CN110133272A - Method for the excretion body in astroglia source to be enriched with or detected from biological fluid - Google Patents

Abstract

The invention discloses a kind of methods of excretion body for being enriched with from biological fluid or detecting astroglia source.Wherein, the excretion body in astroglia source is enriched with method includes the following steps: (a) makes the biological fluid of the excretion body containing astroglia source contact anti-GLT1 antibody to form immune complex, wherein, biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And the excretion body in astroglia source (b) is enriched with using the method for solid phase or liquid phase by immune complex.The biomarker of the excretion body in detectable astroglia source, such as auxiliary diagnosis, antidiastole and monitoring central nervous system disease, Alzheimer disease, Parkinson's disease, multi-system atrophy, prion disease and nervous system neoplasm such as glioma etc. avoids passing through traumatic operation and obtains marker.

Description

For the enrichment or detection of astrocyte-derived exosomes from biological fluids method

technical field

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

Background technique

There is an urgent need for biomarkers for auxiliary 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. Among the current biomarkers, the well-performing markers are all 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, obtaining CSF is an invasive procedure, which severely limits the routine application of CSF biomarkers. Therefore, the development of peripheral biomarkers (for example in blood) of CNS diseases is of great importance. However, CNS (eg, astrocyte)-derived components are difficult to detect in peripheral body fluids due to the presence of the blood-brain barrier. 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). For the above reasons, no peripheral biomarkers have been established for AD, PD, MSA, or prion diseases, despite decades of time and substantial funding. In addition, when disease-associated proteins such as tau, Aβ, and α-syn are measured in peripheral biological fluids using existing techniques, proteins of peripheral origin can significantly affect the signal of central nervous system disease.

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

Contents of the invention

The present invention aims to provide a method for enriching or detecting astrocyte-derived exosomes from biological fluids, so as to solve the existing biological problems for auxiliary diagnosis, differential diagnosis and monitoring of central nervous system diseases. The acquisition of markers is an invasive operation and cannot be used routinely.

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

Further, step (b) includes binding exosomes derived from astrocytes in biological fluids to a solid phase by immune complexes; isolating solid phase-bound exosomes from biological fluids to enrich astrocytes Glial cell-derived exosomes.

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

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

Further, the anti-GLT1 antibody is a labeled antibody, and step (b) includes 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 chemiluminescence agent or a quantum dot.

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

Further, the biomarker is nucleic acid, protein, lipid or astrocyte-derived exosome itself.

Further, the step (b) includes: (b1) binding the astrocyte-derived exosomes in the biological fluid to a solid phase through an immune complex; (b2) isolating the solid phase-bound exosomes from the biological fluid. and (b3) determining the level of an astrocyte-derived biomarker in the solid phase-bound exosomes isolated in step (b2).

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

Further, nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumors; when the nervous system disease is Parkinson's disease, the biomarkers are α-synuclein, phosphorylated α- Synuclein and astrocyte-derived exosomes themselves; when the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the nervous system disease is a nervous system tumor, said nervous system tumor includes glioma, the biomarker is the biomarker level in astrocyte-derived exosomes and the concentration of astrocyte-derived exosomes and particle size distribution, where biomarkers in astrocyte-derived exosomes include α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to still another aspect of the present invention, a use of an anti-GLT1 antibody in preparing a preparation for detecting nervous system diseases in a subject is provided. The use comprises the following steps: (a) contacting a biological fluid containing astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from blood, serum, plasma, saliva and urine and (b) determine the levels of astrocyte-derived biomarkers, and, in subjects from Differences in the levels of biomarkers in astrocyte-derived exosomes significantly indicated that the subjects had neurological diseases.

Further, the biomarker is nucleic acid, protein, lipid or astrocyte-derived exosome itself.

Further, step (b) includes: (b1) binding exosomes derived from astrocytes in biological fluids to a solid phase through an immune complex; (b2) isolating the exosomes bound to the solid phase; and ( b3) determining the level of the astrocyte-derived biomarker in the solid phase-bound exosomes isolated in step (b2), and, compared with the control level from a subject without neurological disease, in the subject Differences in the levels of biomarkers derived from astrocyte-derived exosomes among subjects were significantly indicative of neurological disease.

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

Further, nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumors; when the nervous system disease is Parkinson's disease, the biomarkers are α-synuclein, phosphorylated α- The concentration and particle size distribution of synuclein and astrocyte-derived exosomes; when the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the nervous system disease is a nervous system tumor, the nervous system tumor includes glioma, the biomarker is the biomarker level in astrocyte-derived exosomes and the astrocyte-derived Concentration and particle size distribution of exosomes, in which biomarkers in astrocyte-derived exosomes include α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein and Abeta protein.

According to yet another aspect of the present invention, there is provided a device for detecting astrocyte-derived biomarkers of neurological diseases in a biological fluid. The device includes: an exosome enrichment module, which is used to contact a biological fluid containing exosomes derived from astrocytes with an anti-GLT1 antibody to form an immune complex, and the biological fluid is selected from blood, serum, plasma, saliva and one or more of urine; and a biomarker level determination module for determining a level of an astrocyte-derived biomarker.

Further, the biomarker is nucleic acid, protein, lipid or astrocyte-derived exosome itself.

Further, the exosome enrichment module is used to contact the biological fluid containing exosomes derived from astrocytes with anti-GLT1 antibody to form an immune complex, and the astrocytes in the biological fluid are absorbed by the immune complex. Cell-derived exosomes are bound to a solid phase, and the solid-phase bound exosomes are isolated from biological fluids; and a biomarker level determination module is used to determine the solid-phase bound exosomes isolated in the exosome enrichment module. Levels of biomarkers of astrocyte origin in exosomes.

Furthermore, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody in the exosome enrichment module is a labeled antibody, and the biomarker level determination module directly detects astrocytes in biological fluids by means of the label. Cell-derived exosomes; preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloidal gold; preferably, the biomarker level determination module is passed through a particle size analyzer , gamma counter or small animal live imager to directly detect the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluids; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585 , Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Further, nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumors; when the nervous system disease is Parkinson's disease, the biomarkers are α-synuclein, phosphorylated α- The concentration and particle size distribution of synuclein and astrocyte-derived exosomes; when the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the nervous system disease is a nervous system tumor, the nervous system tumor includes glioma, the biomarker is the biomarker level in astrocyte-derived exosomes and the astrocyte-derived Concentration and particle size distribution of exosomes, in which biomarkers in astrocyte-derived exosomes include α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein and Abeta protein.

According to yet another aspect of the present invention, an apparatus for detecting a neurological disease in a subject is provided. The device includes: an exosome enrichment module, which is used to contact a biological fluid containing exosomes derived from astrocytes with an anti-GLT1 antibody to form an immune complex, and the biological fluid is selected from blood, serum, plasma, saliva and urine; and a comparison module for determining the levels of astrocyte-derived biomarkers and, compared with control levels from subjects without neurological disease, in subjects receiving Significant differences in the levels of biomarkers derived from astrocyte-derived exosomes among the subjects output the information that the subjects suffer from neurological diseases.

Further, the biomarker is nucleic acid, protein, lipid or astrocyte-derived exosome itself.

Further, the exosome enrichment module is used to contact the biological fluid containing exosomes derived from astrocytes with anti-GLT1 antibody to form an immune complex, and the astrocytes in the biological fluid are absorbed by the immune complex. Cell-derived exosomes are bound to a solid phase, and solid-phase-bound exosomes are isolated from biological fluids; and a comparison module is used to determine the star of the solid-phase-bound exosomes isolated by the exosome enrichment module. The levels of biomarkers derived from astrocytes and, compared with the control levels from subjects without neurological diseases, the levels of biomarkers derived from astrocyte-derived exosomes in subjects If the level difference is significant, the information that the subject suffers from a neurological disease is output.

Furthermore, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody in the exosome enrichment module is a labeled antibody, and the comparison module directly detects astrocyte-derived exosomes in biological fluids by means of labels. body, and compared with the control level from a subject without neurological disease; preferably, the anti-GLT1 antibody is an antibody labeled with a fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloidal gold; Preferably, the biomarker level determination module directly detects the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluids through a particle size analyzer, a gamma counter or a small animal live imager; more preferably, the fluorescence The marker is a fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Further, nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumors; when the nervous system disease is Parkinson's disease, the biomarkers are α-synuclein, phosphorylated α- Synuclein and astrocyte-derived exosomes themselves; when the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the nervous system disease is a nervous system tumor, said nervous system tumor includes glioma, the biomarker is the biomarker level in astrocyte-derived exosomes and the concentration of astrocyte-derived exosomes and particle size distribution, where biomarkers in astrocyte-derived exosomes include α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to yet another aspect of the present invention, there is provided a method for detecting astrocyte-derived biomarkers of neurological diseases in a biological fluid. The method comprises the steps of: (a) contacting a biological fluid containing 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 and (b) determining the level of the astrocyte-derived biomarker.

Further, the biomarker is nucleic acid, protein, lipid or astrocyte-derived exosome itself.

Further, the step (b) includes: (b1) binding the astrocyte-derived exosomes in the biological fluid to a solid phase through an immune complex; (b2) isolating the solid phase-bound exosomes from the biological fluid. and (b3) determining the level of an astrocyte-derived biomarker in the solid phase-bound exosomes isolated in step (b2).

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

Further, nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumors; when the nervous system disease is Parkinson's disease, the biomarkers are α-synuclein, phosphorylated α- The concentration and particle size distribution of synuclein and astrocyte-derived exosomes; when the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the nervous system disease is a nervous system tumor, the nervous system tumor includes glioma, the biomarker is the biomarker level in astrocyte-derived exosomes and the astrocyte-derived Concentration and particle size distribution of exosomes, in which biomarkers in astrocyte-derived exosomes include α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein and Abeta protein.

According to yet another aspect of the present invention, a method for detecting a neurological disease in a subject is provided. The method comprises the steps of: (a) contacting a biological fluid containing 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 and (b) determine the levels of astrocyte-derived biomarkers, and, in subjects from Differences in the levels of biomarkers in astrocyte-derived exosomes significantly indicated that the subjects had neurological diseases.

Further, the biomarker is nucleic acid, protein, lipid or astrocyte-derived exosome itself.

Further, step (b) includes: (b) binding exosomes derived from astrocytes in biological fluids to a solid phase through immune complexes; (c) isolating solid phase-bound exosomes; and ( d) determining the level of an astrocyte-derived biomarker in the solid-phase-bound exosomes isolated in step (c), and, compared to control levels from subjects without neurological disease, in the subject Differences in the levels of biomarkers derived from astrocyte-derived exosomes among subjects were significantly indicative of neurological disease.

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

Further, nervous system diseases include Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumors; when the nervous system disease is Parkinson's disease, the biomarkers are α-synuclein, phosphorylated α- Synuclein and astrocyte-derived exosomes themselves; when the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the nervous system disease is a nervous system tumor, said nervous system tumor includes glioma, the biomarker is the biomarker level in astrocyte-derived exosomes and the concentration of astrocyte-derived exosomes and particle size distribution, where biomarkers in astrocyte-derived exosomes include α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to still another aspect of the present invention, an application of GLT1 as a biomarker of exosomes derived from astrocytes is provided.

Further, the application includes: enriching astrocyte-derived exosomes from biological fluids, preparing preparations of astrocyte-derived biomarkers for detecting neurological diseases in biological fluids, Astrocyte-Derived Biomarkers for Detection of Neurological Diseases in Biological Fluids and Detection of Neurological Diseases in Subjects

The exosomes derived from astrocytes enriched by the technical solution of the present invention can be used to detect biomarkers of exosomes derived from astrocytes, assisting in 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, etc., avoid traumatic operations to obtain markers, reduce the pain of patients, and are suitable for routine clinical applications.

Description of drawings

The accompanying drawings constituting a part of the present application are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 shows: A. Western blotting of Alix (universal exosome marker) and GLT1 captured by anti-GLT1 or normal mouse IgG in Example 1; B. plasma cells captured by GLT1 antibody in Example 1 Comparison of extracellular vesicle concentration with plasma extracellular vesicle concentration captured by normal mouse IgG antibody.

Figure 2 shows: A. the assessment of GLT1 fluorescently labeled exosome concentration in clinical plasma samples in Example 3; B. the use of particle size analyzer (Nanosight300) in Example 3 to measure GLT1 fluorescently labeled plasma exosomes concentration and distribution.

Fig. 3 shows: the ROC (receiver operating curve, Receiver Operating Characteristics) analysis of the concentration of GLT1 fluorescently labeled exosomes used in the diagnosis of PD and the differential diagnosis of MSA in Example 3. In the whole cohort (46 MSA patients, 49 PD patients and 50 healthy controls), observe the differential diagnosis efficiency of multiple system atrophy and Parkinson's disease, area under the curve (AUC) = 0.6948, sensitivity = 65.91%, specificity =78.57%; observe the diagnostic efficiency of Parkinson's disease and healthy controls, area under the curve = 0.6750, sensitivity = 66%, specificity = 70.45%.

Fig. 4 shows: the detection result of α-syn in embodiment 5.

Detailed ways

It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and examples.

definition

A "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 directed against said antigens, ligands and their receptors, nucleic acid complementary strands, biotin and avidin, biotin and streptavidin, lectins (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.

"Exosomes" as used herein are 30-150 nm extracellular vesicles that can be released by many different types of cells and perform different cellular functions, including intercellular communication, antigen presentation, and Transfer of proteins and nucleic acids.

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

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

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

The inventors were surprised to find that "GLT1" is a unique, disease-specific biomarker carried by exosomes derived from astrocytes, and can be used as a biomarker for exosomes derived from astrocytes According to this, astrocyte-derived exosomes can be detected in vivo in blood and other peripheral body fluids, astrocyte-derived exosomes can be enriched from biological fluids, and prepared for the detection of neuronal exosomes in biological fluids. Preparations of astrocyte-derived biomarkers of systemic diseases, detection of astrocyte-derived biomarkers of neurological diseases in biological fluids and detection of neurological diseases in subjects.

The present inventors have discovered methods for isolating and labeling astrocyte-derived exosomes in biological fluids such as blood, serum, plasma, saliva or urine.

The present inventors have also found that astrocyte-derived central nervous system biomarkers can be detected and quantified from astrocyte-derived enriched exosomes in biological fluids, and the above results can be used to Detect neurological diseases such as AD, PD, MSA and prion diseases, and 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 Treatment effects in systemic diseases.

Based on the above findings, the inventors of the present invention proposed a method for determining astrocyte-derived substances (biological Marker) level method.

The present invention relates to a method for labeling astrocyte-derived exosomes from a subject's biological fluid, the method comprising the following steps: (a) forming a chemical coupling structure between an anti-GLT1 antibody and a fluorescent dye; ( b) Exposing biological fluids containing astrocyte-derived exosomes to a fluorescent dye-conjugated anti-GLT1 antibody to form immune complexes; (c) Analyzing the concentration of exosomes with fluorescent dyes in biological fluids using Nanosight and distribution.

The present invention also relates to a method for enriching astrocyte-derived exosomes from a biological fluid of a subject, the method comprising the following steps: (a) making an astrocyte-derived exosome Exposure of biological fluids to anti-GLT1 antibodies to form immune complexes; (b) binding of astrocyte-derived exosomes in biological fluids to a solid phase via immune complexes; and (c) isolation from biological fluids Solid-phase bound exosomes to enrich for 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 biological fluids, the present invention uses an immunoaffinity capture method to isolate GLT1-containing exosomes from biological fluids of subjects. GLT1 is a marker on the surface of exosomes derived from astrocytes, which specifically express GLT1, and are absent in other organ systems (so far), including blood cells and platelets , so that exosomes originating from non-astrocyte sources will not be captured non-specifically.

Anti-GLT1 antibodies used to capture astrocyte-derived exosomes can be polyclonal antibodies, monoclonal antibodies, single-chain antibodies, or antibody fragments containing GLT1 antigen-binding domains such as Fab or F(ab')2 fragments . Anti-GLT1 antibodies can be immobilized on the solid phase in advance, or the anti-GLT1-bound exosomes can be bound to the solid phase after contacting astrocyte-derived exosomes in biological fluids to form immune complexes, the above method are immobilized with the aid of reagents capable of capturing anti-GLT1 antibodies. The preferred method is to bind the anti-GLT1 to the solid phase before contacting it with the biological fluid. Methods of immobilization on solid phases involve covalent, hydrophobic or electrostatic bonds. 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 binding to aminopropylsilane coated on the solid surface. An anti-GLT1 antibody labeled with a second member of the binding pair (e.g., biotin, fluorescein, etc.) can then be bound to the solid surface. After specific capture of astrocyte-derived exosomes by means of a solid phase and by GLT1 and anti-GLT1 antibody immune complexes, exosomes were isolated from biological fluids to enrich astrocyte-derived exosomes. secretion body. Exosomes bound to the solid phase can also be used directly or they can be eluted from the solid phase for further use and measurement. Because the steps of the present invention do not involve ultracentrifugation, the method of the present invention is more likely to be used in routine clinical examinations after further optimization.

The present inventors have found that measurements of astrocyte-derived exosomes in biological fluids can be used to detect neurological diseases. Biomarkers can also be proteins, nucleic acids such as DNA or RNA, lipids or exosomes themselves. For example, α-syn or phosphorylated α-syn in astrocyte-derived exosomes (e.g., serine 129-α-syn, phosphorylation of α-syn at residues serine-129 or ps129) is a biomarker for PD. In astrocyte-derived exosomes, tau or acidified tau, for example, p-tau ¹⁸¹ , is a biomarker for AD. Prion protein or phosphorylated prion protein in astrocyte-derived exosomes is a biomarker for prion diseases such as Creutzfeldt-Jakob Disease. RNAs contained in astrocyte-derived exosomes as biomarkers for AD include 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 , AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1, BPTF, FLG, POM121L10P, NXPH3, H3F3A, SH3TC2, GGCX, and GREB1-associated RNAs. RNAs contained in astrocyte-derived exosomes as biomarkers of PD include 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-associated RNAs. Protein biomarkers can be measured in enriched exosomes when captured on a solid phase, or after elution from the solid phase. For protein biomarkers exposed on the surface of exosomes, it can be measured without exosome lysis. For protein biomarkers contained within exosomes, they can be measured after exosome lysis. Protein biomarkers can be measured by any method known to those skilled in the art, for example immunoassays such as ELISA, Luminex and more recently Quanterix are preferred methods for measuring protein biomarkers. For nucleic acid biomarkers, captured exosomes need to be lysed prior to nucleic acid biomarker measurement. Nucleic acids can be detected by any method known to those skilled in the art, eg, DNA or RNA probes, or any known sequencing technique.

The invention also relates to methods for detecting neurological disorders 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) passing the immune complex, Combining astrocyte-derived exosomes in a biological fluid to a solid phase; (c) isolating solid-phase-bound exosomes from biological fluids to enrich astrocyte-derived exosomes; ( d) Determining the levels of biomarkers from enriched astrocyte-derived exosomes, and, compared to normal subjects (for diagnosis) or from subjects with another neurological disease Compared with the control level of patients (for differential diagnosis), the level difference of biomarkers from astrocyte-derived exosomes in the subjects is significantly different. Significant differences indicate that the subjects suffer from neurological diseases (such as AD, PD or prion disease). In one embodiment, the biomarker is α-syn, phosphorylated α-syn, and astrocyte-derived marker concentration itself, and the neurological disorder is PD. In another embodiment, the biomarker is tau, phosphorylated tau or Aβ species and the neurological disease is AD. In another embodiment, the biomarker is prion protein and the neurological disorder is prion disease.

The invention further relates to methods for monitoring the progression of neurological diseases in a subject. The above method comprises the steps of: (a) obtaining biological fluid samples from the subject at different time points (e.g., at time zero, 6 months, 1 year, or 2 years), wherein the biological fluid is blood, serum, plasma , saliva, or urine; (b) each sample was exposed to anti-GLT1 antibody to form an immune complex; (c) the astrocyte-derived exosomes in each biological fluid were bound by the immune complex (d) isolating solid-phase-bound exosomes from each sample to enrich astrocyte-derived exosomes; (e) enriching astrocyte-derived exosomes from each sample Levels of biomarkers were determined in the derived exosomes, where further differences in levels in samples at later time points significantly indicated that the disease was progressive. In one embodiment, the biomarker is α-syn or phosphorylated α-syn and the neurological disorder is PD. In another embodiment. The biomarker is tau or phosphorylated tau or Aβ species and the neurological disease is AD. In another embodiment, the biomarker is prion protein or phosphorylated prion protein and the neurological disorder is a prion disease.

The invention further relates to methods for monitoring drug treatment of neurological disorders in a subject. The above method comprises the steps of: (a) obtaining samples of biological fluid from the subject before and after drug treatment, wherein the biological fluid is blood, serum, plasma, saliva or urine; (b) contacting each sample with an anti-GLT1 antibody to Formation of immune complexes; (c) binding of astrocyte-derived exosomes in each biological fluid to the solid phase by immune complexes; (d) isolation of solid phase-bound exosomes from each sample exosomes to enrich for astrocyte-derived exosomes; (e) determine the levels of biomarkers in the enriched astrocyte-derived exosomes from each sample, where after drug treatment Decreased levels in the samples indicate that the drug treatment is effective. In one embodiment, the biomarker is α-syn or phosphorylated α-syn and the neurological disorder is PD. In another embodiment, the biomarker is tau or phosphorylated tau or Aβ species and the neurological disease is AD. In another embodiment, the biomarker is prion protein or phosphorylated prion protein and the neurological disorder is a prion disease.

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

According to a typical embodiment 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 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 and (b) enriching exosomes derived from astrocytes by means of solid phase or liquid phase through immune complexes.

The exosomes derived from astrocytes enriched by the technical solution of the present invention can be used to detect biomarkers of exosomes derived from astrocytes, assisting in 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, etc., avoid traumatic operations to obtain markers, reduce the pain of patients, and are suitable for routine clinical applications.

Enrichment of exosomes derived from astrocytes through immune complexes can be performed through solid phase separation or liquid phase separation. According to a typical embodiment of the present invention, step (b) includes, through the immune complex Astrocyte-derived exosomes in biological fluids were bound to a solid phase; solid-phase-bound exosomes were isolated from biological fluids to enrich 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 exosomes, the method may further comprise a step (d) of eluting bound exosomes from the solid phase. According to another typical embodiment of the present invention, the anti-GLT1 antibody is a labeled antibody, and step (b) includes enriching astrocyte-derived exosomes by flow cytometry; preferably, the anti-GLT1 antibody Antibodies labeled with fluorescent labels, isotopes, enzyme labels, chemiluminescent agents or quantum dots.

According to a typical embodiment of the present invention, a use of an anti-GLT1 antibody in the preparation of an astrocyte-derived biomarker for detecting neurological diseases in biological fluids is provided. The use comprises the following steps: (a) contacting a biological fluid containing astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from blood, serum, plasma, saliva and urine and (b) determining the level of the astrocyte-derived biomarker. Among them, the biomarker can be nucleic acid, protein, lipid or astrocyte-derived exosome itself, and of course it can also be other substances in the astrocyte-derived exosome that can be used as biomarkers.

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

In another typical embodiment of the present invention, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody is a labeled antibody, and step (b) includes: directly detecting Astrocyte-derived exosomes; the labeling of anti-GLT1 antibodies includes but is not limited to fluorescent labels, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, through corresponding instruments, such as particle size analyzers, A gamma counter or a small animal in vivo imager directly detects the concentration, particle size distribution and tissue distribution of exosomes derived from astrocytes in biological fluids; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585 , Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Nervous system disease of the present invention includes all diseases related to nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; According to the difference of nervous system disease type, can choose different Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes; When the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the neurological disease is a nervous system tumor, The nervous system tumors include glioma, and the biomarkers are the level of biomarkers in exosomes derived from astrocytes and the concentration and particle size distribution of exosomes derived from astrocytes, wherein the exosomes derived from astrocytes are Biomarkers include, but are not limited to, α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to a typical embodiment of the present invention, a use of an anti-GLT1 antibody in preparing a preparation for detecting nervous system diseases in a subject is provided. The use comprises the following steps: (a) contacting a biological fluid containing astrocyte-derived exosomes with an anti-GLT1 antibody to form an immune complex, wherein the biological fluid is selected from blood, serum, plasma, saliva and urine and (b) determine the levels of astrocyte-derived biomarkers, and, in subjects from Differences in the levels of biomarkers in astrocyte-derived exosomes significantly indicated that the subjects had neurological diseases. Among them, the biomarker can be nucleic acid, protein, lipid or astrocyte-derived exosome itself, and of course it can also be other substances in the astrocyte-derived exosome that can be used as biomarkers.

According to a typical embodiment of the present invention, step (b) includes: (b1) binding exosomes derived from astrocytes in biological fluids to a solid phase through an immune complex; (b2) separating the solid phase binding and (b3) determining the level of astrocyte-derived biomarkers in the solid-phase-bound exosomes isolated in step (b2), wherein, compared with those from subjects without neurological diseases Compared with the control level of , the difference in the level of biomarkers from astrocyte-derived exosomes in the subjects significantly indicates that the subjects suffer from neurological diseases.

In another typical embodiment of the present invention, the anti-GLT1 antibody is a fluorescently labeled antibody, and step (b) includes: directly detecting astrocyte-derived exosomes in biological fluids by means of fluorescent labeling; preferably, through corresponding Instruments, such as particle size analyzers, gamma counters, or small animal live imagers, directly detect the concentration, particle size distribution, and tissue distribution of astrocyte-derived exosomes in biological fluids; more preferably, the fluorescent marker is a fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Nervous system disease of the present invention includes all diseases related to nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; According to the difference of nervous system disease type, can choose different Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes; When the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the neurological disease is a nervous system tumor, The nervous system tumors include glioma, and the biomarkers are the level of biomarkers in exosomes derived from astrocytes and the concentration and particle size distribution of exosomes derived from astrocytes, wherein the exosomes derived from astrocytes are Biomarkers include, but are not limited to, α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to a typical embodiment of the present invention, a device for detecting astrocyte-derived biomarkers of neurological diseases in biological fluids is provided. The device includes: an exosome enrichment module, which is used to contact a biological fluid containing exosomes derived from astrocytes with an anti-GLT1 antibody to form an immune complex, and the biological fluid is selected from blood, serum, plasma, saliva and one or more of urine; and a biomarker level determination module for determining a level of an astrocyte-derived biomarker. Among them, the biomarker can be nucleic acid, protein, lipid or astrocyte-derived exosome itself, and of course it can also be other substances in the astrocyte-derived exosome that can be used as biomarkers.

According to a typical embodiment of the present invention, the exosome enrichment module is used to contact the biological fluid containing exosomes derived from astrocytes with anti-GLT1 antibody to form an immune complex, and the biological Astrocyte-derived exosomes in body fluids are bound to a solid phase, and the solid-phase bound exosomes are isolated from biological fluids; and a biomarker level determination module for determining the exosome enrichment module Levels of astrocyte-derived biomarkers in isolated solid-phase-bound exosomes.

In another typical embodiment of the present invention, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody in the exosome enrichment module is a labeled antibody, and the label of the anti-GLT1 antibody includes but Not limited to fluorescent markers, isotopes, enzyme markers, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800 The biomarker level determination module directly detects astrocyte-derived exosomes in biological fluids by means of labels; preferably, the biomarker level determination module uses corresponding instruments, such as particle size analyzers, gamma counters or small animal live imagers directly detect the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluids.

Nervous system disease of the present invention includes all diseases related to nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; According to the difference of nervous system disease type, can choose different Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes; When the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the neurological disease is a nervous system tumor, The nervous system tumors include glioma, and the biomarkers are the level of biomarkers in exosomes derived from astrocytes and the concentration and particle size distribution of exosomes derived from astrocytes, wherein the exosomes derived from astrocytes are Biomarkers include, but are not limited to, α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to an exemplary embodiment of the present invention, a device for detecting neurological diseases in a subject is provided. The device includes: an exosome enrichment module, which is used to contact a biological fluid containing exosomes derived from astrocytes with an anti-GLT1 antibody to form an immune complex, and the biological fluid is selected from blood, serum, plasma, saliva and urine; and a comparison module for determining the levels of astrocyte-derived biomarkers and, compared with control levels from subjects without neurological disease, in subjects receiving Significant differences in the levels of biomarkers derived from astrocyte-derived exosomes among the subjects output the information that the subjects suffer from neurological diseases. Among them, the biomarker can be nucleic acid, protein, lipid or astrocyte-derived exosome itself, and of course it can also be other substances in the astrocyte-derived exosome that can be used as biomarkers.

According to a typical embodiment of the present invention, the exosome enrichment module is used to contact the biological fluid containing exosomes derived from astrocytes with anti-GLT1 antibody to form an immune complex, and the biological Astrocyte-derived exosomes in body fluids are bound to a solid phase, and solid-phase-bound exosomes are isolated from biological fluids; and a comparison module for determining the solid phase isolated by the exosome enrichment module The levels of astrocyte-derived biomarkers of bound exosomes, and, compared with the control levels from subjects without neurological diseases, exosomes derived from astrocytes in subjects Significant differences in the levels of secretory biomarkers output the information that the subject suffers from neurological diseases.

In another typical embodiment of the present invention, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody in the exosome enrichment module is a labeled antibody, and the label of the anti-GLT1 antibody includes but Not limited to fluorescent markers, isotopes, enzyme markers, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800 The comparison module directly detects astrocyte-derived exosomes in biological fluids by means of labels, and compares them with the control level from subjects without neurological diseases; preferably, the comparison module uses corresponding instruments, such as particle size analyzers, Gamma counters or small animal in vivo imagers directly detect the concentration, particle size distribution, and tissue distribution of astrocyte-derived exosomes in biological fluids, and compare them with control levels from subjects without neurological diseases.

Nervous system disease of the present invention includes all diseases related to nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; According to the difference of nervous system disease type, can choose different Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes; When the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the neurological disease is a nervous system tumor, The nervous system tumors include glioma, and the biomarkers are the level of biomarkers in exosomes derived from astrocytes and the concentration and particle size distribution of exosomes derived from astrocytes, wherein the exosomes derived from astrocytes are Biomarkers include, but are not limited to, α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to an exemplary embodiment 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 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 and (b) determining the level of the astrocyte-derived biomarker. Among them, the biomarker can be nucleic acid, protein, lipid or astrocyte-derived exosome itself, and of course it can also be other substances in the astrocyte-derived exosome that can be used as biomarkers.

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

In another typical embodiment of the present invention, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody is a labeled antibody, and step (b) includes: directly detecting the astrocytes in the biological fluid by means of the label Stemocyte-derived exosomes; the labeling of anti-GLT1 antibodies includes but is not limited to fluorescent markers, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, through corresponding instruments, such as particle size analyzers, Gamma Equine counters or small animal living imagers directly detect the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluids; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Nervous system disease of the present invention includes all diseases related to nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; According to the difference of nervous system disease type, can choose different Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes; When the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the neurological disease is a nervous system tumor, The nervous system tumors include glioma, and the biomarkers are the level of biomarkers in exosomes derived from astrocytes and the concentration and particle size distribution of exosomes derived from astrocytes, wherein the exosomes derived from astrocytes are Biomarkers include, but are not limited to, α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

According to an exemplary embodiment of the present invention, a method for detecting neurological diseases in a subject is provided. The method comprises the steps of: (a) contacting a biological fluid containing 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 and (b) determine the levels of astrocyte-derived biomarkers, and, in subjects from Differences in the levels of biomarkers in astrocyte-derived exosomes significantly indicated that the subjects had neurological diseases. Among them, the biomarker can be nucleic acid, protein, lipid or astrocyte-derived exosome itself, and of course it can also be other substances in the astrocyte-derived exosome that can be used as biomarkers.

According to a typical embodiment of the present invention, the step (b) includes: (b) binding the astrocyte-derived exosomes in the biological fluid to a solid phase through an immune complex; (c) separating the solid phase binding exosomes and (d) determining the level of astrocyte-derived biomarkers in the solid-phase-bound exosomes isolated in step (c), and, compared with those from subjects without neurological disease Compared with the control level of , the difference in the level of biomarkers from astrocyte-derived exosomes in the subjects significantly indicates that the subjects suffer from neurological diseases.

In another typical embodiment of the present invention, when the biomarker is astrocyte-derived exosomes themselves, the anti-GLT1 antibody is a labeled antibody, and step (b) includes: directly detecting the astrocytes in the biological fluid by means of the label Stemocyte-derived exosomes; the labeling of anti-GLT1 antibodies includes but is not limited to fluorescent markers, isotopes, enzyme labels, chemiluminescent agents, quantum dots or colloidal gold labels; preferably, through corresponding instruments, such as particle size analyzers, Gamma Equine counters or small animal living imagers directly detect the concentration, particle size distribution and tissue distribution of astrocyte-derived exosomes in biological fluids; more preferably, the fluorescent markers are fluorescent markers Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705, or Qdot800, and the levels of biomarkers from astrocyte-derived exosomes differ significantly in subjects compared to control levels from subjects without neurological disease Indicates that the subject suffers from a neurological disorder.

Nervous system disease of the present invention includes all diseases related to nervous system, including but not limited to Parkinson's disease, Alzheimer's disease, prion disease and nervous system tumor; According to the difference of nervous system disease type, can choose different Biomarkers, for example, when the neurological disease is Parkinson's disease, the biomarkers are the concentration and particle size distribution of α-synuclein, phosphorylated α-synuclein, and astrocyte-derived exosomes; When the neurological disease is Alzheimer's disease, the biomarkers are tau protein and phosphorylated tau protein; when the neurological disease is prion disease, the biomarker is prion protein; when the neurological disease is a nervous system tumor, The nervous system tumors include glioma, and the biomarkers are the level of biomarkers in exosomes derived from astrocytes and the concentration and particle size distribution of exosomes derived from astrocytes, wherein the exosomes derived from astrocytes are Biomarkers include, but are not limited to, α-synuclein, α-synuclein oligomers, PS129, tau protein, phosphorylated tau protein, and Abeta protein.

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

Example 1

Exosome Isolation

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

Brief description: 10 μg of anti-GLT1 antibody (ab178401, Abcam, Cambridge, MA, USA), or normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) (as a negative control) was coated on a group (1 mg) of M -270 epoxy beads using an antibody conjugation 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 2,000ⅹg for 15 minutes, followed by 12,000ⅹg for 30 minutes, and then washed with phosphate-buffered saline (PBS) (pH 7.4 ) to dilute the supernatant 1:3. One set of antibody-coated beads and 900 [mu]L diluted plasma were incubated for approximately 24 hours at 4[deg.] 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 with the aid of 60 μL of a 1:1 mixture of 0.1% BSA/PBS (pH 7.4) and fixation buffer (4% paraformaldehyde/5% glutaraldehyde) for electron microscopy . Alternatively, at room temperature, with the aid of gentle shaking, by adding 1% Triton X-100 plus 10% protease inhibitor cocktail (P2714, Sigma-Aldrich, St Louis , MO, USA; prepared in 10ml of H ₂ O) for 1 hour to lyse exosomes by incubating beads for Luminex measurement and other analysis, the results are shown in Figure 1, where A shows capture by anti-GLT1, or Normal mouse IgG capture, Western blot of Alix (universal exosome marker) and GLT1; B shows the difference between the concentration of plasma EVs captured by GLT1 antibody and the concentration of plasma EVs captured by normal mouse IgG antibody In contrast, this result demonstrates that anti-GLT1 antibodies can specifically enrich astrocyte-derived exosomes 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 variation.

Example 2

Characterization of anti-GLT1-trapped exosomes from human plasma

Luminex assay: α-syn, oligomer a-syn, PS129, tau, Aβ protein concentration.

Western Blot Analysis: Western blots were performed following standard protocols. Exosome samples (~10 μg protein) were lysed with Laemmli sample buffer and separated on 1D SDS-PAGE gels before transferring to polyvinylidene fluoride membranes. Proteins on the above membranes were detected with the aid of the following primary antibodies: mouse anti-human GLT1 (Abcam, 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 a clinical cohort (46 MSA patients, 49 PD patients, and 50 age- and sex-matched healthy controls) were collected and the concentration of GLT1-containing exosomes in plasma was measured using the Nanosight assay. The results are shown in Figure 2. A in Figure 2 is the use of fluorescently labeled GLT1 antibody to label multiple system atrophy (MSA), Parkinson's disease (PD), healthy control (CT) clinical cohort plasma astrocyte-derived exosomes, and calculate the exosomes by Nanosight The results showed that compared with healthy controls, the concentration of exosomes containing GLT1 fluorescent labeling in plasma was significantly higher in PD patients (p<0.01). B in Figure 2 shows the size distribution of astrocyte-derived exosomes labeled with GLT1 antibody in the plasma of the clinical cohort observed by Nanosight. **, p<0.01, the results show that there is no significant difference in the distribution of GLT1 fluorescently labeled exosomes between PD and control.

In order to further evaluate the potential of GLT1 fluorescently labeled exosome concentration for PD diagnosis, ROC (receiver operating curve) was generated in 46 MSA patients, 49 PD patients and 50 healthy controls to characterize the difference between Their sensitivities (sensitivity and specificity) in PD and healthy control subjects. Figure 3 is the receiver operating curve analysis of the diagnosis and differential diagnosis of multiple system atrophy (MSA), Parkinson's disease (PD) and healthy controls (CT) using the concentration of astrocyte-derived exosomes labeled with GLT1 antibody in plasma ( Receiver Operating Characteristic Analysis, ROCanalysis). Observing the differential diagnosis efficiency of multiple system atrophy and Parkinson's disease, it was found that the PD diagnostic efficiency of plasma GLT1 fluorescently labeled exosome concentration was moderate (AUC=0.6750, sensitivity=66.00%, specificity=70.45%), and the difference between 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 following the method described by Devic et al (Brain.134(Pt 7):el78.doi:10.1093brain/awr015.Epub2011 Feb 24). Saliva samples were collected from healthy individuals between 9-11 AM. Samples were centrifuged at 4 °C and 15,000 g for 15 min to remove insoluble material 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 15,000 g for 15 minutes at 4 °C. Remove the supernatant and wash the pellet twice with ice-cold acetone. Protein concentrations were assessed by means of the BCA protein assay kit (Thermo Scientific Pierce) using BSA as a standard and samples were stored at -20°C until analysis. According to the protocol described in Example 1, supernatants were incubated with anti-GLT1 antibody-coated beads or normal mouse IgG-coated beads (negative control). Captured exosomes were lysed for Western blot analysis according to the protocol described in Example 2. Membranes were probed with the aid of mouse anti-human Alix. Alix, a common exosomal marker, was clearly detectable in exosomes captured by anti-GLT1 but not in exosomes captured by normal mouse IgG (negative control). Apart from the known heavy and light chains of human IgG with appropriate molecular weights of 50 kDa and 25 kDa, respectively, no other non-specific bands are present.

Example 5

According to the method described in Example 1, GLT1-exosomes and normal mouse IgG negative controls were enriched from the plasma of the subjects. With the help of the established Luminex protocol (Brain133:713-726, 2010), 100 μL of exosome preparation (extracted from 300 μL plasma) was used to quantify α-syn, and the results are shown in Figure 4.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (28)

1. a kind of method for being enriched with the excretion body in astroglia source from biological fluid, which is characterized in that including Following steps:

(a) biological fluid of the excretion body containing astroglia source is made to contact anti-GLT1 antibody with formed be immunized it is compound Object, wherein the biological fluid is to be selected from one of blood, serum, blood plasma and saliva or a variety of；And

(b) excretion in the astroglia source is enriched with using the method for solid phase or liquid phase by the immune complex Body.

2., will be biological by the immune complex the method according to claim 1, wherein step (b) includes The excretion body in the astroglia source in body fluid is incorporated into solid phase；From biological fluid separate solid phase binding excretion body with It is enriched with the excretion body in astroglia source.

3. according to the method described in claim 2, it is characterized in that, the anti-GLT1 antibody in step (a) is fixed on In the solid phase.

4. according to the method described in claim 2, it is characterized in that, the method further includes from the solid phase elution of bound The excretion body the step of (c).

5. the method according to claim 1, wherein the anti-GLT1 antibody is by the antibody of label, step It (b) include that the excretion body in the astroglia source is enriched with by flow cytometer.

6. according to the method described in claim 5, it is characterized in that, the anti-GLT1 antibody is by fluorescent marker, same to position Element, enzyme marker, chemiluminescent agent or quantum dot-labeled antibody.

7. a kind of anti-GLT1 antibody is in preparation for detecting the astroglia source of the nervous system disease in biological fluid Biomarker preparation in purposes, which comprises the following steps:

(a) biological fluid of the excretion body containing astroglia source is made to contact anti-GLT1 antibody with formed be immunized it is compound Object, wherein the biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And

(b) level of the biomarker in the astroglia source is determined.

8. purposes according to claim 7, which is characterized in that the biomarker is nucleic acid, protein, lipid or star Shape cell origin excretion body itself.

9. purposes according to claim 8, which is characterized in that step (b) includes:

(b1) the excretion body in the astroglia source in the biological fluid is incorporated by the immune complex solid Phase；

(b2) the excretion body of the solid phase binding is separated from the biological fluid；And

(b3) life in the astroglia source of the excretion body of the isolated solid phase binding of step (b2) is determined The level of substance markers object.

10. purposes according to claim 8, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody are by the antibody of label, and step (b) includes: by means of described in the label directly detection Star cell origin excretion body in biological fluid；

Preferably, the anti-GLT1 antibody be by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or The antibody of colloid gold label；

Preferably, it is directly detected in the biological fluid by Particle Size Analyzer, gamma counter or small animal living body imager Concentration, particle diameter distribution and the Tissue distribution of astrocyte source excretion body；

It is furthermore preferred that the fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

11. purposes according to claim 8, which is characterized in that the nervous system disease includes Parkinson's disease, A Erci The silent disease in sea, prion disease and nervous system neoplasm；

When the nervous system disease is Parkinson's disease, the biomarker is the alpha-synapse of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of nucleoprotein, astrocyte source excretion body；

When the nervous system disease is Alzheimer disease, the biomarker is the tau egg of Protein tau and phosphorylation It is white；

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

When the nervous system disease is nervous system neoplasm, the nervous system neoplasm includes glioma, the biomarker Object is the concentration and particle diameter distribution of astrocyte source excretion intracorporal biomarker level and astrocyte source excretion body, Wherein, the intracorporal biomarker of astrocyte source excretion include alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, the Protein tau of phosphorylation and Abeta albumen.

12. a kind of anti-GLT1 antibody is in preparation for the use in the preparation of detection the nervous system disease or tumour in subject On the way, which comprises the following steps:

(a) biological fluid of the excretion body containing astroglia source is made to contact anti-GLT1 antibody with formed be immunized it is compound Object, wherein the biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And

(b) determine the level of the biomarker in the astroglia source, and with come from no nervous system disease The control level of the subject of disease compares, described in the excretion body from the astroglia source in the subject The level difference of biomarker significantly shows the subject with the nervous system disease.

13. purposes according to claim 12, which is characterized in that the biomarker be nucleic acid, protein, lipid or Astrocyte source excretion body itself.

14. purposes according to claim 13, which is characterized in that step (b) includes:

(b1) the excretion body in the astroglia source in the biological fluid is incorporated by the immune complex solid Phase；

(b2) the excretion body of the solid phase binding is separated；And

(b3) life in the astroglia source of the excretion body of the isolated solid phase binding of step (b2) is determined The level of substance markers object, and compared with the control level of the subject from no the nervous system disease, described tested The level difference of the biomarker of excretion body from the astroglia source in person significantly show it is described by Examination person suffers from the nervous system disease.

15. purposes according to claim 13, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody are by the antibody of label, and step (b) includes: by means of described in the label directly detection Star cell origin excretion body in biological fluid；

Preferably, the anti-GLT1 antibody be by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or The antibody of colloid gold label；

Preferably, it is directly detected in the biological fluid by Particle Size Analyzer, gamma counter or small animal living body imager Concentration, particle diameter distribution and the Tissue distribution of astrocyte source excretion body；

It is furthermore preferred that the fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

16. purposes according to claim 13, which is characterized in that the nervous system disease includes Parkinson's disease, A Er Ci Haimo disease, prion disease and nervous system neoplasm；

When the nervous system disease is Parkinson's disease, the biomarker is the alpha-synapse of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of nucleoprotein and astrocyte source excretion body；

When the nervous system disease is Alzheimer disease, the biomarker is the tau egg of Protein tau and phosphorylation It is white；When the nervous system disease is prion disease, the biomarker is prion protein；

When the nervous system disease is nervous system neoplasm, the nervous system neoplasm includes glioma, the biomarker Object is the concentration and particle diameter distribution of astrocyte source excretion intracorporal biomarker level and astrocyte source excretion body, Wherein, the intracorporal biomarker of astrocyte source excretion include alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, the Protein tau of phosphorylation and Abeta albumen.

17. a kind of for detecting the dress of the biomarker in the astroglia source of the nervous system disease in biological fluid It sets characterized by comprising

Excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source contact anti-GLT1 antibody To form immune complex, the biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；With And

Biomarker level determination module, the level of the biomarker for determining the astroglia source.

18. device according to claim 17, which is characterized in that the biomarker be nucleic acid, protein, lipid or Astrocyte source excretion body itself.

19. device according to claim 18, which is characterized in that

The excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source contact anti-GLT1 Antibody, will be outside the astroglia source in the biological fluid by the immune complex to form immune complex It secretes body and is incorporated into solid phase, and separate the excretion body of the solid phase binding from the biological fluid；And

The biomarker level determination module, for determining the solid phase isolated in the excretion body enrichment module In conjunction with excretion body the astroglia source biomarker level.

20. device according to claim 18, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody in the excretion body enrichment module are by the antibody of label, and the biomarker is horizontal really Cover half block directly detects star cell origin excretion body in the biological fluid by means of the label；

Preferably, the anti-GLT1 antibody be by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or The antibody of colloid gold label；

Preferably, the biomarker level determination module by Particle Size Analyzer, gamma counter or small animal living body at As instrument directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in the biological fluid；

It is furthermore preferred that the fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

21. device according to claim 18, which is characterized in that the nervous system disease includes Parkinson's disease, A Er Ci Haimo disease, prion disease and nervous system neoplasm；

When the nervous system disease is Parkinson's disease, the biomarker is the alpha-synapse of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of nucleoprotein and astrocyte source excretion body；

When the nervous system disease is Alzheimer disease, the biomarker is the tau egg of Protein tau and phosphorylation It is white；

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

When the nervous system disease is nervous system neoplasm, the nervous system neoplasm includes glioma, the biomarker Object is the concentration and particle diameter distribution of astrocyte source excretion intracorporal biomarker level and astrocyte source excretion body, Wherein, the intracorporal biomarker of astrocyte source excretion include alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, the Protein tau of phosphorylation and Abeta albumen.

22. a kind of for detecting the device of the nervous system disease in subject characterized by comprising

Excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source contact anti-GLT1 antibody To form immune complex, the biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；With And

Comparison module, the level of the biomarker for determining the astroglia source, also, with come from no institute The control level for stating the subject of the nervous system disease compares, from the astroglia source in the subject The level difference of the biomarker of excretion body significantly then exports the letter that the subject suffers from the nervous system disease Breath.

23. device according to claim 22, which is characterized in that the biomarker be nucleic acid, protein, lipid or Astrocyte source excretion body itself.

24. device according to claim 23, which is characterized in that

The excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source contact anti-GLT1 Antibody, will be outside the astroglia source in the biological fluid by the immune complex to form immune complex It secretes body and is incorporated into solid phase, and separate the excretion body of the solid phase binding from the biological fluid；And

The comparison module, the institute of the excretion body for determining the isolated solid phase binding of the excretion body enrichment module State the level of the biomarker in astroglia source, also, with the subject from no the nervous system disease Control level compare, the biomarker of the excretion body from the astroglia source in the subject Level difference significantly then export the subject suffer from the nervous system disease information.

25. device according to claim 23, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody in the excretion body enrichment module are by the antibody of label, and comparison module is by means of the mark Note directly detects star cell origin excretion body in the biological fluid, also, with from no the nervous system disease The control level of subject compares；

Preferably, the anti-GLT1 antibody be by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or The antibody of colloid gold label；

Preferably, the biomarker level determination module by Particle Size Analyzer, gamma counter or small animal living body at As instrument directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in the biological fluid；

It is furthermore preferred that the fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

26. device according to claim 23, which is characterized in that the nervous system disease includes Parkinson's disease, A Er Ci Haimo disease, prion disease and nervous system neoplasm；

When the nervous system disease is Parkinson's disease, the biomarker is the alpha-synapse of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of nucleoprotein and astrocyte source excretion body；

When the nervous system disease is Alzheimer disease, the biomarker is the tau egg of Protein tau and phosphorylation It is white；

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

When the nervous system disease is nervous system neoplasm, the nervous system neoplasm includes glioma, the biomarker Object is the concentration and particle diameter distribution of astrocyte source excretion intracorporal biomarker level and astrocyte source excretion body, Wherein, the intracorporal biomarker of astrocyte source excretion include alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, the Protein tau of phosphorylation and Abeta albumen.

Application of the 27.GLT1 as the excretion body biomarker in astroglia source.

28. application according to claim 27, which is characterized in that the application includes: to be enriched with star from biological fluid Astroglia source of the excretion body, preparation in spongiocyte source for detecting the nervous system disease in biological fluid The preparation of biomarker, in biological fluid detect the nervous system disease astroglia source biomarker and The nervous system disease is detected in subject.