WO2019153748A1

WO2019153748A1 - Method for enriching or detecting exosome of astrocyte source in biological fluid

Info

Publication number: WO2019153748A1
Application number: PCT/CN2018/106030
Authority: WO
Inventors: 章京; 于震维; 兰国钰
Original assignee: 北京大学
Priority date: 2018-02-09
Filing date: 2018-09-17
Publication date: 2019-08-15
Also published as: CN110133272A

Abstract

A method for enriching or detecting, in biological fluid, exosomes of an astrocyte source. The method for enriching exosomes of an astrocyte source comprises the following steps: (a) causing biological fluid that contains exosomes of an astrocyte source to come into contact with an anti-GLT1 antibody so as to form an immune complex, wherein the biological fluid is selected from one or more of blood, serum, plasma, saliva and urine; and (b) using a solid phase or liquid phase method by means of the immune complex to enrich the exosomes of the astrocyte source. A biological marker of exosomes of an astrocyte source may be detected and used for auxiliary diagnoses, differential diagnoses and monitoring central nervous system diseases such as Alzheimer's disease, Parkinson's disease, multiple system atrophy, prion diseases, and nervous system tumors such as glioma and the like so as to prevent acquiring markers by means of a traumatic operation.

Description

Method for enriching or detecting astrocyte-derived exosomes from biological fluids

Technical field

The present invention relates to the field of biomedical technology, and in particular to a method for enriching or detecting astrocyte-derived exosomes from biological fluids.

Background technique

The need for biomarkers for assisted diagnosis, differential diagnosis, and monitoring of central nervous system diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple system atrophy (MSA), and prion diseases is urgent. Among the current biomarkers, the better performing markers were developed based on cerebrospinal fluid (CSF). CSF directly contacts the brain and spinal cord and is better able to respond to changes in the central nervous system. However, acquisition of CSF is a traumatic procedure that severely limits the routine use of cerebrospinal fluid biomarkers. Therefore, the development of peripheral biomarkers for central nervous system diseases, such as in the blood, is important. However, due to the presence of the blood-brain barrier, it is difficult to detect components derived from the central nervous system (such as astrocytes) in peripheral body fluids. For example, only a small fraction of human brain/CSF-derived proteins can be readily detected in plasma (Pan et al, J. Proteome Res., 13:4535-4545, 2014). In view of the above reasons, although it took several decades and a large amount of funding, peripheral biomarkers for AD, PD, MSA or prion diseases have not yet been established. In addition, when existing disease-related proteins such as tau, Aβ, and α-syn in peripheral biological fluids are measured using existing techniques, peripheral-derived proteins significantly affect signals of central nervous system diseases.

Therefore, how to enrich and label biomarkers that reflect changes in central nervous system diseases from peripheral biological fluids is the focus of the present invention.

Summary of the invention

The present invention aims to provide a method for enriching or detecting astrocyte-derived exosomes from biological fluids to solve existing organisms for assisting diagnosis, differential diagnosis and monitoring of central nervous system diseases. The marker acquires a technical problem that is a traumatic operation and cannot be routinely applied.

In order to achieve the above object, according to an aspect of the present invention, a method for enriching astrocyte-derived exosomes from a biological fluid is provided. The method comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, and saliva. One or more; and (b) enriching astrocyte-derived exosomes by a method of using the immune complex in a solid phase or a liquid phase.

Further, the step (b) comprises: binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; separating the solid-phase-bound exosomes from the biological fluid to enrich the star Glial cell-derived exosomes.

Further, the anti-GLT1 antibody in step (a) was immobilized on a solid phase.

Further, the method further comprises the step (c) of eluting the bound exosomes from the solid phase.

Further, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises enriching astrocyte-derived exosomes by flow cytometry.

Further, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, or a quantum dot.

According to another aspect of the present invention, there is provided the use of an anti-GLT1 antibody for the preparation of an astrocyte-derived biomarker for detecting a neurological disease in a biological fluid. The use comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers.

Further, the biomarker is a nucleic acid, protein, lipid or astrocyte derived exosomes per se.

Further, the step (b) comprises: (b1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; (b2) separating the solid phase-bound exud from the biological fluid And (b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2).

Further, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises: directly detecting astrocyte-derived exosomes in the biological fluid by means of the biomarker; Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold; preferably, directly detected by a particle size analyzer, a gamma counter or a small animal in vivo imager The concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Further, the nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; when the nervous system disease is Parkinson's disease, the biomarker is α-synuclein, phosphorylated α- Synuclein and astrocytes are derived from exosomes themselves; when the nervous system disease is Alzheimer's disease, the biomarker is tau and phosphorylated tau; when the nervous system disease is prion, biomarker Is a prion protein; when the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level of the astrocyte-derived exosome and the concentration of the astrocyte-derived exosome And particle size distribution, wherein the astrocyte-derived exogenous biomarkers include α-synuclein, α-synuclein oligomer, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to still another aspect of the present invention, there is provided the use of an anti-GLT1 antibody for the preparation of a preparation for detecting a nervous system disease in a subject. The use comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers and, in comparison with the control levels from subjects without neurological disease, from the subject Differences in the levels of biomarkers of astrocyte-derived exosomes significantly indicate that the subject has a neurological disorder.

Further, step (b) comprises: (b1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; (b2) separating the solid phase-bound exosomes; and B3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2), and comparing with the control level from the subject without the neurological disease, Differences in the levels of biomarkers from astrocyte-derived exosomes in the tester significantly indicate that the subject has a neurological disorder.

Further, the nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; when the nervous system disease is Parkinson's disease, the biomarker is α-synuclein, phosphorylated α- Concentration and particle size distribution of synuclein and astrocyte-derived exosomes; when the neurological disease is Alzheimer's disease, the biomarker is tau and phosphorylated tau; when the nervous system disease is prion Disease, the biomarker is a prion protein; when the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level of astrocyte-derived exosomes and a source of astrocytes Concentration and particle size distribution of exosomes, wherein biomarkers derived from astrocyte-derived exosomes include α-synuclein, α-synuclein oligomer, PS129, tau, phosphorylated tau Protein and Abeta protein.

According to still another aspect of the present invention, an apparatus for detecting an astrocyte-derived biomarker of a nervous system disease in a biological fluid is provided. The device comprises: an exosome enrichment module for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, the biological fluid being selected from the group consisting of blood, serum, plasma, and saliva. And one or more of the urine; and a biomarker level determining module for determining the level of the astrocyte-derived biomarker.

Further, an exosome enrichment module is used for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, and a star colloid in the biological fluid through the immune complex. The cell-derived exosomes bind to the solid phase and separate the solid phase-bound exosomes from the biological fluid; and the biomarker level determining module is used to determine the solid phase binding separated in the exosome enrichment module The level of astrocyte-derived biomarkers in exosomes.

Further, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody in the exosome enrichment module is a labeled antibody, and the biomarker level determining module directly detects the star in the biological fluid by means of the label Cell-derived exosomes; preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold; preferably, the biomarker level determining module passes the particle size analyzer The gamma counter or the small animal living imager directly detects the concentration, particle size distribution and tissue distribution of the astrocyte-derived exosomes in the biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585 , Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to still another aspect of the present invention, an apparatus for detecting a nervous system disease in a subject is provided. The device comprises: an exosome enrichment module for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, the biological fluid being selected from the group consisting of blood, serum, plasma, and saliva. And one or more of the urine; and a comparison module for determining the level of the astrocyte-derived biomarker and comparing it to the control level from the subject without the neurological disease Significant differences in the levels of biomarkers from astrocyte-derived exosomes in the tester are indicative of the subject's information on neurological disorders.

Further, an exosome enrichment module is used for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, and a star colloid in the biological fluid through the immune complex. The cell-derived exosomes bind to the solid phase and separate the solid phase-bound exosomes from the biological fluid; and the comparison module is used to determine the solid-bound exosomes of the exosomal enrichment module The level of a biomarker derived from a glial cell and, compared to a control level from a subject without a neurological disease, a biomarker from an astrocyte-derived exosome in the subject Significant differences in levels indicate that the subject has information about a neurological disorder.

Further, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody in the exosome enrichment module is a labeled antibody, and the comparison module directly detects the astrocyte-derived exudation in the biological fluid by means of the label. And, compared to a control level from a subject without a neurological disease; preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold; Preferably, the biomarker level determining module directly detects the concentration, particle size distribution and tissue distribution of the astrocyte-derived exosomes in the biological fluid by a particle size analyzer, a gamma counter or a small animal living imager; more preferably, the fluorescence The markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to still another aspect of the present invention, a method for detecting an astrocyte-derived biomarker of a nervous system disease in a biological fluid is provided. The method comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers.

Further, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises: directly detecting astrocyte-derived exosomes in the biological fluid by means of the biomarker; Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold; preferably, the biomarker level determining module passes a particle size analyzer, a gamma counter or a small The animal living body imager directly detects the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in the biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655 , Qdot705 or Qdot800.

According to still another aspect of the present invention, a method for detecting a nervous system disease in a subject is provided. The method comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers and, in comparison with the control levels from subjects without neurological disease, from the subject Differences in the levels of biomarkers of astrocyte-derived exosomes significantly indicate that the subject has a neurological disorder.

Further, step (b) comprises: b1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; (b2) separating the solid phase-bound exosomes from the biological fluid And (b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2).

Further, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises: directly detecting astrocyte-derived exosomes in the biological fluid by means of the biomarker, And the level difference of biomarkers from astrocyte-derived exosomes in the subject significantly indicates that the subject has a nervous system disease compared to a control level from a subject without a neurological disease; Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold; preferably, directly detected by a particle size analyzer, a gamma counter or a small animal in vivo imager The concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to still another aspect of the present invention, there is provided the use of GLT1 as an astrocyte-derived exosome biomarker.

Further, the application comprises: enriching astrocyte-derived exosomes from a biological fluid, preparing an astrocyte-derived biomarker for detecting a nervous system disease in a biological fluid, Detection of astrocyte-derived biomarkers for neurological diseases in biological fluids and detection of neurological diseases in subjects

The astrocyte-derived exosomes enriched by the technical solution of the present invention can be used for detecting biomarkers of astrocyte-derived exosomes, assisting diagnosis, differential diagnosis and monitoring of central nervous system diseases such as Alzheimer's disease, Parkinson's disease, multiple system atrophy, prion disease and nervous system tumors such as glioma, to avoid traumatic manipulation to obtain markers, reduce the pain of patients, suitable for routine clinical applications.

DRAWINGS

The accompanying drawings, which are incorporated in the claims of the claims In the drawing:

Figure 1 shows: A. Western blotting of Alix (general exosome label) and GLT1 by anti-GLT1 capture, or normal mouse IgG capture in Example 1, B. Plasma cell captured by GLT1 antibody in Example 1. Comparison of the concentration of the outer vesicles with the plasma extracellular vesicles captured by normal mouse IgG antibodies.

Figure 2 shows: A. Evaluation of the concentration of GLT1 fluorescently labeled exosomes in clinical plasma samples in Example 3; B. Measurement of GLT1 fluorescently labeled plasma exosomes using a particle size analyzer (Nanosight 300) in Example 3 Concentration and distribution.

Figure 3 shows the ROC (Receiver Operating Characteristics) analysis of GLT1 fluorescently labeled exosomal concentrations for PD diagnosis and differential diagnosis with MSA in Example 3. In the entire cohort (46 MSA patients, 49 PD patients, and 50 healthy controls), the differential diagnosis efficiency of multiple system atrophy and Parkinson's disease was observed. The area under the curve (AUC) = 0.6948, sensitivity = 65.91%, specificity. =78.57%; observe the diagnostic efficiency of Parkinson's disease and healthy controls, the area under the curve = 0.6750, sensitivity = 66%, specificity = 70.45%.

Fig. 4 shows the results of detection of α-syn in Example 5.

Detailed ways

It should be noted that the embodiments in the present application and the features in the embodiments may be combined with each other without conflict. The invention will be described in detail below with reference to the drawings in conjunction with the embodiments.

definition

"Binding pair" as used herein refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but are not limited to, antigens and antibodies to the above antigens, ligands and their receptors, nucleic acid complementary strands, biotin and avidin, biotin and streptavidin, phytohemagglutinin (lectin) , lectin) and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, digioxigenin/anti-digoxigenin.

"Astrocyte-derived exosomes" as used herein refers to exosomes derived from astrocytes.

An "exosome" as used herein is a 30-150 nm extracellular vesicle that can be released by many different types of cells and performs different cellular functions, including intercellular communication, antigen presentation, and Transfer of proteins and nucleic acids.

"Fixed" as used herein means that the reagent is immobilized to a solid surface. When the reagent is immobilized to a solid surface, it is non-covalently bound or covalently bound to the surface.

"GLT1" as used herein refers to a glutamate transporter 1 expressed by astrocytes.

The inventors have found that although the mechanism has not been determined, astrocyte-derived exosomes can cross the blood-brain barrier and enter the peripheral blood.

The inventors have surprisingly discovered that "GLT1" is a unique, disease-specific biomarker carried by astrocyte-derived exosomes and can be used as a biomarker for astrocyte-derived exosomes. According to this, astrocyte-derived exosomes can be detected in vivo in blood and other peripheral body fluids, astrocyte-derived exosomes are enriched from biological fluids, and nerves are prepared for detecting nerves in biological fluids. A formulation of an astrocyte-derived biomarker for a systemic disease, an astrocyte-derived biomarker for detecting a neurological disease in a biological fluid, and detection of a neurological disease in a subject.

The present inventors have found a method for isolating and labeling exosomes derived from astrocytes in biological fluids such as blood, serum, plasma, saliva or urine.

The present inventors have also discovered that astrocyte-derived central nervous system biomarkers can be detected and quantified by astrocyte-derived exosomes derived from biological fluids, and the above results can be used for Detection of neurological diseases such as AD, PD, MSA and prion diseases, these astrocyte-specific markers based on peripheral body fluids can also be used for differential diagnosis of related diseases, monitoring disease progression and objectively evaluating central nervous system The therapeutic effect of systemic diseases.

Based on the above findings, the inventors of the present invention have proposed to determine astrocyte-derived substances by measuring biomarker levels in astrocyte-derived exosomes enriched from biological fluids (biological The method of the level of the marker).

The present invention relates to a method for labeling astrocyte-derived exosomes from a biological fluid of a subject, the method comprising the steps of: (a) forming a chemically coupled structure with an anti-GLT1 antibody and a fluorescent dye; b) contacting a biological fluid containing an astrocyte-derived exosomes with a fluorescent dye-conjugated anti-GLT1 antibody to form an immune complex; (c) analyzing the concentration of exosome with a fluorescent dye in the biological fluid using Nanosight And distribution.

The invention further relates to a method for enriching astrocyte-derived exosomes from a biological fluid of a subject, the method comprising the steps of: (a) exosome containing astrocyte-derived exosomes The biological fluid contacts the anti-GLT1 antibody to form an immune complex; (b) binds the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; and (c) separates from the biological fluid Solid-phase bound exosomes are enriched with astrocyte-derived exosomes. Biological fluids suitable for use in the present invention include blood, serum, plasma, saliva, and urine, with blood, serum, plasma, or saliva being preferred.

In order to specifically enrich exosomes derived from astrocytes in a biological fluid, the present invention uses an immunoaffinity capture method to separate exosomes containing GLT1 from a biological fluid of a subject. GLT1 is a marker on the surface of exosomes derived from astrocytes, which specifically express GLT1 and have no GLT1 expression in other organ systems (so far), including blood cells and platelets. Thus, exosomes derived from non-astrocyte sources are not non-specifically captured.

The anti-GLT1 antibody used to capture astrocyte-derived exosomes may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, or an antibody fragment containing a GLT1 antigen binding domain such as a Fab or F(ab')2 fragment. . The anti-GLT1 antibody may be immobilized on the solid phase in advance, or the GLT1-binding exosomes may be bound to the solid phase after contacting the astrocyte-derived exosomes in the biological fluid to form an immune complex, the above method It is immobilized by means of an agent capable of capturing an anti-GLT1 antibody. The preferred method is to bind the anti-GLT1 to the solid phase before contacting it with the biological fluid. The method of immobilization on a solid phase involves covalent, hydrophobic or electrostatic bonding. For example, the first member of the binding pair (eg, streptavidin, anti-fluorescein, etc.) can be first immobilized by adsorption to a solid surface or by covalent attachment to an aminopropyl silane coated on a solid surface. The anti-GLT1 antibody labeled with a second member of the binding pair (eg, biotin, fluorescein, etc.) can then be bound by biotin-streptavidin or binding of fluorescein and anti-fluorescein (binding pair) Solid surface. After specifically capturing astrocyte-derived exosomes by means of a solid phase and immunizing the complex by GLT1 and anti-GLT1 antibodies, the exosomes are isolated from the biological fluid to enrich the astrocyte-derived source. Colloids. Solid phase bound exosomes can also be used directly or they can be eluted from the solid phase for further use and measurement. Since the steps of the present invention do not involve ultracentrifugation, the method of the present invention is further optimized for routine clinical examination.

The inventors have found that measurements of astrocyte-derived exosomes in biological fluids can be used to detect neurological diseases. The biomarker can also be a protein, a nucleic acid such as DNA or RNA, a lipid or the exosomes themselves. For example, α-syn or phosphorylated α-syn in astrocyte-derived exosomes (eg, serine 129-α-syn, phosphorylation of α-syn at residue serine-129 or ps129) It is a biomarker for PD. In astrocyte-derived exosomes, tau or acidified tau, for example, p-tau ¹⁸¹ , is a biomarker for AD. The prion protein or phosphorylated prion protein in astrocyte-derived exosomes is a biomarker for prion diseases such as Creutzfeldt-Jakob Disease. RNA contained as a biomarker for AD in astrocyte-derived exosomes includes 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, MGAT5, NBPF14, FBXO31, WDR52, TBC1D2B, ZNF648, NBPF16, PAGR1 RNA associated with AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1, BPTF, FLG, POM121L10P, NXPH3, H3F3A, SH3TC2, GGCX, and GREB1. RNA contained as a biomarker for PD in astrocyte-derived exosomes includes genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP, HNRNPAB, NXN, SLC9A4, EIF2AK1 RNA associated with PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5. Protein biomarkers can be measured in enriched exosomes when captured on a solid phase, or after elution from a solid phase. For protein biomarkers that are exposed on the surface of the exosomes, it can be measured without exosome lysis. For protein biomarkers contained in the exosomes, they can be measured after exosomes have been lysed. Protein biomarkers can be measured by any method known to those skilled in the art, for example immunoassays such as ELISA, Luminex and the latest Quanterix are preferred methods for measuring protein biomarkers. For nucleic acid biomarkers, the captured exosomes need to be cleaved prior to measurement of the nucleic acid biomarkers. Nucleic acids can be detected by any method known to those skilled in the art, for example, DNA or RNA probes, or any known sequencing technique.

The invention also relates to methods for detecting a neurological disease in a subject or for differential diagnosis. The above method comprises the steps of: (a) contacting a biological fluid from a subject with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is blood, serum, plasma, saliva or urine; (b) by immune complex, The astrocyte-derived exosomes in the biological fluid are bound to the solid phase; (c) the solid phase-bound exosomes are separated from the biological fluid to enrich the astrocyte-derived exosomes; d) determining the level of biomarkers from enriched astrocyte-derived exosomes, and with subjects from normal subjects (for diagnosis) or from patients with another nervous system disease A significant difference in the level of biomarkers from astrocyte-derived exosomes in the subject compared to the control level (for differential diagnosis) indicates that the subject has a neurological disorder (eg, AD, PD or prion disease). In one embodiment, the biomarker is alpha-syn, phosphorylated alpha-syn, and astrocyte-derived marker concentration itself, and the neurological disease is PD. In another embodiment, the biomarker is tau, phosphorylated tau or A[beta] material and the nervous system disease is AD. In another embodiment, the biomarker is a prion protein and the nervous system disorder is a prion disease.

The invention further relates to a method for monitoring the progression of a neurological disease in a subject. The above method comprises the steps of: (a) obtaining a biological fluid sample from a subject at different time points (eg, at time zero, 6 months, 1 year, or 2 years), wherein the biological fluid is blood, serum, plasma , saliva or urine; (b) contacting each sample with an anti-GLT1 antibody to form an immune complex; (c) binding the astrocyte-derived exosomes in each biological fluid through the immune complex (d) Separation of solid-phase-bound exosomes from each sample to enrich astrocyte-derived exosomes; (e) enrichment of astrocytes from each sample The level of biomarkers is determined in the exosomes of the source, with further differences in levels in the samples at later time points significantly indicating that the disease is progressive. In one embodiment, the biomarker is alpha-syn or phosphorylated alpha-syn and the nervous system disorder is PD. In another embodiment. The biomarker is tau or phosphorylated tau or Aβ material and the nervous system disease is AD. In another embodiment, the biomarker is a prion protein or a phosphorylated prion protein and the nervous system disease is a prion disease.

The invention further relates to a method of medical treatment for monitoring a neurological condition in a subject. The above method comprises the steps of: (a) obtaining a biological fluid sample from a subject before or after the medical treatment, wherein the biological fluid is blood, serum, plasma, saliva or urine; (b) contacting each sample with an anti-GLT1 antibody. Forming an immune complex; (c) binding the astrocyte-derived exosomes in each biological fluid to the solid phase via the immune complex; (d) separating the solid phase-bound exosomes from each sample Enrichment of astrocyte-derived exosomes; (e) determination of biomarker levels in enriched astrocyte-derived exosomes from each sample, after drug treatment A decrease in the level in the sample indicates that the drug treatment is effective. In one embodiment, the biomarker is alpha-syn or phosphorylated alpha-syn and the nervous system disorder is PD. In another embodiment, the biomarker is tau or phosphorylated tau or A[beta] material and the nervous system disease is AD. In another embodiment, the biomarker is a prion protein or a phosphorylated prion protein and the nervous system disease is a prion disease.

Subjects of the invention are mammalian subjects such as humans, horses, and dogs, with humans being preferred subjects.

According to an exemplary embodiment of the present invention, there is provided a method for enriching astrocyte-derived exosomes from a biological fluid. The method comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the liquids; and (b) enriching the astrocyte-derived exosomes by the method of using the immune complex in a solid phase or a liquid phase.

Enrichment of astrocyte-derived exosomes by immune complexes may be by solid phase separation or by liquid phase separation. According to a typical embodiment of the invention, step (b) comprises passing the immune complexes The astrocyte-derived exosomes in the biological fluid are bound to the solid phase; the solid-phase-bound exosomes are separated from the biological fluid to enrich the astrocyte-derived exosomes. Preferably, the anti-GLT1 antibody in step (a) is immobilized on a solid phase, which facilitates subsequent operations. In order to further enrich the exosomes, the method may further comprise the step (d) of eluting the bound exosomes from the solid phase. According to another exemplary embodiment of the present invention, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises enriching astrocyte-derived exosomes by flow cytometry; preferably, anti-GLT1 antibody An antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, or a quantum dot.

According to an exemplary embodiment of the present invention, there is provided the use of an anti-GLT1 antibody for the preparation of an astrocyte-derived biomarker for detecting a neurological disease in a biological fluid. The use comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers. Wherein, the biomarker may be a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself, and of course may be other substances in the astrocyte-derived exosomes that can serve as biomarkers.

According to an exemplary embodiment of the present invention, step (b) comprises: (b1) binding an astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; (b2) from the biological fluid Separating the solid phase-bound exosomes; and (b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2).

In another exemplary embodiment of the present invention, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises: directly detecting the biological fluid by means of a fluorescent label Astrocyte-derived exosomes; markers for anti-GLT1 antibodies include, but are not limited to, fluorescent labels, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, by corresponding instruments, such as particle size analyzers, A gamma counter or a small animal living imager directly detects the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in a biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585 , Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

The nervous system diseases of the present invention include all diseases associated with the nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion diseases, and nervous system tumors; depending on the type of nervous system diseases, different ones may be selected. Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of alpha-synuclein, phosphorylated alpha-synuclein, and astrocyte-derived exosomes; When the nervous system disease is Alzheimer's disease, the biomarker is tau protein and phosphorylated tau protein; when the nervous system disease is prion disease, the biomarker is prion protein; when the nervous system disease is nervous system tumor, The nervous system tumor includes a glioma, the biomarker is a biomarker level in the astrocyte-derived exosome, and the concentration and particle size distribution of the astrocyte-derived exosomes, wherein the astrocyte-derived exosomes are Biomarkers include, but are not limited to, alpha-synuclein, alpha-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to an exemplary embodiment of the invention, there is provided the use of an anti-GLT1 antibody for the preparation of a preparation for detecting a neurological disorder in a subject. The use comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers and, in comparison with the control levels from subjects without neurological disease, from the subject Differences in the levels of biomarkers of astrocyte-derived exosomes significantly indicate that the subject has a neurological disorder. Wherein, the biomarker may be a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself, and of course may be other substances in the astrocyte-derived exosomes that can serve as biomarkers.

According to an exemplary embodiment of the present invention, step (b) comprises: (b1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; (b2) separating the solid phase binding And (b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2), wherein the subject is from a subject having no neurological disease A comparison of the levels of the biomarkers from astrocyte-derived exosomes in the subject significantly indicates that the subject has a neurological disorder.

In another exemplary embodiment of the present invention, the anti-GLT1 antibody is a fluorescently labeled antibody, and the step (b) comprises: directly detecting the astrocyte-derived exosomes in the biological fluid by means of fluorescent labeling; preferably, by corresponding The apparatus, such as a particle size analyzer, a gamma counter or a small animal living imager, directly detects the concentration, particle size distribution and tissue distribution of the astrocyte-derived exosomes in the biological fluid; more preferably, the fluorescent marker is a fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to an exemplary embodiment of the present invention, an apparatus for detecting an astrocyte-derived biomarker of a nervous system disease in a biological fluid is provided. The device comprises: an exosome enrichment module for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, the biological fluid being selected from the group consisting of blood, serum, plasma, and saliva. And one or more of the urine; and a biomarker level determining module for determining the level of the astrocyte-derived biomarker. Wherein, the biomarker may be a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself, and of course may be other substances in the astrocyte-derived exosomes that can serve as biomarkers.

According to an exemplary embodiment of the present invention, an exosome enrichment module is used for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, and the organism is passed through the immune complex. The astrocyte-derived exosomes in the body fluid bind to the solid phase and separate the solid phase-bound exosomes from the biological fluid; and the biomarker level determining module is used to determine the exosome enrichment module The level of the astrocyte-derived biomarker of the solid phase-bound exosomes obtained is isolated.

In another exemplary embodiment of the present invention, when the biomarker is an astrocyte-derived exosomes themselves, the anti-GLT1 antibody in the exosomal enrichment module is a labeled antibody, and the anti-GLT1 antibody marker includes Not limited to fluorescent labels, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, fluorescent labels are fluorescent labels Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800 The biomarker level determining module directly detects the astrocyte-derived exosomes in the biological fluid by means of the label; preferably, the biomarker level determining module passes the corresponding instrument, such as a particle size analyzer, a gamma counter or a small animal live imager Directly detect the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluids.

According to an exemplary embodiment of the invention, an apparatus for detecting a nervous system disorder in a subject is provided. The device comprises: an exosome enrichment module for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, the biological fluid being selected from the group consisting of blood, serum, plasma, and saliva. And one or more of the urine; and a comparison module for determining the level of the astrocyte-derived biomarker and comparing it to the control level from the subject without the neurological disease Significant differences in the levels of biomarkers from astrocyte-derived exosomes in the tester are indicative of the subject's information on neurological disorders. Wherein, the biomarker may be a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself, and of course may be other substances in the astrocyte-derived exosomes that can serve as biomarkers.

According to an exemplary embodiment of the present invention, an exosome enrichment module is used for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, and the organism is passed through the immune complex. The astrocyte-derived exosomes in the body fluid bind to the solid phase and separate the solid phase-bound exosomes from the biological fluid; and the comparison module is used to determine the solid phase separated by the exosome enrichment module The level of the astrocyte-derived biomarker of the bound exosomes, and compared to the control level from subjects without neurological disease, from the source of astrocytes in the subject Significant differences in the levels of biomarkers in the body of the body are indicative of the subject's information on neurological disorders.

In another exemplary embodiment of the present invention, when the biomarker is an astrocyte-derived exosomes themselves, the anti-GLT1 antibody in the exosomal enrichment module is a labeled antibody, and the anti-GLT1 antibody marker includes Not limited to fluorescent labels, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, fluorescent labels are fluorescent labels Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800 The comparison module directly detects the astrocyte-derived exosomes in the biological fluid by means of the label and compares it with the control level from the subject without the neurological disease; preferably, the comparison module passes the corresponding instrument, such as a particle size analyzer, A gamma counter or a small animal living imager or the like directly detects the concentration, particle size distribution, and tissue distribution of astrocyte-derived exosomes in a biological fluid, and is compared with a control level from a subject without a neurological disease.

According to an exemplary embodiment of the present invention, there is provided a method for detecting an astrocyte-derived biomarker of a nervous system disease in a biological fluid. The method comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers. Wherein, the biomarker may be a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself, and of course may be other substances in the astrocyte-derived exosomes that can serve as biomarkers.

In another exemplary embodiment of the present invention, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises: directly detecting the star in the biological fluid by means of the label Cell-derived exosomes; markers for anti-GLT1 antibodies include, but are not limited to, fluorescent labels, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, by corresponding instruments, such as particle size analyzers, gamma The horse counter or the small animal living imager directly detects the concentration, particle size distribution and tissue distribution of the astrocyte-derived exosomes in the biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to an exemplary embodiment of the invention, a method for detecting a nervous system disorder in a subject is provided. The method comprises the steps of: (a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from the group consisting of blood, serum, plasma, saliva, and urine. One or more of the fluids; and (b) determining the level of astroglial-derived biomarkers and, in comparison with the control levels from subjects without neurological disease, from the subject Differences in the levels of biomarkers of astrocyte-derived exosomes significantly indicate that the subject has a neurological disorder. Wherein, the biomarker may be a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself, and of course may be other substances in the astrocyte-derived exosomes that can serve as biomarkers.

According to an exemplary embodiment of the present invention, step (b) comprises: (b) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; (c) separating the solid phase binding Exosomes; and (d) determining the level of astrocyte-derived biomarkers of the solid phase-bound exosomes isolated in step (c), and with subjects from no neurological disease A comparison of the levels of the biomarkers from astrocyte-derived exosomes in the subject significantly indicates that the subject has a neurological disorder.

In another exemplary embodiment of the present invention, when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, and step (b) comprises: directly detecting the star in the biological fluid by means of the label Cell-derived exosomes; markers for anti-GLT1 antibodies include, but are not limited to, fluorescent labels, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, by corresponding instruments, such as particle size analyzers, gamma The horse counter or the small animal living imager directly detects the concentration, particle size distribution and tissue distribution of the astrocyte-derived exosomes in the biological fluid; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800, and the level of biomarkers from astrocyte-derived exosomes differs significantly in subjects compared to control levels from subjects without neurological disease The subject was shown to have a neurological disorder.

Advantageous effects of the present invention will be further described below in conjunction with the examples. These examples are only intended to illustrate the invention and should not be construed as limiting.

Example 1

Exosomal separation

Exosomes were isolated from mouse or human plasma according to protocols modified from Tauro et al (Methods 56: 293-304, 2012) and using antibody-coated superparamagnetic microbeads.

Brief Description: 10 μg of anti-GLT1 antibody (ab178401, Abeam, Cambridge, MA, USA), or normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) (as a negative control) was coated in one set (1 mg) M -270 epoxy beads, using an antibody coupling kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. After rapid thawing (within 2 minutes) at 37 ° C, plasma samples (>300 μL) were centrifuged at 2,000 x g for 15 minutes, then centrifuged at 12,000 xg for 30 minutes, then phosphate buffered saline (PBS) (pH 7.4) ) Dilute the supernatant 1:3. A set of antibody-coated beads and 900 μL of diluted plasma were incubated for about 24 hours at 4 °C with gentle rotation. The beads were then washed four times with 1 mL of 0.1% bovine serum albumin (BSA) / PBS (pH 7.4) and transferred to a new tube.

Exosomes were eluted from the beads by means of a 1:1 mixture of 60 μL of 0.1% BSA/PBS (pH 7.4) and fixing buffer (4% paraformaldehyde/5% glutaraldehyde) for electron microscopy . Alternatively, by gently shaking, by adding 10% protease inhibitor mixture in 110 μL of 1% Triton X-100 in 0.1% BSA/PBS (pH 7.4) (P2714, Sigma-Aldrich, St Louis) , MO, USA; prepared in 10 ml of H ₂ O) medium temperature beads for 1 hour to lyse exosomes for Luminex measurements and other analyses, the results are shown in Figure 1, where A shows capture by anti-GLT1, or Normal mouse IgG capture, Alix (general exosome label) and Western blot of GLT1; B shows plasma extracellular vesicle concentration captured by GLT1 antibody and plasma extracellular vesicle concentration captured by normal mouse IgG antibody In contrast, this result demonstrates that the anti-GLT1 antibody is capable of specifically enriching exosomes derived from astrocytes in biological fluids.

Exosomes in clinical plasma samples were extracted in batches and PD and control samples were assigned to each batch. Two reference plasma samples pooled from 30 healthy controls were added to each batch to help eliminate batch changes.

Example 2

Characterization of anti-GLT1 captured exosomes from human plasma

Luminex assay: 100 μL of exosomal preparation (extracted from 300 μL plasma) was used to quantify α-syn, oligomer a-syn, PS129, tau, by means of the established Luminex protocol (Brain 133: 713-726, 2010). Aβ protein concentration.

Western blot analysis: Western blotting was performed following standard protocols. Laemmli sample buffer was used to dissolve the exosomal sample (~10 μg protein) and separated on a 1D SDS-PAGE gel prior to transfer to the polyvinylidene fluoride membrane. The protein on the above membrane was probed by the following primary antibodies: mouse anti-human GLT1 (Abeam, 1:500) and mouse anti-human Alix (Cat# ABC40, Millipore, Billerica, MA, USA; 1:1000).

Example 3

Evaluation of plasma GLT1 fluorescently labeled exosomes in clinical samples

Plasma samples from clinical cohorts (46 MSA patients, 49 PD patients and 50 age- and sex-matched healthy controls) were collected and the concentrations of exosomes containing GLT1 in plasma were measured using the Nanosight assay. The results are shown in Figure 2. In Figure 2, A is a fluorescent-labeled GLT1 antibody labeled with multi-system atrophy (MSA), Parkinson's disease (PD), healthy control (CT) clinical cohort plasma astrocyte-derived exosomes, and the exosome is calculated by Nanosight Concentrations, the results showed that exogenous concentrations of GLT1 fluorescently labeled exosomes in plasma were significantly higher (p < 0.01) in PD patients compared to healthy controls. Figure 2B shows the distribution of GLT1 antibody-labeled astrocyte-derived exosomes in clinical cohort by Nanosight. **, p < 0.01, the results showed that there was no significant difference in the distribution of GLT1 fluorescently labeled exosomes in PD and controls.

To further assess the potential for exosome concentrations containing GLT1 fluorescent markers for PD diagnosis, ROC (subject work curve) was generated in 46 MSA patients, 49 PD patients, and 50 healthy controls to characterize the difference. Their sensitivity (sensitivity and specificity) in PD and healthy control subjects. Figure 3 is a graph of the working curve of a multi-system atrophy (MSA), Parkinson's disease (PD), and healthy control (CT) diagnosis and differential diagnosis using GLT1 antibody-labeled astrocyte-derived exosome concentrations in plasma ( Receiver Operating Characteristic Analysis, ROC analysis). To observe the differential diagnosis efficiency of multi-system atrophy and Parkinson's disease, it was found that the diagnostic efficiency of plasma GLT1 fluorescently labeled exosomes was moderate (AUC=0.6750, sensitivity=66.00%, specificity=70.45%), PD and MSA. The differential diagnosis efficiency was moderate (AUC=0.6948, sensitivity=65.91%, specificity=78.57%) (Fig. 3).

Example 4

Detection of GLT1 in human saliva Human saliva from normal subjects was collected according to the method described by Devic et al (Brain. 134 (Pt 7): el 78. doi: 10.1 093brain/awr015. Epub 2011 Feb 24). Saliva samples were collected from healthy individuals between 9 and 11 AM in the morning. The samples were centrifuged at 4 ° C and 15,000 g for 15 minutes to remove insoluble materials and debris. A 10% (v/v) protease inhibitor cocktail was immediately added to the supernatant and the protein was precipitated on ice for 1 hour with the aid of 20% (v/v) TCA. The mixture was centrifuged at 4 ° C and 15,000 g for 15 minutes. The supernatant was removed and the precipitate was washed twice with ice cold acetone. Protein concentration was assessed by means of the BCA Protein Assay Kit (Thermo Scientific Pierce) and BSA as a standard, and the samples were then stored at -20 °C until analysis. The supernatant was incubated with anti-GLT1 antibody coated beads or normal mouse IgG coated beads (negative control) according to the protocol described in Example 1. The captured exosomes were lysed according to the protocol described in Example 2 for Western blot analysis. The membrane was probed by means of mouse anti-human Alix. Alix, a common exosomal marker, is clearly detectable in exosomes captured by anti-GLT1, but not in exosomes captured by normal mouse IgG (negative control). There are no other non-specific bands other than the known heavy and light chains of human IgG with appropriate molecular weights of 50 kDa and 25 kDa, respectively.

Example 5

GLT1-exosomes and normal mouse IgG negative controls were enriched from the subject's plasma according to the method described in Example 1. 100 μL of the exosomal preparation (extracted from 300 μL plasma) was used to quantify α-syn by means of the established Luminex protocol (Brain 133: 713-726, 2010), and the results are shown in FIG.

The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims

A method for enriching astrocyte-derived exosomes from biological fluids, comprising the steps of:

(a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is one or more selected from the group consisting of blood, serum, plasma, and saliva. Kind;

(b) enriching the astrocyte-derived exosomes by the method of using the immune complex in a solid phase or a liquid phase.
The method according to claim 1, wherein the step (b) comprises: binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex; separating from the biological fluid Solid-phase bound exosomes are enriched with astrocyte-derived exosomes.
The method according to claim 2, wherein said anti-GLT1 antibody in step (a) is immobilized on said solid phase.
The method of claim 2, wherein the method further comprises the step (c) of eluting the exosomes from the solid phase.
The method of claim 1 wherein said anti-GLT1 antibody is a labeled antibody and step (b) comprises enriching said astrocyte-derived exosomes by flow cytometry.
The method according to claim 5, wherein the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent or a quantum dot.
Use of an anti-GLT1 antibody for the preparation of an astrocyte-derived biomarker for detecting a neurological disease in a biological fluid, comprising the steps of:

(a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is one selected from the group consisting of blood, serum, plasma, saliva, and urine. Species or more;

(b) determining the level of the astrocyte-derived biomarker.
The use according to claim 7, characterized in that the biomarker is a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself.
The use according to claim 8, wherein step (b) comprises:

(b1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex;

(b2) separating the solid phase-bound exosomes from the biological fluid;

(b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2).
The use according to claim 8, wherein when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, step (b) comprises: The biomarker directly detects the astrocyte-derived exosomes in the biological fluid;

Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold;

Preferably, the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in the biological fluid are directly detected by a particle size analyzer, a gamma counter or a small animal living imager;

More preferably, the fluorescent label is fluorescent label Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.
The use according to claim 8, wherein the nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease, and nervous system tumor;

When the neurological disease is Parkinson's disease, the biomarker is a concentration and a particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes;

When the neurological disease is Alzheimer's disease, the biomarker is tau protein and phosphorylated tau protein;

When the nervous system disease is a prion disease, the biomarker is a prion protein;

When the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level in astrocyte-derived exosomes and a concentration of astrocyte-derived exosomes a particle size distribution, wherein the biomarkers of the astrocyte-derived exosomes include α-synuclein, α-synuclein oligomer, PS129, tau protein, phosphorylated tau protein, and Abeta protein .
Use of an anti-GLT1 antibody for the preparation of a preparation for detecting a nervous system disease or tumor in a subject, comprising the steps of:

(a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is one selected from the group consisting of blood, serum, plasma, saliva, and urine. Species or more;

(b) determining the level of said astrocyte-derived biomarker and comparing it from said control level to a subject without said neurological disease, said star-shaped gel in said subject The difference in the level of the biomarker of the cytoplasmic-derived exosomes significantly indicates that the subject has the neurological disease.
The use according to claim 12, characterized in that the biomarker is a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself.
The use according to claim 13, wherein step (b) comprises:

(b1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex;

(b2) separating the solid phase-bound exosomes;

(b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2) and from a subject not having the neurological disease A level difference in the biomarker from the astrocyte-derived exosomes in the subject is significantly indicative of the subject having the neurological disease compared to a control level.
The use according to claim 13, wherein when the biomarker is an astrocyte-derived exosome itself, the anti-GLT1 antibody is a labeled antibody, step (b) comprises: The biomarker directly detects the astrocyte-derived exosomes in the biological fluid;

Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold;

Preferably, the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in the biological fluid are directly detected by a particle size analyzer, a gamma counter or a small animal living imager;

More preferably, the fluorescent label is fluorescent label Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.
The use according to claim 13, wherein the neurological diseases include Parkinson's disease, Alzheimer's disease, prion disease, and nervous system tumor;

When the neurological disease is Parkinson's disease, the biomarker is a concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes;

When the neurological disease is Alzheimer's disease, the biomarker is tau protein and phosphorylated tau protein; when the nervous system disease is prion disease, the biomarker is a prion protein;

When the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level in astrocyte-derived exosomes and a concentration of astrocyte-derived exosomes a particle size distribution, wherein the biomarkers of the astrocyte-derived exosomes include α-synuclein, α-synuclein oligomer, PS129, tau protein, phosphorylated tau protein, and Abeta protein .
An apparatus for detecting an astrocyte-derived biomarker of a nervous system disease in a biological fluid, comprising:

An exosomal enrichment module for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex selected from the group consisting of blood, serum, plasma, saliva, and urine One or more of the liquids;

A biomarker level determining module for determining the level of the astrocyte-derived biomarker.
17. Apparatus according to claim 17 wherein the biomarker is a nucleic acid, protein, lipid or astrocyte derived exosomes per se.
The device of claim 18, wherein

The exosome enrichment module is configured to contact an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, and the star in the biological fluid is passed through the immune complex The exosome derived from the glial cell binds to the solid phase, and separates the solid phase-bound exosomes from the biological fluid;

The biomarker level determining module is configured to determine the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in the exosome enrichment module.
The device according to claim 18, wherein when said biomarker is an astrocyte-derived exosome itself, said anti-GLT1 antibody in said exosomal enrichment module is a labeled antibody, said The biomarker level determining module directly detects the astrocyte-derived exosomes in the biological fluid by means of the label;

Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold;

Preferably, the biomarker level determining module directly detects the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in the biological fluid by a particle size analyzer, a gamma counter or a small animal living imager;

More preferably, the fluorescent label is fluorescent label Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.
The device according to claim 18, wherein said nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease, and nervous system tumor;

When the neurological disease is Parkinson's disease, the biomarker is a concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes;

When the neurological disease is Alzheimer's disease, the biomarker is tau protein and phosphorylated tau protein;

When the nervous system disease is a prion disease, the biomarker is a prion protein;

When the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level in astrocyte-derived exosomes and a concentration of astrocyte-derived exosomes a particle size distribution, wherein the biomarkers of the astrocyte-derived exosomes include α-synuclein, α-synuclein oligomer, PS129, tau protein, phosphorylated tau protein, and Abeta protein .
A device for detecting a nervous system disease in a subject, comprising:

An exosomal enrichment module for contacting an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex selected from the group consisting of blood, serum, plasma, saliva, and urine One or more of the liquids;

a comparison module for determining the level of the astrocyte-derived biomarker and, in the subject, from the control level from a subject without the neurological disease A significant difference in the level of the biomarker of the astrocyte-derived exosomes is significant to output information that the subject has the neurological disease.
The device according to claim 22, wherein the biomarker is a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself.
The device according to claim 23, wherein

The exosome enrichment module is configured to contact an organism fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, and the star in the biological fluid is passed through the immune complex The exosome derived from the glial cell binds to the solid phase, and separates the solid phase-bound exosomes from the biological fluid;

The comparison module is configured to determine a level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated by the exosome enrichment module, and Comparison of control levels of subjects of the neurological disease in which the level of the biomarker from the astrocyte-derived exosomes is significantly different, then the subject is output Information about the neurological disease.
The device according to claim 23, wherein when said biomarker is an astrocyte-derived exosome itself, said anti-GLT1 antibody in said exosomal enrichment module is a labeled antibody, comparison module The astrocyte-derived exosomes in the biological fluid are directly detected by means of the label and compared to a control level from a subject without the neurological disease;

Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold;

Preferably, the biomarker level determining module directly detects the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in the biological fluid by a particle size analyzer, a gamma counter or a small animal living imager;

More preferably, the fluorescent label is fluorescent label Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.
The device according to claim 23, wherein said nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease, and nervous system tumor;

When the neurological disease is Parkinson's disease, the biomarker is a concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes;

When the neurological disease is Alzheimer's disease, the biomarker is tau protein and phosphorylated tau protein;

When the nervous system disease is a prion disease, the biomarker is a prion protein;

When the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level in astrocyte-derived exosomes and a concentration of astrocyte-derived exosomes a particle size distribution, wherein the biomarkers of the astrocyte-derived exosomes include α-synuclein, α-synuclein oligomer, PS129, tau protein, phosphorylated tau protein, and Abeta protein .
The use of GLT1 as an astrocyte-derived exosome biomarker.
The use according to claim 27, wherein the application comprises: enriching astrocyte-derived exosomes from biological fluids, and preparing star glue for detecting nervous system diseases in biological fluids. A preparation of a cytoplasmic-derived biomarker, an astrocyte-derived biomarker for detecting a nervous system disease in a biological fluid, and detecting a nervous system disease in a subject.
A method for detecting a nervous system disease in a subject, comprising the steps of:

(a) contacting a biological fluid containing an astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is one selected from the group consisting of blood, serum, plasma, saliva, and urine. Multiple;

(b) determining the level of astrocyte-derived biomarkers and exosomes derived from astrocytes in the subject compared to control levels from subjects without neurological disease The difference in levels of biomarkers significantly indicates that the subject has a neurological disorder.
The method according to claim 29, wherein the biomarker is a nucleic acid, a protein, a lipid or an astrocyte-derived exosome itself.
The method of claim 29 wherein step (b) comprises:

B1) binding the astrocyte-derived exosomes in the biological fluid to the solid phase by the immune complex;

(b2) separating the solid phase-bound exosomes;

(b3) determining the level of the astrocyte-derived biomarker of the solid phase-bound exosomes isolated in step (b2) and from a subject not having the neurological disease A level difference in the biomarker from the astrocyte-derived exosomes in the subject is significantly indicative of the subject having the neurological disease compared to a control level.
The method according to claim 29, wherein when said biomarker is an astrocyte-derived exosome itself, said anti-GLT1 antibody is a labeled antibody, step (b) comprises: The biomarker directly detects the astrocyte-derived exosomes in the biological fluid;

Preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent label, an isotope, an enzyme label, a chemiluminescent agent, a quantum dot or a colloidal gold;

Preferably, the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in the biological fluid are directly detected by a particle size analyzer, a gamma counter or a small animal living imager;

More preferably, the fluorescent label is fluorescent label Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.
The method according to claim 29, wherein said nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease, and nervous system tumor;

When the neurological disease is Parkinson's disease, the biomarker is a concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes;

When the neurological disease is Alzheimer's disease, the biomarker is tau protein and phosphorylated tau protein; when the nervous system disease is prion disease, the biomarker is a prion protein;

When the nervous system disease is a nervous system tumor, the nervous system tumor includes a glioma, the biomarker is a biomarker level in astrocyte-derived exosomes and a concentration of astrocyte-derived exosomes a particle size distribution, wherein the biomarkers of the astrocyte-derived exosomes include α-synuclein, α-synuclein oligomer, PS129, tau protein, phosphorylated tau protein, and Abeta protein .