelia; The central nervous system infection includes encephalitis and meningitis. The nervous system genetic disease includes neurocutaneous syndrome and spinocerebellar ataxia; The nervous system development disorder includes congenital hydrocephalus and cerebral palsy; the autonomic nervous system disorder is autonomic dysfunction; Item 58. The kit according to Item 57, wherein the nervous system degenerative disease includes degenerative dementia, movement disorder, and prion disease. 59. The degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB); Item 59. The kit according to item 58, wherein the movement disorder disease includes motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), Sydenham's disease, hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Effect of the Invention

[0019] According to the methods, systems, kits and compositions of the present application, it is possible to diagnose whether or not a subject is suffering from a central nervous system disease using only the peripheral body fluids of the subject. The methods, systems, kits, and compositions of the present application make it possible to effectively concentrate and specifically label biomarkers that can reflect changes in central nervous system diseases from peripheral body fluids, providing greater diagnostic efficacy than directly detecting disease-related markers in peripheral body fluids. The methods, systems, kits and compositions of the present application effectively utilize peripheral body fluids for detection, thereby avoiding the need to invasively obtain cerebrospinal fluid from the subject, significantly reducing the risk of sampling from the subject, significantly reducing the difficulty of detection, and greatly facilitating the clinical use and widespread adoption of detection. The methods, systems, kits and compositions of the present application enable the simultaneous detection of central nervous system-derived markers and disease-related markers, shortening the sample detection time and making it more efficient and convenient. The key to the present application is a detection method and system using dual and / or multiple markers, which allows for simultaneous detection of dual and / or multiple markers, i.e., using markers to identify central nervous system-derived portions in biological samples, particularly extracellular vesicles, and based on this, using disease-related markers, thus contributing to disease diagnosis, differential diagnosis, follow-up and drug evaluation as a result of co-detection. [Brief description of the drawings]

[0020] [Figure 1A] FIG. 1A shows that in Example 1, significant differences were observed between the AD group and the healthy control group (CT group) in terms of the ratio of singly labeled SHISA9 protein-positive extracellular vesicles in plasma to the total number of extracellular vesicles in the particle size ranges of 60 to 100 nm, 100 to 150 nm, and the entire particle size range (**: p<0.01). [Figure 1B] FIG. 1B shows that in Example 1, with regard to the ratio of single-labeled p-tau217 protein-positive extracellular vesicles in plasma to the total number of extracellular vesicles, a very large significant difference was observed between the AD group and the CT group in the particle size range of 60 to 100 nm and the entire particle size range (****: p<0.0001), and a significant difference was observed between the AD group and the CT group in the particle size range of 100 to 150 nm (**: p<0.01). [Figure 1C] FIG. 1C shows that in Example 1, with regard to the ratio of SHISA9 protein-p-tau217 protein double-positive extracellular vesicles in plasma to the total number of extracellular vesicles, a very large significant difference was observed in both the AD group and the CT group in the particle size range of 100 to 150 nm and in the entire particle size range (***: p<0.001). [Figure 1D]FIG. 1D shows that in Example 1, the receiver operating characteristic curve (ROC) analysis results showed that the area under the curve (AUC) of the SHISA9 protein single marker in the particle size range of 60 to 100 nm was 0.749, and the AUC of the SHISA9 protein and p-tau217 protein double marker in the particle size range of 100 to 150 nm was 0.890. When the SHISA9 60 to 100 nm and SHISA9 + p-tau217 100 to 150 nm data were input using the logistic regression analysis method, the AUC was 0.891. [Figure 1E] FIG. 1E shows that in Example 1, the results of ROC analysis showed that the AUC of the p-tau217 protein single marker in the particle size range of 60-100 nm was 0.877, and the AUC of the SHISA9 protein and p-tau217 protein double marker in the particle size range of 100-150 nm was 0.890. When the p-tau217 60-100 nm and SHISA9+p-tau217 100-150 nm data were inputted by the logistic regression analysis Enter method, the AUC was 0.910. [Figure 2A] FIG. 2A shows the number of single-labeled GLT1 protein-positive extracellular vesicles and the number of single-labeled α-syn protein (syn211 and MJFR14 are all anti-α-syn protein antibodies)-positive extracellular vesicles in plasma in Example 2. [Figure 2B-C] 2B and C show the number of single-labeled GLT1 protein or α-syn protein-positive extracellular vesicles and the number of GLT1 protein-α-syn protein double-positive extracellular vesicles in plasma in Example 2. [Diagram 3] 3A and 3B show that in Example 2, the number of GLT1 protein-α-syn protein double-positive extracellular vesicles in plasma was significantly higher in the PD group than in the MSA group and CT group (***: p<0.001), and no significant difference was observed between the MSA group and the CT group. [Figure 4A]FIG. 4A shows that in Example 3, no significant difference was observed between the MSA group and the PD group in the number of CNPase protein-positive extracellular vesicles in plasma, and a significant difference was observed between the MSA group and the HC group (*: p<0.05). [Figure 4B] Figure 4B shows that in Example 3, no significant difference was observed among the MSA group, PD group, and HC group in the number of CNPase protein-α-syn protein (anti-MJFR14 antibody labeled) double-positive extracellular vesicles in plasma. [Figure 4C] FIG. 4C shows that in Example 3, when the number of CNPase protein-α-syn protein double-positive extracellular vesicles in plasma was normalized by the total number of extracellular vesicles in plasma, significant differences were observed between the MSA group and the HC group, and between the MSA group and the PD group (*: p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, specific embodiments of the present invention will be described in more detail with reference to the drawings. Although specific embodiments of the present invention are shown in the drawings, it should be understood that the present invention may be embodied in various forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to more completely understand the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0022] In addition, specific terms are used in the specification and claims to refer to specific components. As will be apparent to those skilled in the art, engineers may use different nouns to describe the same components. In this specification and claims, components are not distinguished by noun differences, but by functional differences. The terms "comprise" and "include" used throughout the specification and claims are open terms and should be interpreted as "including, but not limited to". In the following specification, preferred embodiments for carrying out the present invention are described, but the description is intended for the general principles of the specification and does not limit the scope of the present invention. The patent scope of the present invention is defined by the appended claims.

[0023] In one particular embodiment of the present application, the present application relates to a method for diagnosing a central nervous system disease, comprising the steps of detecting a central nervous system-derived marker in a biological fluid of a subject, detecting a central nervous system disease-associated marker in the biological fluid of the subject, and diagnosing whether the subject is suffering from a central nervous system disease based on the detection results of the central nervous system-derived marker and the central nervous system disease-associated marker.

[0024] In one particular embodiment of the present application, the present application relates to a method for diagnosing a central nervous system disease, comprising the steps of detecting a central nervous system-derived marker in a biological fluid of a subject, detecting a central nervous system disease-associated marker in the biological fluid of the subject, and diagnosing whether the subject is suffering from a central nervous system disease based on the detection results of the central nervous system-derived marker and the central nervous system disease-associated marker, wherein the detection of the central nervous system-derived marker in the biological fluid of the subject and the detection of the central nervous system disease-associated marker in the biological fluid of the subject are performed simultaneously.

[0025] In one particular embodiment of the present application, the present application relates to a system for diagnosing a central nervous system disease, the system including: a first detection module for detecting a central nervous system-derived marker in a subject's biological fluid; a second detection module for detecting a central nervous system disease-associated marker in the subject's biological fluid; and a diagnostic module for diagnosing whether or not the subject is suffering from a central nervous system disease based on the detection results of the central nervous system-derived marker and the central nervous system disease-associated marker obtained by the first detection module and the second detection module, respectively.

[0026] In one particular embodiment of the present application, the present application relates to a system for diagnosing a central nervous system disease, the system including a first detection module for detecting a central nervous system-derived marker in a biological fluid of a subject, a second detection module for detecting a central nervous system disease-related marker in the biological fluid of the subject, and a diagnosis module for diagnosing whether the subject suffers from a central nervous system disease based on the detection results of the central nervous system-derived marker and the central nervous system disease-related marker obtained by the first detection module and the second detection module, respectively. The first detection module and the second detection module simultaneously detect the central nervous system-derived marker in the biological fluid of the subject and the central nervous system disease-related marker in the biological fluid of the subject, or the first detection module and the second detection module simultaneously detect the central nervous system-derived marker in the biological fluid of the subject and the central nervous system disease-related marker in the biological fluid of the subject as a single module.

[0027] As described above, in the method and system of the present application, the present inventors have developed a new method and system, namely, detection using dual markers and / or multiple markers having different actions, and in particular simultaneous detection can be performed using such dual markers and / or multiple markers, thereby effectively detecting central nervous system diseases even with peripheral body fluids alone. In such a detection method and system, there is no need to extract cerebrospinal fluid from the patient, which greatly reduces the risk of sampling the patient. In addition, since dual labeling and / or multiple labeling are adopted to label a marker derived from the central nervous system and a marker related to the disease to be detected, the detection accuracy is greatly improved, and when simultaneous detection is performed, the overall detection efficiency can also be improved. Meanwhile, other conventional methods require complicated extraction, concentration, purification steps, etc. to obtain a biological sample for diagnosis. The present application aims to improve the accuracy and efficiency of disease detection by simplifying the operation steps and innovatively utilizing markers derived from the central nervous system to indicate their origin and combining them with disease-related markers.

[0028] Furthermore, the method of the present application further includes the steps of obtaining and pre-treating the subject's biological fluid and concentrating a biological sample in the biological fluid, and detecting central nervous system-derived markers in the subject's biological fluid means detecting central nervous system-derived markers in the concentrated biological sample from the subject, and detecting central nervous system disease-related markers in the subject's biological fluid means detecting central nervous system disease-related markers in the concentrated biological sample from the subject.

[0029] Furthermore, the method of the present application can be used to increase the number of biological samples in which both CNS-derived markers and CNS disease-related markers are detected. count and / or measuring the particle size of the biological samples in which both the central nervous system-derived marker and the central nervous system disease-related marker are detected, and diagnosing whether the subject is suffering from a central nervous system disease based on the number of the biological samples and / or the particle size of the biological samples. Specifically, a biological sample in which both the central nervous system-derived marker and the central nervous system disease-related marker are detected is usually called a double-positive biological sample, and whether the subject is suffering from a central nervous system disease is diagnosed based on the number and / or particle size of the double-positive biological samples.

[0030] Furthermore, the system of the present application further includes a concentration module that obtains and pre-processes the subject's biological fluid and concentrates the biological sample in the biological fluid, and the first detection module and the second detection module detect central nervous system-derived markers and central nervous system disease-related markers in the concentrated biological sample from the subject.

[0031] Furthermore, the system of the present application can estimate the number of biological samples in which both CNS-derived markers and CNS disease-related markers were detected. count Quantity to be countand / or a particle size measurement module for measuring the particle size of a biological sample in which both a central nervous system-derived marker and a central nervous system disease-related marker are detected, and a diagnosis module for diagnosing whether or not a subject suffers from a central nervous system disease based on the number of the biological samples and / or the particle size of the biological samples. Specifically, a biological sample in which both a central nervous system-derived marker and a central nervous system disease-related marker are detected is usually called a double-positive biological sample, and whether or not a subject suffers from a central nervous system disease is diagnosed based on the number and / or particle size of the double-positive biological samples.

[0032] The present application also relates to a composition for detecting a central nervous system disease, comprising an antibody that targets a central nervous system-derived marker and an antibody that targets a central nervous system disease-associated marker.

[0033] The present application also relates to a kit for detecting a central nervous system disease in a subject, comprising a reagent for detecting a central nervous system-derived marker in the biological fluid of the subject, and a reagent for detecting a central nervous system disease-associated marker in the biological fluid of the subject.

[0034] In one particular embodiment, the reagent for detecting a central nervous system-derived marker in a biological fluid of a subject and the reagent for detecting a central nervous system disease-associated marker in a biological fluid of a subject are the compositions described herein above.

[0035] In one particular embodiment, the reagent for detecting a central nervous system derived marker in the subject's biological fluid is a primer or probe targeting a central nervous system derived marker, and the reagent for detecting a central nervous system disease associated marker in the subject's biological fluid is a primer or probe targeting a central nervous system disease associated marker.

[0036] In certain embodiments of the methods, systems, compositions, or kits of the present application, the biological fluid is blood, serum, plasma, saliva, urine, lymph, semen, or milk. Preferred biological fluids are blood, serum, plasma, saliva, or urine. The method of obtaining the biological fluid from the subject may be any method known to those skilled in the art. Those skilled in the art may obtain the biological fluid from the subject by the method of obtaining the biological fluid selected.

[0037] In one particular embodiment, the biological sample of the present application is an extracellular vesicle. The extracellular vesicles (EVs, also called extracellular vesicles) according to the present specification are vesicular bodies with a double membrane structure that are shed from the cell membrane or secreted from the cell, and have a diameter of 40 nm to 1000 nm. Extracellular vesicles are mainly composed of microvesicles (MVs) and exosomes (Ex: Exosomes), and the microvesicles are microvesicles with a diameter of about 100 nm to 1000 nm that are shed from the cell membrane after cell activation, damage, or apoptosis. Exosomes are multivesicular bodies within cells that fuse with the cell membrane and are released outside the cell in the form of exosomes, have a diameter of about 30 to 150 nm, are released by many different types of cells, and can exert various cellular functions such as intercellular communication, antigen presentation, and protein and nucleic acid transfer. Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), and contain multiple associated proteins, lipids, DNA, mRNA, miRNA, etc. derived from cells, and are involved in processes such as cell-cell communication, cell migration, angiogenesis, and immune regulation. In one particular embodiment, the extracellular vesicles are preferably neuron-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles, or microglia-derived extracellular vesicles.

[0038] In certain embodiments of the method, system, composition, or kit of the present application, the method of obtaining and pre-treating a biological fluid from a subject, concentrating a biological sample in the biological fluid, and particularly obtaining extracellular vesicles may be centrifugation, ultracentrifugation, filtration through an ultrafiltration tube, polymerization precipitation, or specific antibody capture. In this specification, centrifugation, ultracentrifugation, filtration through an ultrafiltration tube, polymerization precipitation, or specific antibody capture all have meanings that can be understood by those skilled in the art, and those skilled in the art may select an appropriate method depending on the extracellular vesicles to be obtained.

[0039] In one embodiment of the method or system of the present application, ultracentrifugation methods are used to concentrate extracellular vesicles, in particular neuronal-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles, or microglia-derived extracellular vesicles, from biological fluids.

[0040] In one embodiment of the method or system of the present application, extracellular vesicles, in particular neuron-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles or microglia-derived extracellular vesicles, are concentrated by conventional centrifugation or filtration through an ultrafiltration tube.

[0041] In certain embodiments of the methods, systems, compositions, or kits of the present application, the central nervous system disease is one or more selected from cerebrovascular disorders, spinal cord lesions, central nervous system infections, genetic diseases of the nervous system, developmental disorders of the nervous system, autonomic nervous system diseases, demyelinating lesions of the nervous system, epilepsy, and degenerative diseases of the nervous system.

[0042] In certain embodiments of the method, system, composition, or kit of the present application, the cerebrovascular disorder includes cerebral infarction, cerebral hemorrhage, cerebral blood flow insufficiency, and transient ischemic attack, the spinal cord lesion includes acute myelitis, polio, and syringomyelia, the central nervous system infection includes encephalitis and meningitis, the genetic disease of the nervous system includes neurocutaneous syndrome and spinocerebellar ataxia, the developmental abnormality of the nervous system includes congenital hydrocephalus and cerebral palsy, the autonomic nervous system disease is autonomic dysfunction, and the degenerative disease of the nervous system includes degenerative dementia, movement disorder, and prion disease. Further, the degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB), and the movement disorder disease includes motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), Sydenham's disease, hepatolenticular degeneration, multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD).

[0043] In certain embodiments of the methods, systems, compositions, or kits herein, the central nervous system disease is Alzheimer's disease.

[0044] In certain embodiments of the methods, systems, compositions or kits herein, the central nervous system disorder is Parkinson's disease.

[0045] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived markers and / or central nervous system disease-associated markers are one, two or three selected from proteins, nucleic acids and lipids, respectively.

[0046] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived markers and the central nervous system disease-associated markers are all proteins.

[0047] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived markers and the central nervous system disease-associated markers are all nucleic acids.

[0048] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived markers and the central nervous system disease-associated markers are all lipids.

[0049] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived marker is a protein and the central nervous system disease-associated marker is a nucleic acid.

[0050] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived marker is a nucleic acid and the central nervous system disease-associated marker is a protein.

[0051] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived marker is a protein and the central nervous system disease-associated marker is a lipid.

[0052] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived marker is a nucleic acid and the central nervous system disease-associated marker is a lipid.

[0053] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived marker is a lipid and the central nervous system disease-associated marker is a protein.

[0054] In certain embodiments of the methods, systems, compositions or kits of the present application, the central nervous system-derived marker is a lipid and the central nervous system disease-associated marker is a nucleic acid.

[0055] In certain embodiments of the method, system, composition, or kit of the present application, the central nervous system-derived marker is a biomarker derived from a neuron or cell, such as a mature neuron, an oligodendrocyte, an astrocyte, a microglia, an immature neuron, a Schwann cell, a radial glial cell, an intermediate progenitor cell, a glutamatergic neuron, a GABAergic neuron, a dopaminergic neuron, a serotonergic neuron, or a cholinergic neuron. In certain embodiments of the method, system, composition, or kit of the present application, the central nervous system-derived marker is preferably a biomarker derived from a neuron. In certain embodiments of the method, system, composition, or kit of the present application, the central nervous system-derived marker is preferably a biomarker derived from an astrocyte and / or an oligodendrocyte.

[0056] In certain embodiments of the methods, systems, compositions, or kits of the present application, the central nervous system-derived marker is a marker located on the surface of central nervous system-derived extracellular vesicles.

[0057] In certain embodiments of the methods, systems, compositions, or kits of the present application, the markers from the central nervous system are G Protein-Coupled Receptor 162 (GPR162), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1 (HCN1), Gamma-Aminobutyric Acid Type A Receptor Subunit Delta (GABRD), Neuroligin 3 (NLGN3), SHISA9, contactin-1 (CNTN1), NeuN, MAP2, neurofilament light (NF-L), neurofilament medium (NF-M), neurofilament H (NF-H), or a combination of these. heavy), synaptophysin, syntaxin, PSD95, L1CAM, doublecortin, beta III tubulin, NeuroD1, TBR1, stathmin 1, MPZ, NCAM, GAP43, S100, CNPase, olig 1, olig 2, olig 3, MBP, OSP, MOG, SOX10, NG2, GFAP, GLAST (EAAT1), GLT1 (EAAT2), glutamine synthetase synthetase), S100-β, ALDH1L1, CD11b, CD45, Iba1, F4 / 80, CD68, CD40, vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, TBR2, MASH1 (Ascl1), vGluT1, vGluT2, NMDAR, glutaminase, glutamine synthetase, GABAGABA transporter 1 (GAT1), GABA B Receptor 1 (GABA B receptor 1), GABA B Receptor 2 (GABA B receptor 2), GAD65, GAD67, tyrosine hydroxylase (TH), dopamine transporter (DAT), FOXA2, GIRK2, Nurr1, LMX1B, tryptophan hydroxylase (TPH), serotonin transporter, Pet1, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AchE).

[0058] In certain embodiments of the methods, systems, compositions, or kits of the present application, the central nervous system-derived marker is one or more selected from L1CAM, SHISA9, CNP enzyme, and GLT1 (EAAT2), of which L1CAM and SHISA9 are biomarkers derived from neurons, GLT1 (EAAT2) is a biomarker derived from astrocytes, and CNP enzyme is a biomarker derived from oligodendrocytes.

[0059] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from mature neurons include L1CAM, SHISA9, contactin-1 (CNTN1), NeuN, MAP2, neurofilament-L (NF-L: neurofilament light), neurofilament-M (NF-M: neurofilament medium), neurofilament-H (NF-H: neurofilament heavy), synaptophysin, syntaxin, PSD95, and the like.

[0060] In certain embodiments of the methods, systems, compositions or kits of the present application, the biomarkers derived from oligodendrocytes include CNPase, olig 1, olig 2, olig 3, MBP, OSP, MOG, SOX10, NG2, etc. As used herein, CNP enzyme is a 2',3'-cyclic nucleotide 3'-phosphodiesterase.

[0061] In certain embodiments of the methods, systems, compositions, or kits of the present application, the astrocyte-derived biomarkers include GFAP, GLAST (EAAT1), GLT1 (EAAT2), glutamine synthetase, S100-β, ALDH1L1, etc. As used herein, GLT1 (EAAT2) is glutamate transporter 1. GLT1 (EAAT2) is an astrocyte-specific glutamate transporter in the central nervous system and is one of the markers of astrocytes.

[0062] In certain embodiments of the methods, systems, compositions or kits of the present application, the microglia-derived biomarkers include CD11b, CD45, Iba1, F4 / 80, CD68, CD40, and the like.

[0063] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from immature neurons include doublecortin, beta III tubulin, NeuroD1, TBR1, stathmin 1, and the like.

[0064] In certain embodiments of the methods, systems, compositions or kits of the present application, the biomarkers derived from Schwann cells include MPZ, NCAM, GAP43, S100, and the like.

[0065] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from radial glia include vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, and the like.

[0066] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from intermediate progenitors include TBR2, MASH1 (Ascl1), and the like.

[0067] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from glutamatergic neurons include vGluT1, vGluT2, NMDAR, glutaminase, glutamine synthetase, and the like.

[0068] In certain embodiments of the methods, systems, compositions, or kits of the present application, the biomarkers derived from GABAergic neurons are GABA transporter 1 (GAT1), GABA B Receptor 1 (GABA Breceptor 1), GABA B Receptor 2 (GABA B receptor 2), GAD65, GAD67, etc.

[0069] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from dopaminergic neurons include tyrosine hydroxylase (TH), dopamine transporter (DAT), FOXA2, GIRK2, Nurr1, LMX1B, and the like.

[0070] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from serotonergic neurons include tryptophan hydroxylase (TPH), serotonin transporter, Pet1, and the like.

[0071] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers derived from cholinergic neurons include choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AchE), and the like.

[0072] In certain embodiments of the method, system, composition, or kit of the present application, the central nervous system disease-related marker is a protein, and the protein is one or more selected from Aβ1-40 (Aβ40) protein, Aβ1-42 (Aβ42) protein, oligomeric Aβ (oligo-Aβ) protein, tau protein, phosphorylated tau (p-tau) protein, α-synuclein protein, α-synuclein phosphorylated at position 129 (pS129) protein, oligomeric α-synuclein (oligomeric α-synuclein, oligo-α-syn) protein, and prion protein.

[0073] In certain embodiments of the methods, systems, compositions, or kits of the present application, biomarkers associated with Alzheimer's disease (AD) include, but are not limited to, Aβ1-40 (Aβ40) protein, Aβ1-42 (Aβ42) protein, oligomeric Aβ (oligo-Aβ) protein, tau protein, and phosphorylated tau (p-tau) protein.

[0074] In certain embodiments of the method, system, composition, or kit of the present application, biomarkers associated with Parkinson's disease (PD) and parkinsonian syndromes, including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Parkinson's disease dementia (PDD), and dementia with Lewy bodies (DLB), include, but are not limited to, α-synuclein protein, 129-phosphorylated α-synuclein (pS129) protein, oligomeric α-synuclein (oligo-α-syn) protein, tau protein, and phosphorylated tau (p-tau) protein. α-synuclein protein (α-syn protein) is the main protein component of Lewy bodies, and as has been shown in many studies, is closely related to the development of Parkinson's disease.

[0075] In certain embodiments of the methods, systems, compositions or kits of the present application, the biomarker associated with prion disease is a prion protein.

[0076] In certain embodiments of the method, system, composition, or kit of the present application, the central nervous system disease-related marker is a nucleic acid, and the nucleic acid is one or more selected from DNA or RNA that is a biomarker for Alzheimer's disease (AD), DNA or RNA that is a biomarker for Parkinson's disease (PD) and parkinsonian syndromes (e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB)).

[0077] In certain embodiments of the methods, systems, compositions, or kits of the present application, the DNA or RNA that is a biomarker for AD is selected from the group consisting of the genes UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3, RNF7, RPLP0, SPARC, WTAP, HNRNPU, LINC00265, INMT, SLC35E2, CT60, SYNCRIP, RGS2, SMC6, ARSA, SPDYE7P, SMIM17, TRAF3IP2-AS1, KCNC2, SLC35E2, and CT60. The reagents for detecting a central nervous system-derived marker in a subject's biological fluid include DNA or RNA related to 24A1, HLCS, GOSR1, MN1, MGAT5, NBPF14, FBXO31, WDR52, TBC1D2B, ZNF648, NBPF16, PAGR1, AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1, BPTF, FLG, POM121L10P, NXPH3, H3F3A, SH3TC2, GGCX, and GREB1. The reagents for detecting a central nervous system-derived marker in a subject's biological fluid include primers or probes targeting the central nervous system-derived marker, and primers or probes targeting the above DNA or RNA.

[0078] In certain embodiments of the methods, systems, compositions, or kits of the present application, the DNA or RNA that is a biomarker for PD includes DNA or RNA associated with the genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP, HNRNPAB, NXN, SLC9A4, EIF2AK1, PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5. The reagent for detecting the central nervous system-derived marker in the biological fluid of the subject is a primer or probe targeting the central nervous system-derived marker, and is a primer or probe targeting the above DNA or RNA.

[0079] In certain embodiments of the methods, systems, compositions, or kits of the present application, the central nervous system-derived marker is a protein, and the step of detecting the central nervous system-derived marker in the subject's biological fluid comprises reacting the subject's biological fluid, preferably the subject's extracellular vesicles, with a labeled antibody that specifically reacts with the central nervous system-derived marker, and detecting the intensity of the labeled signal after the reaction to determine the presence and amount of the central nervous system-derived marker; the central nervous system disease-associated marker is a protein, and the step of detecting the central nervous system disease-associated marker in the subject's biological fluid comprises reacting the subject's biological fluid, preferably the subject's extracellular vesicles, with a labeled antibody that specifically reacts with the central nervous system disease-associated marker, and detecting the intensity of the labeled signal after the reaction to determine the presence and amount of the central nervous system disease-associated marker. Furthermore, in certain embodiments, the subject's extracellular vesicles are preferably reacted with both a labeled antibody that specifically reacts with a central nervous system-derived marker and a labeled antibody that specifically reacts with a central nervous system disease-associated marker, thereby allowing efficient tracking of double-marker and / or multi-marker positive extracellular vesicles.

[0080] Additionally, certain embodiments of the methods, systems, compositions, or kits of the present application may be used to determine the number of biological samples (e.g., extracellular vesicles) in which both CNS-derived markers and CNS disease-associated markers are detected. count and / or measuring the particle size of biological samples (e.g., extracellular vesicles) in which both the CNS-derived marker and the CNS disease-related marker are detected, thereby allowing for tracking of dual marker and / or multiple marker positive biological samples, e.g., extracellular vesicles, and then analyzing the subject's risk of disease in combination with the results of these indicators, taking into full consideration the number of such biological samples and / or their particle size.

[0081] In certain embodiments of the methods, systems, compositions, or kits of the present application, the label is one or more selected from a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, or a colloidal gold label.

[0082] In certain embodiments of the methods, systems, compositions or kits of the present application, the fluorescent labels are one or more selected from Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705, Qdot800, Alexa350, Alexa 405, Alexa488, Alexa 532, Alexa546, Alexa555, Alexa 568, Alexa 594, Alexa647, Alexa700, and Alexa750.

[0083] In certain embodiments of the methods, systems, compositions or kits of the present application, isotope labeling, enzyme labeling, chemiluminescent labeling, quantum dot labeling or colloidal gold labeling may all be performed using methods known to those of skill in the art.

[0084] In certain embodiments of the method, system, composition or kit of the present application, the central nervous system-derived marker is a nucleic acid, and the step of detecting the central nervous system-derived marker in the subject's biological fluid comprises dissolving the subject's biological fluid, preferably the subject's extracellular vesicles, and detecting the presence and content of the central nervous system-derived nucleic acid by DNA or RNA probes or sequencing techniques, and the central nervous system disease-related marker is a nucleic acid, and the step of detecting the central nervous system disease-related marker in the subject's biological fluid comprises dissolving the subject's biological fluid, preferably the subject's extracellular vesicles, and detecting the presence and content of the central nervous system-derived nucleic acid by DNA or RNA probes or sequencing techniques. Furthermore, in certain embodiments, preferably, the presence and content of the central nervous system-derived nucleic acid and the presence and content of the central nervous system disease-related nucleic acid are simultaneously detected in the subject's extracellular vesicles using DNA and RNA probes, thereby efficiently tracking dual marker and / or multi-marker positive extracellular vesicles.

[0085] In certain embodiments of the methods, systems, compositions or kits of the present application, detecting the intensity of the label signal after the reaction to determine the presence and content of the CNS-derived marker may employ, for example, an immunoassay, for example ELISA, Luminex, MSD, Quanterix, Singulex, eCL8000, Cobas are suitable methods for measuring protein biomarkers.

[0086] In certain embodiments of the methods, systems, compositions, or kits of the present application, the number of biological samples in which both the CNS-derived marker and the CNS disease-associated marker are detected is determined. count A detection device for detecting and / or measuring the particle size of a biological sample in which both a central nervous system-derived marker and a central nervous system disease-related marker are detected includes, but is not limited to, a granulometer and a nanoflow cytometer. For example, the particle size of extracellular vesicles is detected using nanoparticle tracking technology, and the number of the double-positive extracellular vesicles is detected using nanoflow analysis technology.

[0087] In certain embodiments of the methods, systems, compositions, or kits of the present application, when the biomarker is a nucleic acid, the biological fluid or extracellular vesicles must be lysed prior to measuring the nucleic acid biomarker. Nucleic acid detection may be performed by any method known to those of skill in the art, such as DNA or RNA probes, or any known sequencing technology.

[0088] In one particular embodiment, the present application relates to a composition for detecting a central nervous system disease, comprising an antibody that targets a central nervous system-derived marker and an antibody that targets a central nervous system disease-associated marker.

[0089] In one particular embodiment of the present application, the antibody targeting the central nervous system-derived marker and the antibody targeting the central nervous system disease-associated marker are labeled antibodies, and preferably, the label is one or more selected from a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, or a gold colloid label.

[0090] In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting SHISA9 protein and an antibody targeting phosphorylated tau217 (p-tau217) protein.

[0091] In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting the GLT1 (EAAT2) protein and an antibody targeting the tau protein. In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting the GLT1 (EAAT2) protein and an antibody targeting the α-synuclein protein. In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting the GLT1 (EAAT2) protein and an antibody targeting the oligomeric α-synuclein (oligo-α-syn) protein. In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting the GLT1 (EAAT2) protein and an antibody targeting the 129-phosphorylated α-synuclein (pS129) protein. In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting GLT1 (EAAT2) protein and an antibody targeting phosphorylated tau (p-tau) protein.

[0092] In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting CNP enzyme (CNPase) and an antibody targeting tau protein. In one particular embodiment of the present application, a composition for detecting a central nervous system disease comprises an antibody targeting CNP enzyme (CNPase) and an antibody targeting α-synuclein protein (α-synuclein protein).

[0093] The present application relates to a kit for detecting a central nervous system disease in a subject, comprising a reagent for detecting a central nervous system-derived marker in a biological fluid of a subject, and a reagent for detecting a central nervous system disease-related marker in a biological fluid of a subject, wherein the reagent for detecting a central nervous system-derived marker in a biological fluid of a subject and the reagent for detecting a central nervous system disease-related marker in a biological fluid of a subject are the above-mentioned composition of the present application.

[0094] The present application relates to a kit for detecting a central nervous system disease in a subject, comprising a reagent for detecting a central nervous system-derived marker in a biological fluid of the subject, and a reagent for detecting a central nervous system disease-associated marker in the biological fluid of the subject. The reagent for detecting the central nervous system-derived marker in the biological fluid of the subject is a primer or a probe targeting the central nervous system-derived marker, and the reagent for detecting the central nervous system disease-associated marker in the biological fluid of the subject is a primer or a probe targeting the central nervous system disease-associated marker.

[0095] The kit according to the present application further includes a reagent and an apparatus for obtaining a biological sample from a subject, preferably a reagent and an apparatus for obtaining extracellular vesicles from a subject. The reagent and the apparatus for obtaining extracellular vesicles may include general kits and apparatus known to those skilled in the art.

[0096] According to the method, system, kit and composition of the present application, it is possible to diagnose whether a subject suffers from a central nervous system disease using only the peripheral body fluid of the subject, and to effectively diagnose and differentially diagnose diseases such as Alzheimer's disease, Parkinson's disease, and multiple system atrophy, and to effectively distinguish them from healthy controls and other diseases.

[0097] According to the method, system, kit and composition of the present application, biomarkers that can reflect changes in central nervous system diseases can be effectively concentrated and specifically labeled from peripheral body fluids, and the diagnostic effect is higher than that of directly detecting disease-related markers in peripheral body fluids. For example, extracellular vesicle biomarkers derived from the central nervous system, such as L1CAM protein, SHISA9 protein, GLT1 protein, and CNPase protein, can be combined with biomarkers that can reflect changes in central nervous system diseases, and in a preferred embodiment, extracellular vesicle biomarkers derived from the central nervous system, such as L1CAM protein, SHISA9 protein, GLT1 protein, and CNPase protein, can be simultaneously detected with biomarkers that can reflect changes in central nervous system diseases, such as Aβ1-40 protein, Aβ1-42 protein, oligomeric Aβ (oligo-Aβ) protein, tau protein, phosphorylated tau (p-tau) protein, and α-synuclein (α-syn) protein, and diagnosis can be performed based on the simultaneous detection results. The number of double-positive extracellular vesicles for two markers and / or the particle size of such extracellular vesicles may be detected simultaneously to perform diagnosis. For example, the ratio of the double-positive extracellular vesicles for two markers to the total number of extracellular vesicles or extracellular vesicles positive for a marker may be calculated with the aid of the above, which can further improve the diagnostic effect.

[0098] The method, system, kit and composition of the present application effectively utilize peripheral body fluids for detection, thereby avoiding the need to invasively obtain cerebrospinal fluid from the subject, greatly reducing the risk of sampling from the subject, greatly reducing the difficulty of detection, and greatly promoting the clinical use and widespread use of detection. The method, system, kit and composition of the present application can simultaneously detect central nervous system-derived markers and disease-related markers, shortening the sample detection time and making it more efficient and convenient.

[0099] According to the method, system, composition, or kit of the present application, Alzheimer's disease patients can be distinguished from healthy subjects by effectively detecting SHISA9 protein and phosphorylated tau217 (p-tau217) protein as dual markers.

[0100] According to the method, system, composition, or kit of the present application, GLT1 protein and α-syn protein can be effectively detected as dual markers, making it possible to distinguish Parkinson's disease patients and multiple system atrophy patients from healthy subjects.

[0101] According to the method, system, composition, or kit of the present application, CNPase protein and α-syn protein can be effectively detected as dual markers, making it possible to distinguish Parkinson's disease patients and multiple system atrophy patients from healthy subjects. EXAMPLES

[0102] Experimental Method 1: (a) Peripheral body fluid pretreatment - ultracentrifugation and concentration of extracellular vesicles 1. Thaw plasma from the subject in a 37℃ incubator for 10-15 minutes, then vortex mix evenly. 2. Centrifuge at 10,000 g for 30 minutes at 4°C. 3. After centrifugation, 100 μL of plasma is aspirated into an ultracentrifuge tube, and 900 μL of PBS buffer (0.22 μm filtered) is added and mixed uniformly. 4. Ultracentrifuge at 100,000 g for 1 hour at 4°C. 5. Discard the supernatant, leaving approximately 100 μL of pellet. 6. Add 200 μL of PBS buffer (0.22 μm filtered) and resuspend by pipetting 200 times with a 200 μL pipette. 7. Add 700 μL of PBS buffer (0.22 μm filtered) to the resuspended solution and mix evenly. 8. Ultracentrifuge at 100,000 g for 1 hour at 4°C. 9. Discard the supernatant, leaving approximately 50 μL of pellet. 10. Add 100 μL of PBS buffer (0.22 μm filtered), pipette 200 times using a 200 μL pipette, resuspend, and store until use. (b) Labeling of antibodies for biomarker detection by a labeling method Reagents used: The antibody targeting the extracellular vesicle biomarker from the central nervous system and the antibody targeting the Alzheimer's disease biomarker are labeled with Alexa fluorescent labels. The antibody targeting the extracellular vesicle biomarker from the central nervous system is a SHISA9 polyclonal antibody. The antibody targeting the Alzheimer's disease biomarker is a p-tau217 polyclonal antibody. Here, for the labeled reagents, the SHISA9 polyclonal antibody label is purchased as Zenon™ Alexa Fluor™ 405, and the p-tau217 polyclonal antibody label is purchased as Zenon™ Alexa Fluor™ 488. 1. Sample Block 1.1 Place 10 μL of extracellular vesicles ultracentrifuged by the above method into a 1.5 mL centrifuge tube or PCR tube. 1.2 Add an equal volume of 2% BSA solution (0.22 μm filtered) and mix evenly. 1.3 Incubate at room temperature to block for 1 hour. 1.4 Add 10 μL of PBS buffer (0.22 μm filtered) to dilute the blocked sample. 2. Labeling of samples In this experiment, the neurospecific marker antibody SHISA9 and the disease-related marker antibody p-tau217 are labeled with Zenon™ Alexa Fluor™ 405 and Zenon™ Alexa Fluor™ 488, respectively. 2.1 Prepare 1 μg of antibody in PBS buffer (0.2 μg / μL in 5 μL PBS). 2.2 Add 5 μL of dye to the antibody (1 μg), mix evenly, and then incubate at room temperature (25°C) in the dark for 20 minutes. 2.3 Add 3 μL (3 μg) of blocking agent (IgG blocking agent diluted to 1 μg / μL) to the solution in step 3.2, mix evenly, and then incubate at room temperature (25°C) in the dark for 10 minutes. 2.4 Dilute the above mixture to a total volume of 50 μL with PBS buffer (0.22 μm filtered). 2.5 Add 5 μL of Zenon™ Alexa Fluor™ 488-labeled p-tau217 to the sample prepared in step (a), mix uniformly, and then incubate at room temperature (25° C.) in the dark for 5 minutes. 2.6 Add 3 μL of Zenon™ Alexa Fluor™ 405-labeled SHISA9 to the sample from step 2.5, mix thoroughly, and then incubate overnight at 4° C. in the dark. 3. Fixation of samples 3.1 Add 20 μL of 4% PFA to the labeled sample, mix evenly, and then incubate at room temperature (25°C) in the dark for 20 minutes. 3.2 Dilute the immobilized sample with 170 μL of PBS buffer (0.22 μm filtered) and store until use. (c) Detection The NanoFCM nanoflow analyzer detects the label signal intensity and particle size distribution to confirm the presence and content of relevant biomarkers.

[0103] Example 1 Plasma samples were collected from 98 sex- and age-matched Alzheimer's disease subjects (AD group) and 12 healthy controls (CT group), and experiments were performed according to Experimental Method 1 described above. The numbers of extracellular vesicles single fluorescently labeled with SHISA9 antibody, single fluorescently labeled with p-tau217 antibody, and dual fluorescently labeled with SHISA9 and p-tau217 antibodies were detected using the NanoFCM nanoflow analyzer, and the ratios of these to the total number of extracellular vesicles were calculated. The differences between groups for each particle size range were analyzed. Regarding the ratio of single-labeled SHISA9 protein-positive extracellular vesicles in plasma to the total number of extracellular vesicles, significant differences were observed in the 60-100 nm, 100-150 nm, and entire size ranges in both the AD and CT groups (**: p<0.01) (Figure 1A). Regarding the ratio of single-labeled p-tau217 protein-positive extracellular vesicles in plasma to the total number of extracellular vesicles, very significant differences were observed in the 60-100 nm and entire size ranges in both the AD and CT groups. A large significant difference was observed (****: p<0.0001), and in the particle size range of 100-150 nm, a significant difference was observed between the AD group and the CT group (**: p<0.01) (Figure 1B). Regarding the ratio of SHISA9 protein-p-tau217 protein double-positive extracellular vesicles in plasma to the total number of extracellular vesicles, a very large significant difference was observed in both the AD group and the CT group in the particle size range of 100-150 nm and in the entire particle size range (***: p<0.001) (Figure 1C). Diagnostic efficiency was evaluated by receiver operating characteristic curve (ROC) analysis. The area under the curve (AUC) of the SHISA9 protein single marker was 0.749 in the particle size range of 60-100 nm, and the AUC of the SHISA9 protein and p-tau217 protein double marker was 0.890 in the particle size range of 100-150 nm. When the SHISA9 60-100 nm and SHISA9+p-tau217 100-150 nm data were introduced by the logistic regression analysis method, the AUC was 0.891 (Figure 1D). The p-tau217 protein single marker has an AUC of 0.877 in the particle size range of 60-100 nm, and the SHISA9 protein and p-tau217 protein double marker has an AUC of 0.890 in the particle size range of 100-150 nm. When the p-tau217 60-100 nm and SHISA9+p-tau217 100-150 nm data were introduced by the logistic regression analysis Enter method, the AUC was 0.910 (Figure 1E). From the results of Example 1, SHISA9 protein-positive extracellular vesicles, p-tau217 protein-positive extracellular vesicles, and SHISA9 protein-p-tau217 protein double-positive extracellular vesicles all have high potential for diagnosing Alzheimer's disease, especially when p-tau217 protein-positive extracellular vesicles and SHISA9 protein + p-tau217 protein double-positive extracellular vesicles are used in combination for diagnosis.

[0104] Experimental Method 2: (a) Peripheral body fluid pretreatment - ultracentrifugation and concentration of extracellular vesicles 1. The subject's plasma was rapidly thawed in a 37°C water bath for 1 minute, centrifuged at 2,000g for 15 minutes at 4°C, transferred to a separate 1.5mL centrifuge tube, and then centrifuged at 12,000g for 30 minutes at 4°C. 2. 100 μL of plasma is transferred to a 1.5 ml ultracentrifuge tube, diluted with 900 μL of 0.22 μm filtered PBS, and centrifuged at 100,000 g for 30 minutes at 4° C. 3. Carefully discard 800 μL of the supernatant, add an equal volume of 0.22 μm filtered PBS, mix uniformly, and then centrifuge at 100,000 g for 30 minutes at 4°C. Leave 200 μL of the supernatant and repeatedly pipette the tube wall using a gun head to resuspend and mix the extracellular vesicles uniformly. Individually package and store at -80°C. 4. Transfer 20 μL of extracellular vesicles to a 100 kDa ultrafiltration tube, add 200 μL of 0.1% Triton X-100 / PBS, and incubate at room temperature for 30 minutes. 5. Centrifuge the ultrafiltration tube at 12,000 g for 3 minutes at 4°C, add 200 μL of 0.22 μm filtered PBS, and further centrifuge. 6. Transfer the liquid remaining in the ultrafiltration tube to a new 1.5 ml centrifuge tube and store until use. (b) Labeling of antibodies for biomarker detection by a labeling method The antibody targeting the extracellular vesicle biomarker from the central nervous system and the antibody targeting the Alzheimer's disease biomarker are labeled with Alexa fluorescent labels. The antibody targeting the extracellular vesicle biomarker from the central nervous system is a GLT1 monoclonal antibody, and the antibody targeting the Parkinson's disease biomarker is an α-synuclein (α-syn) monoclonal antibody (syn211 and MJFR14). Here, for the labeled reagents, the label for the GLT1 monoclonal antibody is purchased as Zenon™ Alexa Fluor™ 405, and the label for the α-synuclein (α-syn) monoclonal antibody is purchased as Zenon™ Alexa Fluor™ 488. 1. Label the GLT1 antibody using the Zenon™ Alexa Fluor™ 405 kit according to the product instructions: incubate with 5 μL fluorescent secondary antibody + 1 μg GLT1 antibody for 5 minutes at room temperature, protected from light, then add 5 μL IgG and incubate for 5 minutes at room temperature, protected from light. 2. Add 0.2 μg of GLT1 antibody to each sample and incubate at room temperature in the dark for 30 minutes. 3. Label α-synuclein monoclonal antibody (syn211 or MJFR14) using Zenon™ Alexa Fluor™ 488 kit according to the product instructions: incubate with 5 μL fluorescent secondary antibody + 1 μg α-syn antibody for 5 minutes at room temperature, protected from light, then add 5 μL IgG and incubate for 5 minutes at room temperature, protected from light. 4. Add 0.2 μg of α-syn antibody (syn211 or MJFR14) to each sample and incubate at room temperature in the dark for 30 minutes. 5. Dilute the sample to 400 μL with 0.22 μm filtered PBS and store until use. (c) Detection The label signal intensity is detected by an Apogee nanoflow analyzer to confirm the presence and content of CNS-derived disease-related biomarkers in peripheral body fluids.

[0105] Example 2 Using the above experimental method 2, plasma samples were collected from a clinical cohort (103 Parkinson's disease (PD) subjects, 45 multiple system atrophy (MSA) subjects, and 106 age- and sex-matched healthy controls (CT)) and detected by immunofluorescence labeling technique using an Apogee nano flow cytometer. Immunofluorescence labeling technique using Apogee nanoflow analyzer can detect GLT1 protein-positive extracellular vesicles and α-syn protein (syn211 and MJFR14 are all anti-α-syn protein antibodies) positive extracellular vesicles (Figure 2A), respectively, and can simultaneously detect GLT1 protein-α-syn protein double positive extracellular vesicles, with excellent reliability and reproducibility (Figure 2B and C). Using the above experimental method 2, plasma samples were collected from a clinical cohort (103 Parkinson's disease (PD) subjects, 45 multiple system atrophy (MSA) subjects, and 106 age- and sex-matched healthy controls (CT)). The number of GLT1 protein-α-syn protein double-positive extracellular vesicles in plasma was detected by immunofluorescence labeling technique using an Apogee nano flow cytometer. The results showed that the number of GLT1 protein-α-syn protein double-positive extracellular vesicles in the PD group was significantly higher than that in the MSA and CT groups (***: p<0.001), while no significant difference was observed between the MSA and CT groups (Figures 3A and B). The number of GLT1-α-syn double-positive extracellular vesicles, when used in clinical diagnosis, could effectively distinguish PD patients from healthy controls, and PD patients from MSA patients, and had clinical potential in the diagnosis of Parkinson's disease and differential diagnosis of MSA. In CN106062559B, it has been reported that exosomes (a type of extracellular vesicle with a diameter ranging from approximately 30 to 150 nm) derived from the central nervous system are first enriched using L1CAM as a marker, and then related markers in the exosomes are detected using a Luminex detection platform. Using a cohort of 267 Parkinson's disease patients (PD group) and 215 age- and sex-matched healthy controls (CT group), we performed detection of the plasma-associated protein marker α-syn, i.e., direct detection of α-syn protein in plasma. As a result, there was no statistical difference between the PD group and the CT group (see Figures 1 and 2 of CN106062559B), whereas the α-syn protein in plasma exosomes captured by anti-L1CAM was significantly higher than that in the control group. The quality of α-syn and the ratio of this α-syn protein to total α-syn protein in plasma were statistically significantly elevated in the PD group (**: p<0.01) (see Figures 1 and 2 in CN106062559B), and the significance of the difference was comparable when compared with α-syn protein in cerebrospinal fluid (clinical cohort: 100 Parkinson's disease patients and 100 healthy controls) (**: p<0.01) (see Figures 1 and 2 in CN106062559B). Furthermore, we evaluated the potential of α-syn protein in plasma exosomes captured by anti-L1CAM for the clinical diagnosis of Parkinson's disease by performing Pearson correlation analysis and Receiver Operating Characteristic Curve (ROC) analysis of this α-syn protein. As a result, we found that the level of α-syn protein in plasma exosomes captured by anti-L1CAM was correlated with Parkinson's UPDRS motor score and disease severity (r=0.176, p=0.004, Pearson correlation) (see Figure 3C in CN106062559B). The concentration of α-syn protein in exosomes (AUC=0.654, sensitivity=70.1%, specificity=52.9%) (see Figure 3A in CN106062559B) and the ratio of this concentration to total α-syn protein in plasma (AUC=0.657, sensitivity=71.2%, specificity=50.0%) could all provide moderate diagnostic efficiency, which was equivalent to the diagnostic efficiency of α-syn protein in cerebrospinal fluid (AUC=0.724, sensitivity=76.8%, specificity=53.5%) (see Figure 3B in CN106062559B). In the Chinese invention patent application with application number 201810136747.8, plasma samples from a clinical cohort (46 patients with multiple system atrophy (MSA group), 49 patients with Parkinson's disease (PD group), and 50 age- and sex-matched healthy controls (CT group)) were collected and the concentration of fluorescently labeled GLT1 (EAAT2) extracellular vesicles was measured using a Nanosight nanoparticle tracking analyzer to evaluate the potential of the concentration of extracellular vesicles containing GLT1 fluorescent label in diagnosing PD. Receiver operating characteristic curve analysis (ROC analysis) was performed. Observation of the differential diagnostic efficiency between multiple system atrophy and Parkinson's disease showed that the diagnostic efficiency for Parkinson's disease was moderate (AUC=0.6750, sensitivity=66.00%, specificity=70.45%), and the differential diagnostic efficiency between Parkinson's disease and multiple system atrophy was moderate (AUC=0.6948, sensitivity=65.91%, specificity=78.57%) (see Figure 3 of Chinese Application No. 201810136747.8). As described above, compared with the reported methods, the system or method of the present application achieves a similar PD diagnostic effect, while the method of the present application simultaneously detects two or more markers in biological fluids, thereby shortening the time required for detection of multiple markers, improving efficiency, and enabling wider clinical application and usability.

[0106] Experimental Method 3: (a) Peripheral fluid pretreatment - ultrafiltration and centrifugation 1. Thaw approximately 100 μL of the subject's plasma in a 37°C water bath, centrifuge at 2,000 g for 15 minutes at 4°C, transfer to a new 1.5 mL centrifuge tube, and then centrifuge at 12,000 g for 30 minutes at 4°C. 2. After centrifugation, transfer the plasma supernatant to a new 1.5 ml centrifuge tube and mix thoroughly. 3. Add 200 μL of 0.1% Triton X-100 / PBS after 0.22 μm filtration to a 100 kDa ultrafiltration tube, then add 50 μL of plasma after centrifugation, and incubate at room temperature for 30 minutes. 4. After incubation, centrifuge at 12,000 g for 3.5 minutes at 4°C. 5. Add 200 μL of 0.1% Triton X-100 / PBS after 0.22 μm filtration to the ultrafiltration tube, and then centrifuge at 12,000 g for 3.5 minutes at 4° C. 6. Aspirate the remaining part after filtration (the part of the inner core of the ultrafiltration tube) with a pipette and dispense the required volume for each reaction unit at 20 μL per pipette. Store until use. (b) Labeling of antibodies for biomarker detection by a labeling method Reagents used: The antibody targeting the extracellular vesicle biomarker from the central nervous system and the antibody targeting the Alzheimer's disease biomarker are labeled with Alexa fluorescent labels. The antibody targeting the extracellular vesicle biomarker from the central nervous system is a CNPase monoclonal antibody. The antibody targeting the multiple system atrophy biomarker is an alpha-synuclein (alpha-syn) monoclonal antibody. For the labeling reagents, the label for the CNPase monoclonal antibody is purchased as Zenon™ Alexa Fluor™ 488, and the label for the alpha-synuclein (alpha-syn) monoclonal antibody is purchased as Zenon™ Alexa Fluor™ 405. 1. Label CNPase antibody using Zenon™ Alexa Fluor™ 488 kit according to the product instructions: incubate with 5 μL fluorescent secondary antibody + 1 μg CNPase antibody for 15 minutes at room temperature, protected from light, then add 5 μL IgG and incubate for 5 minutes at room temperature, protected from light. 2. Add 0.2 μg of CNPase antibody to each sample (20 μL) and incubate at room temperature in the dark for 30 minutes. 3. Label α-synuclein monoclonal antibody using Zenon™ Alexa Fluor™ 405 kit according to the product instructions: incubate with 5 μL fluorescent secondary antibody + 1 μg α-syn antibody for 15 minutes at room temperature, protected from light, then add 5 μL IgG and incubate for 5 minutes at room temperature, protected from light. 4. Add 0.2 μg of α-syn antibody to each sample (20 μL) and incubate at room temperature in the dark for 30 minutes. 5. Dilute the sample to 400 μL with 0.22 μm filtered PBS and store until use. (c) Detection The label signal intensity is detected by an Apogee nanoflow analyzer to confirm the presence and content of CNS-derived disease-related biomarkers in peripheral body fluids.

[0107] Example 3 Using immunofluorescence labeling technology with an Apogee nano flow cytometer, plasma samples from a small clinical cohort (15 Parkinson's disease (PD) subjects, 17 multiple system atrophy (MSA) subjects, and 17 age- and sex-matched healthy controls (HC)) were detected according to the above experimental method 3. As a result, CNPase protein-positive extracellular vesicles were detected in the plasma, and the number of CNPase protein-α-syn protein (anti-MJFR14 antibody labeled) double-positive extracellular vesicles was not significantly different between the MSA group and the PD group, while a significant difference was observed between the MSA group and the HC group (*: p<0.05) (Figure 4A). The number of CNPase protein-α-syn protein (anti-MJFR14 antibody labeled) double-positive extracellular vesicles was not significantly different between the MSA group, the PD group, and the HC group (Figure 4B), and when normalized by the total number of extracellular vesicles in plasma, a significant difference was observed between the MSA group and the HC group, and between the MSA group and the PD group (*: p<0.05) (Figure 4C). The number of CNPase protein-α-syn protein double-positive extracellular vesicles is expected to be useful in the diagnosis of multiple system atrophy (MSA) and in the differential diagnosis between MSA and Parkinson's disease (PD).

[0108] The above is merely a preferred embodiment of the present invention, and does not limit the present invention in any way, and those skilled in the art may modify the above technical content or convert it into an equivalent embodiment with equivalent changes. All simple modifications, equivalent changes and modifications made to the above embodiments based on the technical spirit of the present invention without departing from the content of the technical solution of the present invention are within the patent scope of the technical solution of the present invention.

Claims

1. 1. A system for diagnosing a central nervous system disease, comprising: a first detection module configured to detect a central nervous system-derived marker in a biological fluid of a subject; a second detection module configured to detect a central nervous system disease associated marker in a biological fluid of the subject; and a diagnosis module configured to diagnose whether or not the subject is suffering from a central nervous system disease based on the detection results of the central nervous system-derived marker and the central nervous system disease-related marker obtained by the first detection module and the second detection module, respectively. the first detection module and the second detection module simultaneously detect a central nervous system-derived marker in the biological fluid of the subject and a central nervous system disease-related marker in the biological fluid of the subject; or the first detection module and the second detection module are identical modules configured to simultaneously detect a central nervous system-derived marker in the subject's biological fluid and a central nervous system disease-related marker in the subject's biological fluid; The central nervous system-derived marker is SHISA9, and the central nervous system disease-related marker is phosphorylated tau (p-tau), or The central nervous system-derived marker is GLT1 (EAAT2) and the central nervous system disease-associated marker is oligomeric α-synuclein (oligo-α-syn); or The central nervous system-derived marker is CNP enzyme, and the central nervous system disease-related marker is oligomeric α-synuclein (oligo-α-syn), The system, wherein the central nervous system disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD) and multiple system atrophy (MSA).

2. and a concentration module configured to obtain and pre-treat a biological fluid from the subject and concentrate the biological sample in the biological fluid; The system of claim 1 , wherein the first detection module and the second detection module detect central nervous system-derived markers and central nervous system disease-associated markers in an enriched biological sample from a subject.

3. 3. The system of claim 2, wherein the concentration module has sub-modules configured to perform one or more of the steps of centrifugation, ultracentrifugation, filtration through ultrafiltration tubes, polymerization precipitation, and specific antibody capture.

4. A quantity counting module configured to count the number of biological samples in which both the CNS-derived marker and the CNS disease-associated marker are detected; and / or A particle size measurement module configured to measure particle size of a biological sample in which both a central nervous system-derived marker and a central nervous system disease-related marker are detected; The system of any one of claims 1 to 3, further comprising a diagnostic module configured to diagnose whether a subject is suffering from a central nervous system disease based on the number of biological samples and / or the particle size of the biological samples.

5. the biological sample is an extracellular vesicle; or The biological fluid is one or more selected from blood, serum, plasma, saliva, urine, lymph, semen, and milk; 4. A system according to claim 2 or 3.

6. The extracellular vesicles are neuron-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles, or microglia-derived extracellular vesicles. The system of claim 5.

7. The system according to any one of claims 1 to 6, wherein the central nervous system-derived marker is a biomarker derived from neurons or cells of mature neurons, oligodendrocytes, astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate progenitor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons.

8. The system according to any one of claims 1 to 7, wherein the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system.

9. The system of claim 1, wherein the first detection module is configured to react the subject's biological fluid with a labeled antibody that specifically reacts with the central nervous system-derived marker, and detect the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system-derived marker.

10. The system of claim 9, wherein the first detection module is configured to react the subject's extracellular vesicles with a labeled antibody that specifically reacts with the central nervous system-derived marker, and detect the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system-derived marker.

11. The system of claim 1, wherein the central nervous system disease-associated marker is a protein, and the second detection module is configured to react the subject's biological fluid with a labeled antibody that specifically reacts with the central nervous system disease-associated marker, and detect the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system disease-associated marker.

12. The system of claim 11, wherein the central nervous system disease-associated marker is a protein, and the second detection module is configured to react the subject's extracellular vesicles with a labeled antibody that specifically reacts with the central nervous system disease-associated marker, and detect the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system disease-associated marker.

13. The system according to any one of claims 9 to 12, wherein the label is one or more selected from a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, and a gold colloid label.

14. A composition for detecting a central nervous system disease, comprising: an antibody used to target a central nervous system derived marker; and an antibody used to target a central nervous system disease associated marker; The central nervous system-derived marker is SHISA9, and the central nervous system disease-related marker is phosphorylated tau (p-tau), or The central nervous system-derived marker is GLT1 (EAAT2) and the central nervous system disease-associated marker is oligomeric α-synuclein (oligo-α-syn); or The central nervous system-derived marker is CNP enzyme, and the central nervous system disease-related marker is oligomeric α-synuclein (oligo-α-syn), The composition, wherein the central nervous system disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD) and multiple system atrophy (MSA).

15. The central nervous system-derived markers are biomarkers derived from neurons or cells of mature neurons, oligodendrocytes, astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate progenitor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons; The composition of claim 14.

16. The central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system.

16. The composition according to claim 14 or 15.

17. The composition of any one of claims 14 to 16, wherein the antibody used to target the central nervous system derived marker and the antibody used to target the central nervous system disease associated marker are labeled antibodies.

18. The composition according to claim 17 , wherein the label is one or more selected from the group consisting of a fluorescent label, an isotope label, an enzyme label, a chemiluminescent label, a quantum dot label, and a gold colloid label.

19. A kit for detecting a central nervous system disease in a subject, comprising: a reagent used to detect a central nervous system-derived marker in a biological fluid of a subject; and a reagent used to detect a central nervous system disease-associated marker in a biological fluid of a subject, The central nervous system-derived marker is SHISA9, and the central nervous system disease-related marker is phosphorylated tau (p-tau), or The central nervous system-derived marker is GLT1 (EAAT2) and the central nervous system disease-associated marker is oligomeric α-synuclein (oligo-α-syn); or The central nervous system-derived marker is CNP enzyme, and the central nervous system disease-related marker is oligomeric α-synuclein (oligo-α-syn), The kit, wherein the central nervous system disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD) and multiple system atrophy (MSA).

20. The kit according to claim 19, wherein the reagent used to detect a central nervous system-derived marker in the biological fluid of the subject and the reagent used to detect a central nervous system disease-related marker in the biological fluid of the subject are compositions according to any one of claims 14 to 18.

21. 21. The kit of claim 19 or 20, further comprising reagents and equipment used to obtain a biological sample from a subject.

22. The kit of claim 19 or 20, further comprising reagents and equipment used to obtain extracellular vesicles from a subject.

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