Application of body fluid extracellular vesicle membrane protein in preparation of product for diagnosing glaucoma disease

Abstract

The invention discloses an application of a body fluid extracellular vesicle membrane protein in preparation of a product for diagnosing glaucoma disease, the outer vesicle membrane protein is selected from Tau protein and / or GFAP protein on outer vesicles, the invention firstly proposes that NDEVs in peripheral blood are captured through specific membrane proteins (GLAST and L1CAM); therefore, pathological changes of the central nervous system and the retina tissue can be reflected more accurately.

Description

Technical Field

[0001] This invention relates to the field of biomarker detection technology, and more particularly to a field of detection technology for extravesicular proteins. Background Technology

[0002] Glaucoma, or optic nerve degeneration, is a leading cause of irreversible blindness worldwide. Its etiology is complex, influenced by a combination of genetic and environmental factors. Primary open-angle glaucoma (POAG) is the most common type. POAG is characterized by a slow progression and insidious onset, primarily manifesting as chronic, progressive optic nerve damage and visual field defects. Due to its insidious onset and complex pathological mechanisms, many patients are only diagnosed when their visual function has been severely impaired or they are on the verge of blindness. Studies show that 30-50% of retinal ganglion cells (RGCs) are already lost at the time of POAG diagnosis. Furthermore, even after treatment, 40% of patients experience progressive optic nerve damage, and 10% eventually go blind. Therefore, early diagnosis and intervention for glaucoma are crucial.

[0003] Currently, glaucoma screening and early diagnosis still face multiple technical bottlenecks. The difficulty in diagnosis lies in the limitations of existing methods in terms of sensitivity, standardization, and applicability. Although high intraocular pressure is a significant risk factor, intraocular pressure measurements are easily affected by physiological fluctuations and individual differences, often failing to accurately reflect the condition. Routine visual field examinations (such as static threshold visual field testing) have limited ability to detect early lesions, often relying on the presence of significant visual field defects for diagnosis. While fundus imaging combined with hemodynamic monitoring shows potential, its complex equipment, high operational requirements, and patient compliance issues limit its widespread application. The scarcity of primary healthcare resources, low patient compliance, and insufficient public awareness of glaucoma further restrict the widespread implementation of early glaucoma screening. Therefore, developing a highly sensitive, easily deployable, and non-invasive biomarker detection method is a crucial breakthrough urgently needed for the early diagnosis and dynamic monitoring of POAG.

[0004] Extracellular vesicles (EVs) are nanovesicles actively released by cells under normal or stress conditions. They are widely distributed in bodily fluids such as blood, aqueous humor, urine, and saliva, and can carry a variety of bioactive molecules, including proteins, mRNA, miRNA, lncRNA, and DNA. Their double-membrane structure endows them with good stability and resistance to degradation, enabling them to effectively transmit cellular signals and reflect the pathophysiological state of the cells from which they originate. Numerous studies have confirmed that the content and molecular composition of EVs exhibit specific changes in various diseases, demonstrating their potential as biomarkers. Summary of the Invention

[0005] The purpose of this invention is to provide the first discovery of the use of plasma extravesicular protein Tau protein and / or GFAP protein in the diagnosis of glaucoma disease, and to provide a highly sensitive, non-invasive biomarker.

[0006] This invention involves extracting external vesicles from peripheral blood samples of patients with porcine angina pectoris (POAG) and cataracts, performing mass spectrometry sequencing, and then separating different types of non-invasive vesicle (NDEV) subsets using immunoprecipitation. ELISA is then used to detect POAG plasma, total plasma EVs, and NDEVs (L1CAM). + EVs and GLAST + By screening differentially expressed proteins in exosomes, two protein targets with high clinical value, Tau protein and GFAP protein, were identified. Based on this, a product was developed that uses Tau protein and GFAP protein on exosomes as biomarkers to assist in the diagnosis of glaucoma.

[0007] Tau protein is a protein primarily found in neurons of the brain. It belongs to the "microtubule-associated protein" family and plays a crucial role in the health and maintenance of brain nerve cells.

[0008] GFAP, short for glial fibrillary acidic protein, is a type III intermediate filament protein. Intermediate filaments are one of the three main components of the cytoskeleton, primarily serving to provide structural support and resist mechanical stress. They are also a hallmark protein of astrocytes. As a major cytoskeletal protein of astrocytes, GFAP's core functions include: maintaining cell morphology and structural integrity, providing mechanical strength, participating in cell movement and morphological changes, supporting the blood-brain barrier, and participating in signal transduction.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The use of a humoral extracellular vesicle membrane protein in the preparation of products for diagnosing glaucoma, wherein the extracellular vesicle membrane protein is selected from Tau protein and / or GFAP protein on extracellular vesicles.

[0010] Preferably, the product is a detection reagent for detecting the content of Tau protein and / or GFAP protein.

[0011] Preferably, the glaucoma is primary open-angle glaucoma.

[0012] Preferably, the body fluid is selected from plasma or serum.

[0013] Preferably, the extracellular vesicles in the body fluid are extracellular vesicles derived from nerve cells and enriched with specific proteins on the membrane surface.

[0014] Preferably, the membrane surface-specific protein is GLAST, or GLAST and L1CAM.

[0015] Preferably, the method for enriching extracellular vesicles derived from nerve cells includes at least the following steps: Step (1) preparing magnetic beads coupled with labeled antibodies, adding ultrafiltered body fluid extracellular vesicles and protease inhibitors for incubation, wherein the labeled antibody is a labeled GLAST antibody, or a labeled GLAST antibody and a labeled L1CAM antibody; Step (2): Adsorbing magnetic beads to separate external vesicles under the action of a magnetic field to enrich external vesicles derived from nerve cells in body fluids; Step (3): Elute the outer vesicles by repeatedly blowing and eluting with an elution buffer, wherein the elution buffer is a mild antigen / antibody elution buffer or glycine hydrochloride.

[0016] Preferably, the labeled antibody is a biotin-labeled antibody, and the magnetic beads are streptavidin magnetic beads.

[0017] The present invention also provides a method for enriching extracellular vesicles of peripheral body fluids derived from the nervous system, wherein the extracellular vesicles of peripheral body fluids include Tau protein and / or GFAP protein, and the enrichment method includes at least the following steps: Step (1) Preparing magnetic beads coupled with labeled antibodies, adding ultrafiltered extracellular vesicles of peripheral body fluids and protease inhibitors for incubation, wherein the labeled antibody is a labeled GLAST antibody, or a labeled GLAST antibody and a labeled L1CAM antibody; Step (2): Adsorbing magnetic beads to separate external vesicles under the action of a magnetic field to enrich external vesicles derived from nerve cells in body fluids; Step (3): Elute the outer vesicles by repeatedly blowing and eluting with an elution buffer, which is either Gentle Ag / Abelution buffer or glycine hydrochloride.

[0018] Preferably, the labeling antibody is a biotin-labeled antibody, and the magnetic beads are streptavidin magnetic beads.

[0019] Preferably, the body fluid is plasma or serum.

[0020] Preferably, the specific steps for preparing magnetic beads coupled with labeled antibodies include: rinsing the magnetic beads with buffer, discarding the supernatant after adsorption by a magnetic rack, adding 3% BSA, adding GLAST antibody, or adding labeled GLAST antibody and labeled L1CAM antibody, incubating at room temperature, and the volume ratio of magnetic beads to labeled antibody is 5-20:1.

[0021] Through experimental verification using examples, we conclude that extracellular vesicles (NDEVs) of the nervous system originating in peripheral blood have diagnostic value in primary open-angle glaucoma (POAG). This invention is the first to propose capturing NDEVs in peripheral blood using specific membrane proteins (GLAST and L1CAM), thereby more accurately reflecting the pathological changes in the central nervous system and retinal tissue. The innovations of this invention are mainly reflected in the following aspects: (1) Identifying GLAST as a novel target for capturing NDEVs. (2) Demonstrating the advantages of NDEVs in the non-invasive diagnosis of primary open-angle glaucoma. (3) Analyzing the proteins (Tau, GFAP) in NDEVs, revealing that the proteins (Tau, GFAP) in NDEVs have higher diagnostic efficacy for primary open-angle glaucoma. Attached Figure Description

[0022] Figure 1 In the diagram, A represents the flowchart for extracting NDEVs from plasma; Figure 1 In the diagram, B represents the expression of GLAST on the EV membrane surface as detected by electron microscopy (indicated by the black arrow; CD81 is the positive control), and L1CAM... + EVs and GLAST + Transmission electron microscopy of EVs (scale bar: 200 nm). Figure 1 In this context, C represents the particle size of NDEVs detected by NTA. Figure 1 DWB detection of marker proteins of NDEVs; Figure 2 In the figure, A represents the total EVs and L1CAM in the cataract and glaucoma experimental groups. + Concentrations of α-syn, Tau, CRYM, and RLBP1 proteins in EVs; Figure 2 Total EVs and GLAST in the B cataract and glaucoma experimental groups + Concentrations of GFAP, Tau, CRYM, and RLBP1 proteins in EVs (all data are presented as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001). Figure 3 The expression differences of Tau in the glaucoma experimental group and the control group were detected using different detection methods. Figure 3 In the figure, B represents the ROC curve of Tau. Figure 3 C in the figure represents the difference in GFAP expression between the experimental group and the control group in glaucoma, detected by different detection methods; Figure 3D in the figure represents the ROC curve of GFAP (all data are shown as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001); Figure 4 Mendelian randomization analysis showed that changes in Tau, GFAP, RLABP1, and CRYM expression were risk factors for POAG. Detailed Implementation

[0023] As mentioned above, in view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have been able to propose the technical solution of this invention. The technical solution of this invention will be further disclosed and explained below with reference to examples. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. It should be noted that the following embodiments are intended to facilitate understanding of this invention and are not intended to limit it in any way. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] Patient specimen collection Fifty patients with primary open-angle glaucoma (POAG) and 50 age-matched cataract patients hospitalized at the Eye Hospital of Wenzhou Medical University between October 2023 and December 2024 were selected. Patients meeting the inclusion and exclusion criteria were selected. This study was approved by the Medical Ethics Committee of the Eye Hospital of Wenzhou Medical University (2023-126-K-101-02), and complies with the purposes of the Declaration of Helsinki. Informed consent was obtained from each enrolled patient.

[0025] POAG inclusion criteria: (1) Confirm POAG diagnosis: Baseline untreated intraocular pressure ≥21 mmHg (measured with a Goldmann applanation tonometer) or stable intraocular pressure controlled at 10-21 mmHg after treatment (≥4 weeks); gonioscopy confirms open angle (Shaffer grade ≥III); and there is evidence of glaucomatous optic nerve injury (cup-to-disc ratio ≥0.6 / binocular difference >0.2, OCT shows localized RNFL defect); visual field abnormalities are present: typical visual field defects include paracentral scotomas, nasal step, arcuate scotomas, or temporal wedge-shaped defects. Abnormal visual field indicators (Humphrey perimeter 24-2 / 30-2 procedure): MD (mean deviation): MD≥-6 dB (early stage), -12dB≤MD<-6 dB (mid stage), MD<-12 dB (late stage); (2) Age 50-75 years old; (3) Voluntarily sign the informed consent form.

[0026] Exclusion criteria: (1) Angle-closure glaucoma, secondary glaucoma (such as uveitis, neovascularization), ocular hypertension (without visual field / optic nerve damage); (2) Underwent intraocular surgery or laser treatment (such as SLT or trabeculectomy) within the past 3 months. (3) Systemic and other eye diseases: Alzheimer's disease, Parkinson's disease, mental illness and other diseases affecting the central nervous system, severe heart, liver and kidney diseases, active eye infections, corneal lesions, and patients with only one eye.

[0027] Cataract surgery inclusion criteria: (1) No signs of glaucoma: Baseline intraocular pressure ≤21 mmHg (measured with a Goldmann applanation tonometer); gonioscopy showed an open angle (Shaffer grade ≥III); no evidence of optic nerve injury (cup-to-disc ratio ≤0.5 and binocular difference ≤0.1, OCT showed RNFL thickness ≥95 μm without localized defects); normal visual field (Humphrey 24-2 procedure, MD value >-2 dB and no glaucomatous defect pattern); age 50–75 years.

[0028] (2) Diagnosis of simple cataract: The lens opacity meets the LOCS III classification criteria (nuclear / cortical / posterior subcapsular opacity ≥ grade 2), with no history of other eye surgeries; best corrected visual acuity ≥ 0.1 (LogMAR), and the visual acuity loss is mainly caused by cataracts.

[0029] (3) Exclude mixed lesions: No family history of glaucoma, uveitis, high myopia (greater than -6.0 D), diabetic retinopathy, macular degeneration or other diseases affecting visual function; no use of glucocorticoids or glaucoma medications in the past 6 months.

[0030] (4) Can assist in completing follow-up and examinations (visual acuity, intraocular pressure, OCT, visual field, etc.).

[0031] (5) Voluntarily sign the informed consent form.

[0032] Example 1: Extraction of extracellular vesicles from plasma of patients with primary glaucoma 1) Plasma pretreatment Plasma separation: Centrifuge blood sample at low speed (1000 g, 10 min, 4 °C) to remove cells and cell debris; transfer supernatant to new tubes; remove large extracellular particles: centrifuge at 10000 g for 30 min, 4 °C to remove larger particles and microparticles; filtration: filter using a 0.22 µm filter membrane to remove large particles.

[0033] 2) Size exclusion chromatography (SEC) was used to collect total plasma EVs.

[0034] Column packing: Use a dedicated EV separation column (such as a qEV column) and perform each step of the experiment according to the instruction manual.

[0035] Sample loading: Loading plasma samples and PBS.

[0036] Separation and collection: Collect a specific volume of EVs enriched components (typically components 1-6).

[0037] Purification: Centrifuge at 3000 g for about 30 minutes using an ultrafiltration tube to concentrate the EVs to about 500 µL.

[0038] Example 2. Capture of NDEVs in peripheral blood using GLAST and L1CAM antibodies. First, total plasma EVs are collected, and then NDEVs are captured from the total plasma EVs through immunization.

[0039] 1) Preparation of antibody-conjugated magnetic beads: Each batch of magnetic beads is 50 µL. Wash once with 200 µL PBS. After adsorption on a magnetic rack, discard the supernatant. Add 100 µL of 3% BSA, 5 µL each of L1CAM and GLAST, and incubate at room temperature for 1 hour before use.

[0040] 2) Mixing EVs with antibody-conjugated magnetic beads: Add approximately 500 µL of EV sample to antibody-conjugated magnetic beads, add 1% protease inhibitor, gently swirl to mix, and incubate at room temperature for 3 hours. At the end of incubation, wash the captured NDEVs, wash the magnetic beads three times with cold PBS, remove the supernatant after magnetic adsorption, and retain the bound EVs.

[0041] 3) Elution of NDEVs: Elution Method 1: Glycine-hydrochloric acid elution method Add 100 µL of glycine hydrochloride (pH=3) to the magnetic beads, repeatedly pipet for 1 minute, let stand for 15 minutes, add 10 µL of Tis-HCl (pH=8.6), mix well, adjust the pH to 7, and collect the supernatant into a new EP tube using a magnetic rack. Repeat the operation 3 times. This solution can be used for ELISA experiments.

[0042] Elution Method 2: Gentle Ag / Ab elution buffer (mild elution solution) Add 200 µL of Gentle Ag / Ab elution buffer, repeatedly pipette for 1 minute, let stand for 5 minutes, collect in an empty labeled EP tube, repeat 3 times, add 3 mL of sterile TBS, centrifuge in an ultrafiltration tube to obtain 100 µL of concentrate, for electron microscopy and nanoparticle detection, and Western blotting verification experiment.

[0043] Total EVs were extracted from peripheral blood plasma using the SEC method, and then NDEVs (L1CAM) were separated using immunoprecipitation. + EVs and GLAST + The process and method are shown in Figure 1(A). Immunogold electron microscopy results from GLAST further validated its localization characteristics on the membrane surface of EVs, meeting the basic requirements for a target protein (Figure 1(B)). L1CAM + EVs and GLAST + Transmission electron microscopy (TEM) images of EVs show a typical cup-shaped structure (B in Figure 1). NTA analysis of L1CAM + EVs and GLAST + The diameter range of EVs is around 100 nm at its peak, and the average particle diameter is around 130 nm. Figure 1 (C in the text). Western blotting validated L1CAM. + EVs and GLAST + All EVs expressed the marker proteins of small extracellular vesicles, ALIX, TSG101, and CD63 (D in Figure 1).

[0044] Example 1: Enrichment of characteristic proteins by NDEVs L1CAMA ELISA for detecting cataracts and POAG + The levels of α-syn, Tau protein, RLBP1, and CRYM in EVs were measured, and GLAST was used to detect cataracts and POAG. +The levels of GFAP, Tau protein, RLBP1, and CRYM in EVs were measured. Specific procedures were performed according to the ELISA kit's instruction manual.

[0045] ① α-syn protein ELISA detection steps: Prepare reagents and samples: Quantify total EVs and NDEVs at the same concentration of 5 µg / 100 µL, and dilute plasma 1:5 (20 µL to 100 µL). Equilibrate all reagents and samples to room temperature (20-25°C).

[0046] Add the standard and sample to the wells pre-coated with anti-α-syn antibody. Incubate: After sealing, incubate at 37°C for 1 hour.

[0047] Washing: Wash the plate 3 times with washing solution, 250 μL / well each time.

[0048] Add biotin-labeled secondary antibody: Add biotin-labeled anti-α-syn antibody and incubate at 37°C for 1 hour. Wash again: Repeat the washing step 3 times.

[0049] Adding enzyme-labeled avidin: Add horseradish peroxidase (HRP)-labeled avidin and incubate at 37°C for 1 hour.

[0050] Wash again: Repeat the washing steps 5 times.

[0051] Add substrate TMB: Add substrate TMB and incubate at 37°C in the dark for 15 minutes. Terminate the reaction: Add acidic stop solution; the color changes from blue to yellow.

[0052] OD value determination: Measure the absorbance (OD value) of each well at a wavelength of 450 nm. Concentration calculation: The concentration of α-syn in the sample was calculated based on the standard curve.

[0053] The detection steps for CRYM and RLBP1 are the same as for α-syn.

[0054] ②Tau protein ELISA detection steps: Prepare reagents and samples: Quantify total EVs and NDEVs at the same concentration of 2 µg / 100 µL. Dilute plasma 1:5 (20 µL to 100 µL). Equilibrate all reagents and samples to room temperature (20-25°C). Add samples: Add standards and samples to microwells pre-coated with anti-Tau antibody.

[0055] Incubation: After sealing, incubate at 37°C for 30 minutes. Discard the sample solution.

[0056] Add enzyme-labeled secondary antibody: Add HRP-labeled Tau antibody and incubate at 37°C for 30 minutes. Repeat the washing step 3 times, 200 µL each time.

[0057] Add substrate TMB: Add substrate TMB and incubate at 37°C in the dark for 15 minutes. Terminate the reaction: Add stop solution.

[0058] OD value determination: The absorbance (OD value) of each well was measured at a wavelength of 450 nm. Concentration calculation: The concentration of Tau protein in the sample was calculated based on the standard curve.

[0059] In addition to exhibiting typical EV characteristics, NDEVs should also demonstrate enrichment of neurotrophic substances. ELISA detection of equivalent concentrations of L1CAM... + EVs and Total-EVs, of which L1CAM + The higher levels of α-syn and Tau in EVs indicate that L1CAM + EVs have an enrichment effect on characteristic proteins of the central nervous system. Figure 2 A in (the same concentration of GLAST). + Compared to Total-EVs, EVs showed higher levels of GFAP and Tau proteins (Figure 2, B). We examined the characteristic retinal proteins CRYM and GLAST, and the results showed that L1CAM... + EVs and GLAST + The levels of CRYM and GLAST in EVs were significantly higher than those in total plasma EVs.

[0060] Example 2: Diagnostic role of total plasma EVs and NDEVs To assess whether plasma Tau and GFAP proteins are potential causal risk factors for primary open-angle glaucoma (POAG), this study employed Mendelian randomization. This method, based on the principle of random allocation of genetic variations at birth, can infer the causal relationship between exposure factors (such as biomarkers) and the disease with minimal confounding factors. First, we screened single nucleotide polymorphisms (SNPs) significantly associated with the expression levels of Tau protein (encoding gene MAPT) and GFAP protein (encoding gene GFAP) from publicly available genome association study databases as instrumental variables. Screening criteria included a p-value less than 5 × 10⁻⁶. -8The study identified independence (LD r² < 0.01) and SNPs that were significantly associated with exposure but did not directly affect the outcome variable (POAG). Subsequently, using large-scale GWAS summary data of POAG as the data source for the outcome variable, a two-sample MR analysis framework was constructed, and the Inverse-Variance Weighted (IVW) method was employed for primary causal inference. Furthermore, to ensure the robustness of the results, sensitivity analysis was conducted using MR-Egger regression and weighted median method.

[0061] To detect the presence of multi-level effects among the instrumental variables, the MR-Egger intercept test and Cochran's Q statistic were used to assess heterogeneity. Furthermore, to avoid weak instrumental bias, an F-statistic was calculated for each instrumental variable to ensure its strength (F>10). All MR analyses were performed using the "TwoSampleMR" package in R. The statistical significance level was set at P<0.05.

[0062] Statistical data were analyzed using Graphpad Prism 9.0 software. Data are presented as mean ± standard deviation (mean ± SD), and all data were tested for normality. The t-test was used for comparisons between two groups. One-way ANOVA was used for univariate multi-group data, and two-way ANOVA was used for two-way multi-group data comparisons. ROC analysis was performed to plot the relationship between sensitivity and specificity (1-specificity). The results are shown below. Figure 3 As shown, P < 0.05 is considered statistically significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. “ns” indicates no statistically significant difference.

[0063] Detection of plasma, total-EVs, and NDEVs (L1CAM) in POAG and control groups. + EVs and GLAST + The concentrations of Tau and GFAP in different EV samples were measured. After quantification of the same type of samples, the concentrations of Tau and GFAP were detected by ELISA. Results showed that the levels of Tau and GFAP in NDEVs were significantly higher than in the control cataract group, while there were no significant differences in these two proteins in plasma and total EVs between the two groups (A and C in Figure 3). Further analysis of their diagnostic value was confirmed by the area under the receiver operating characteristic (ROC) curve (AUC) for Tau and L1CAM. +EVs (AUC: 0.8163) and GLAST + EVs (AUC: 0.8980) all showed high diagnostic efficacy in distinguishing POAG from the control group (Figure 3, B); for GFAP protein, L1CAM + EVs (AUC: 0.9375) and GLAST + EVs (AUC: 0.8750) all showed high diagnostic efficacy in distinguishing POAG from the control group (D in Figure 3). Compared with plasma and Total-EVs, the marker proteins in NDEVs were more effective in distinguishing POAG.

[0064] Mendelian randomization analysis of the causal relationship between differentially expressed proteins in NDEVs and the risk of POAG showed that Tau, GFAP, RLABP1, and CRYM were significant risk factors for POAG. Figure 4 ).

[0065] Finally, it is necessary to state that the above embodiments are only used to further illustrate the technical solution of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention shall fall within the scope of protection of the present invention.

Claims

1. The use of a humoral extracellular vesicle membrane protein in the preparation of products for diagnosing glaucoma, characterized in that: The extravesicular membrane proteins are selected from Tau protein and / or GFAP protein on the extravesicular membrane.

2. The use of the extracellular vesicle membrane protein of body fluids according to claim 1 in the preparation of products for diagnosing glaucoma, characterized in that: The product is a detection reagent for detecting the content of Tau protein and / or GFAP protein.

3. The use of the extracellular vesicle membrane protein of body fluids according to claim 1 in the preparation of products for diagnosing glaucoma, characterized in that: The glaucoma mentioned is primary open-angle glaucoma.

4. The use of the extracellular vesicle membrane protein of body fluids according to claim 1 in the preparation of products for diagnosing glaucoma, characterized in that: The body fluid is selected from blood, plasma, or serum.

5. The use of the extracellular vesicle membrane protein of body fluids according to claim 1 in the preparation of products for diagnosing glaucoma, characterized in that: Extracellular vesicles in body fluids are extracellular vesicles derived from nerve cells and enriched with specific proteins on their membrane surface.

6. The use of the extracellular vesicle membrane protein of body fluids according to claim 5 in the preparation of products for diagnosing glaucoma, characterized in that: The membrane surface-specific protein is GLAST, or GLAST and L1CAM.

7. The use of the extracellular vesicle membrane protein of body fluids according to claim 5 in the preparation of products for diagnosing glaucoma, characterized in that: The method for enriching neural cell-derived extravesicles includes at least the following steps: a) Exposing a biological fluid containing nerve cell-derived extravesicles to GLAST antibodies, or GLAST and L1CAM antibodies, to form an immune complex, wherein the biological fluid is selected from one or more of blood, serum, and plasma; and b) Enrich the nerve cell-derived extracellular vesicles using solid-phase or liquid-phase methods with the immune complex.

8. The use of the extracellular vesicle membrane protein of body fluids according to claim 7 in the preparation of products for diagnosing glaucoma, characterized in that: The antibody is a labeled antibody, and the labeled antibody is a biotinylated antibody.

9. A method for enriching peripheral fluid extravesicles derived from the nervous system, characterized in that, The peripheral body fluid extravesicles include Tau protein and / or GFAP protein, and the enrichment method includes at least the following steps: Step (1) Prepare magnetic beads coupled with labeled antibodies, add ultrafiltered peripheral body fluid extravesicles and protease inhibitors for incubation, wherein the labeled antibody is a labeled GLAST antibody, or a labeled GLAST antibody and a labeled L1CAM antibody; Step (2): Adsorbing magnetic beads to separate external vesicles under the action of a magnetic field to enrich external vesicles derived from nerve cells in body fluids; Step (3): Elute the outer vesicles by repeatedly blowing and eluting with an elution buffer, wherein the elution buffer is a mild antigen / antibody elution buffer or glycine hydrochloride.

10. The method for enriching peripheral fluid extravesicles of the nervous system according to claim 9, characterized in that, The labeled antibody is a biotin-labeled antibody, and the magnetic beads are streptavidin magnetic beads.

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