Capture microsphere, preparation method thereof and high-purity exosome enrichment method

Abstract

The invention discloses a capture microsphere, which is a compound obtained by coupling a microsphere and an exosome marker antibody, and the average particle size of the microsphere is more than 1 [mu] m. The invention also discloses a high-purity exosome enrichment method, which comprises the following steps: crude extraction: taking a fresh body fluid sample, and carrying out ultracentrifugation to preliminarily remove impurities so as to obtain the exosome subjected to crude extraction; and performing fine purification: adding the capture microspheres into the exosome subjected to crude extraction, performing mixed incubation, performing centrifugation, removing supernatant, and performing resuspension so as to obtain the high-purity exosome. The method is lower in cost and more efficient, the obtained exosome sample is high in purity and clinical application degree, and the method can cover multiple body fluid samples at the same time and has good application prospects.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a capture microsphere and a preparation method thereof, as well as a high-purity exosome enrichment method. Background Art

[0002] Exosomes are an extracellular membrane structure that is actively secreted by cells, with a diameter ranging from 30 to 150 nm and a phospholipid bilayer structure. As a conservative process, the secretion of exosomes can be found in most body fluids in the body. These body fluids include semen, blood, urine, saliva, breast milk, amniotic fluid, ascites, cerebrospinal fluid and bile.

[0003] Exosome biogenesis is a mechanism of protein quality control. Once released, exosomes have multiple activities such as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This intercellular vesicle transport pathway plays an important role in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer and neurodegenerative diseases. In 2015, Melo et al. found that a cell surface proteoglycan, glypian-1 (GPC1), was particularly enriched in cancer cell-derived exosomes, which helped to distinguish healthy subjects and patients with benign pancreatic diseases from patients with early and late stage pancreatic cancer. According to the description of CN 113637736A, pTau217, one of the biomarkers of Alzheimer's disease (AD) in plasma exosomes, can be used alone as a marker to distinguish AD patients from healthy people, and its AUC can reach 0.877. In 2013, Bijnsdorp et al. found that ITGA3 in urine exosomes can be used as a marker for diagnosing patients with metastatic prostate cancer. In 2016, Stuendl et al. published an article supporting the distinction between Parkinson's disease and Lewy body dementia by α-syn in cerebrospinal fluid exosomes. According to a review by Wang et al., miRNA-34, miRNA-21, and lncRNA-p21 in blood exosomes are also excellent markers for distinguishing a variety of tumor diseases. In 2019, Jia et al. confirmed that Aβ42, T-tau, and P-Tau-181 in serum and cerebrospinal fluid exosomes have the same AD and aMCI diagnostic capabilities as the corresponding CSF markers. Therefore, exosomes have great application prospects in disease diagnosis and efficacy evaluation.

[0004] At present, exosome-related disease markers have not been widely used in clinical practice. The main reason is that the current exosome enrichment technology still has various shortcomings. Differential ultracentrifugation is a classic method for exosome enrichment. This method is based on the sequential separation of different extracellular components of fluid samples by density, size and shape under a certain centrifugal force. Based on this principle, this method may enrich some impurities with similar parameters such as exosome particle size, resulting in a decrease in the purity of the target exosomes. Density gradient centrifugation can rely on gradient density to distinguish different components in more detail, but its sample processing capacity is low and the operation requirements are relatively strict, which is not suitable for scale-up applications. Ultrafiltration centrifugation and size exclusion methods rely on the use of ultrafine nanomembranes with different molecular weight cutoff values ​​to separate exosomes from small rods according to size. They have the advantages of fast speed and convenience, but due to the potential membrane clogging and capture caused by the interaction between the membrane and the vesicles, this may shorten the life of the expensive membrane used and cause sample loss, and inevitably introduce impurities with similar particle sizes. The use of polyethylene glycol (PEG) precipitants can promote the aggregation and precipitation of a large number of polymer-embedded vesicles. Although it is easy to use, it will also precipitate microvesicles of other particle sizes. At the same time, PEG is not easy to remove, and its presence will also affect downstream applications. Based on this, the exosome enrichment method based on particle size as a distinction or using precipitants generally has the problem of insufficient purity.

[0005] In addition, there are a large number of proteins and receptors in the exosome membrane, which provides an excellent opportunity to develop highly specific exosome separation technology. Exosomes can be efficiently separated by utilizing the immunoaffinity interaction between these proteins and their antibodies, as well as the specific interaction between receptors and ligands. However, the current technology of exosome separation based on capture magnetic beads has the following disadvantages:

[0006] (1) It takes a long time

[0007] Shorter enrichment time is helpful for large-scale application in non-scientific research environments. However, the magnetic bead capture method mentioned in the authorized patent CN106062559B takes about 24 hours to obtain exosomes, and the microsphere capture method used in CN107893051A, CN111505264A and CN113652388A takes no less than 12 hours.

[0008] (2) Low purity

[0009] Unlike the amplification system of molecular biology, immunological detection is susceptible to interference. For example, high-abundance proteins such as hemoglobin and lipoprotein in the blood may cover the immune sites and significantly interfere with the detection. At the same time, some membrane fragments in the blood also contain exosome membrane marker proteins such as CD63 and CD9, which are difficult to remove by a single magnetic bead capture method. In addition, some impurities that are similar to the particle size of exosomes are also difficult to remove by enrichment methods based on particle size.

[0010] (3) High cost

[0011] The current magnetic bead capture method for exosome purification generally requires a large amount of antibodies, and the high price of antibodies leads to a high application cost of this method. According to the embodiment provided by CN110133272A, the amount of capture antibody used is 10μg / T. The capture antibody used in the technical solution mentioned in CN111505264A is 0.5μg / T. Low-cost reagents are conducive to the promotion and application of terminals and can effectively improve production efficiency.

[0012] (4) Limited application scope

[0013] The secretion of exosomes can be found in most body fluids in the body. However, there is still a lack of universal means to enrich exosomes from a variety of body fluids. For example, CN106062559B, CN109082400A and CN114467030A can directly enrich exosomes from plasma, while CN113652399B can separate exosomes from cerebrospinal fluid. Summary of the invention

[0014] Based on the above problems in the prior art, there is an urgent need to develop a high-purity, improved exosome enrichment method based on capture magnetic beads. The present application provides a high-purity exosome enrichment method based on capture magnetic beads. The method of the present application is short in time, low in cost and suitable for complex systems.

[0015] The specific technical methods of this application are as follows:

[0016] 1. A capture microsphere, wherein the microsphere is a complex obtained by coupling the microsphere with an exosome marker antibody, and the average particle size of the microsphere is greater than 1 μm.

[0017] 2. The capture microspheres according to item 1, wherein the average particle size of the microspheres is 3.5 μm or more.

[0018] 3. The capture microsphere according to item 1 or 2, wherein the microsphere is a magnetic microsphere, a latex microsphere or a silica microsphere, preferably a magnetic microsphere.

[0019] 4. The capture microsphere according to any one of items 1 to 3, wherein the exosome marker antibody is an anti-CD63 antibody, an anti-CD9 antibody or an anti-CD81 antibody, preferably an anti-CD63 antibody;

[0020] Preferably, the mass ratio of the exosome marker antibody to the microspheres is 0.005-0.2:1.

[0021] 5. A method for preparing capture microspheres, comprising the following steps:

[0022] Microspheres with an average particle size of more than 1 μm were taken, and exosome marker antibodies were added and mixed and incubated to obtain capture microspheres.

[0023] 6. The preparation method according to item 5, wherein the average particle size of the microspheres is greater than 3.5 μm.

[0024] 7. The preparation method according to item 5 or 6, wherein the microspheres are magnetic microspheres, latex microspheres or silica microspheres, preferably magnetic microspheres.

[0025] 8. The preparation method according to any one of items 5 to 7, wherein the exosome marker antibody is an anti-CD63 antibody, an anti-CD9 antibody or an anti-CD81 antibody, preferably an anti-CD63 antibody;

[0026] Preferably, the mass ratio of the exosome marker antibody to the microspheres is 0.005-0.2:1.

[0027] 9. The preparation method according to any one of items 5 to 8, wherein the preparation method comprises the following steps:

[0028] The magnetic microspheres are magnetically adsorbed and the supernatant is removed;

[0029] The magnetic microspheres were then equilibrated and the supernatant was removed again;

[0030] Then add coupling agent for activation, magnetic adsorption, and remove the supernatant;

[0031] Wash the activated magnetic microspheres and resuspend them;

[0032] adding the exosome marker antibody and incubating;

[0033] Add blocking agent, incubate, and remove supernatant after magnetic adsorption;

[0034] Add the second buffer, magnetically adsorb, and remove the supernatant;

[0035] Add a second buffer and resuspend to obtain the capture microspheres;

[0036] Preferably, the preparation method comprises the following steps:

[0037] Add magnetic microspheres into centrifuge tubes, magnetically adsorb, and remove supernatant;

[0038] Then, the first buffer or distilled water was added to equilibrate the magnetic microspheres, and the supernatant was removed again;

[0039] Add the first buffer or distilled water again, shake and mix, then add the coupling agent for activation, magnetic adsorption, and remove the supernatant;

[0040] Wash the activated magnetic microspheres with the first buffer and resuspend them with the first buffer or distilled water;

[0041] adding the exosome marker antibody and incubating;

[0042] Add blocking agent, incubate, and remove supernatant after magnetic adsorption;

[0043] Add the second buffer, shake and mix, magnetically adsorb, remove the supernatant, and repeat 3 to 8 times;

[0044] Add a second buffer, resuspend, and shake to mix, to obtain the capture microspheres;

[0045] Preferably, the first buffer is MES buffer;

[0046] Preferably, EDC and NHS are used as coupling agents;

[0047] Preferably, the activation temperature is 35-37°C and the activation time is 10-30 min;

[0048] Preferably, the activated magnetic microspheres are washed at least 3 times with the first buffer;

[0049] Preferably, after adding the exosome marker antibody, incubate at 30-37° C. for 1-2 h;

[0050] Preferably, after adding the blocking agent, incubate at 30-37°C for 1-2h;

[0051] Preferably, the second buffer is PBS buffer.

[0052] 10. Use of the capture microspheres described in any one of items 1 to 4 or the capture microspheres prepared by the preparation method described in any one of items 5 to 9 in enriching high-purity exosomes.

[0053] 11. A method for enriching high-purity exosomes, comprising the following steps:

[0054] Crude extraction: Take a body fluid sample, remove impurities by ultracentrifugation, and obtain crudely extracted exosomes;

[0055] Purification: Add the capture microspheres described in any one of items 1 to 4 or the capture microspheres prepared by the preparation method described in any one of items 5 to 9 to the crudely extracted exosomes, mix and incubate, centrifuge, remove the supernatant, and resuspend to obtain high-purity exosomes.

[0056] 12. The method according to item 11, wherein the body fluid sample is a plasma sample, a serum sample, a cerebrospinal fluid sample or a urine sample.

[0057] 13. The method according to item 11 or 12, wherein, in the purification step, the volume ratio of the crudely extracted exosomes to the capture microspheres is 1:0.005 to 0.1, preferably 1:0.005 to 0.025.

[0058] 14. The method according to any one of items 11 to 13, wherein in the purification step, the incubation conditions are shaking incubation at 2 to 8°C for 12 to 16 hours or incubation at 30 to 37°C for 1 to 2 hours, preferably incubation at 37°C for 1 hour.

[0059] 15. The method according to any one of items 11 to 14, wherein in the crude extraction step, the ultracentrifugation comprises:

[0060] Centrifuge at 5000-15000 g for 20-50 min at 2-5 °C and aspirate the supernatant;

[0061] Then filter using a 0.22 μm filter membrane;

[0062] Centrifuge again at 80,000 to 150,000 g for 1 to 3 h at 2 to 5 °C, remove the supernatant and resuspend the precipitate;

[0063] Then centrifuge again at 80,000 to 150,000 g at 2 to 5° C. for 1 to 3 h, remove the supernatant and resuspend the precipitate.

[0064] 16. The method according to any one of items 11 to 15, wherein in the purification step, the centrifugation is performed at 3000 to 5000 rpm for 5 to 10 min.

[0065] Effects of the Invention

[0066] The high-purity exosome enrichment method first uses ultracentrifugation to crudely extract exosomes, and then uses the microsphere capture method to combine specific microspheres with exosome marker antibodies and then mix and incubate with the crudely extracted exosomes to obtain high-purity exosomes, with membrane particles accounting for up to 88.3%. The method of this application is time-consuming, has high purity of exosomes, low cost, and can cover a variety of samples, which helps to strengthen the industrialization capabilities of related products and further promote the application of exosomes in clinical testing and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is a graph showing the percentage of membrane-bearing particles in the high-purity exosomes obtained in Example 8.

[0068] Figure 2 This is a graph showing the percentage of membrane-bearing particles in the high-purity exosomes obtained in Comparative Example 1.

[0069] Figure 3 This is a graph showing the percentage of membrane-bearing particles in the high-purity exosomes obtained in Comparative Example 2.

[0070] Figure 4 This is the BCA standard curve.

[0071] Figure 5 This is the NTA test result of exosomes in clinical samples.

[0072] Figure 6 This is a graph showing the percentage of CD9-positive particles in exosomes of clinical samples.

[0073] Figure 7 This is a graph showing the percentage of CD81-positive particles in exosomes of clinical samples.

[0074] Figure 8 These are the WB results of exosomes from clinical samples.

[0075] Fig. 9 TEM image of exosomes from clinical samples.

[0076] Fig.10 The results of blood exosome pTau217 marker detection in Alzheimer's disease patients and healthy controls.

[0077] Fig.11 The results of blood exosome GPC1 marker detection in pancreatic cancer patients and healthy controls.

[0078] Fig.12 The results of urine exosome ITGA3 marker detection in prostate cancer patients and healthy controls. DETAILED DESCRIPTION

[0079] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0080] It should be noted that the words "including" or "comprising" mentioned throughout the specification and claims are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of illustrating the general principles of the present application and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the definition of the attached claims.

[0081] On the one hand, the present application provides a capture microsphere, which is a complex obtained by coupling the microsphere with an exosome marker antibody, and the average particle size of the microsphere is greater than 1 μm.

[0082] The average particle size of the microspheres of the present application is obtained by first measuring the diameter of each microsphere separately, and then averaging the diameters of all the microspheres. For example, Nanosight (model NS300) can be used for measurement. The Nanosight NS300 nanoparticle tracking analyzer uses nanoparticle tracking analysis (NTA) technology, which uses a laser light source to illuminate a particle suspension and forms a completely black background with the help of a metal coating. The Brownian motion of particles with scattered light can be clearly observed, and the diameter of each particle with scattered light can be measured, thereby averaging the diameters of all particles with scattered light, that is, obtaining the average particle size of the microspheres.

[0083] The average particle size of the microspheres of the present application can be, for example, 1 μm, 1.2 μm, 1.5 μm, 1.8 μm, 2 μm, 2.3 μm, 2.5 μm, 2.7 μm, 3 μm, 3.2 μm, 3.5 μm, 3.8 μm, 4 μm, 4.2 μm, 4.5 μm, 4.7 μm, 5 μm, 5.2 μm, 5.5 μm, 5.7 μm, 6 μm, 6.3 μm, 6.5 μm, 6.8 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, etc. In some embodiments, the average particle size of the microspheres is 3.5 μm or more.

[0084] In some embodiments, the microspheres are magnetic microspheres, latex microspheres or silica microspheres, preferably magnetic microspheres. For example, the magnetic microspheres may be polystyrene magnetic microspheres, silica magnetic microspheres, ferroferric oxide magnetic microspheres, urea-formaldehyde resin magnetic microspheres, γ-Fe2O3 magnetic microspheres, etc., the latex microspheres may be non-magnetic polystyrene latex microspheres, etc., and the silica microspheres refer to non-magnetic silica microspheres.

[0085] In some embodiments, the microspheres are carboxyl-modified magnetic microspheres.

[0086] In some embodiments, the exosome marker antibody is an anti-CD63 antibody, an anti-CD9 antibody, or an anti-CD81 antibody, preferably an anti-CD63 antibody.

[0087] In some embodiments, the microspheres are magnetic microspheres, and the exosome marker antibody is an anti-CD63 antibody. In some embodiments, the microspheres are carboxyl-modified magnetic microspheres, and the exosome marker antibody is an anti-CD63 antibody.

[0088] CD63 in this application is a member of the tetraspanin superfamily and is considered to be a key factor in regulating the production of extracellular vesicles and the sorting of endosomal cargo. It can interact with a variety of cell surface proteins and intracellular signal transduction molecules to mediate a series of biological processes such as movement, adhesion and signal transduction. It can be detected in platelets (at the protein level). CD63 protein is located on the cell membrane, endosomes, and lysosomes.

[0089] CD9 in this application is a member of the four-type transmembrane protein superfamily. It is also an integral membrane protein associated with integrins, which can regulate different processes, such as sperm-egg fusion, platelet activation and aggregation, and cell adhesion. In macrophages, CD9 function is associated with CD81 and β-1 and β-2 integrins, and prevents macrophages from fusing into multinuclear giant cells, which specialize in engulfing complement-conditioned large particles. CD9 participates in platelet activation and aggregation, and it also participates in cell adhesion, cell movement and tumor metastasis. CD9 is expressed in a variety of hematopoietic and non-hematopoietic cells, such as stromal cells, megakaryocytes, platelets, B and T lymphocytes, dendritic cells, endothelial cells, mast cells, eosinophils and basophils. CD9 protein is located extracellularly or in exosomes, cell membranes.

[0090] The CD81 in this application is a member of the tetratransmembrane protein superfamily, a tetratransmembrane protein with a molecular weight of 25kD. It is widely expressed and expressed in 90% of Burkitt lymphoma cell lines. It crosses the cell membrane four times to form two large and small ring structures outside the cell. The components of CD81 in the intracellular and transcellular membrane regions are highly conserved, while its large extracellular loop region (EC2) is variable, and the amino acid sequence is different in different species, but the extracellular large loop region of CD81 is highly conserved in humans and chimpanzees, which may be related to the species-specific characteristics of HCV infection. CD81 is widely distributed in a variety of cells, such as T lymphocytes, B cells, macrophages, dendritic cells, NK cells, and eosinophils, which can express CD81 molecules.

[0091] In some embodiments, the mass ratio of the exosome marker antibody to the microspheres is 0.005 to 0.2:1, for example, 0.005:1, 0.01:1, 0.02:1, 00.3:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, etc.

[0092] In some embodiments, the average particle size of the microspheres is greater than 3.5 μm, and the microspheres are magnetic microspheres.

[0093] In some embodiments, the average particle size of the microspheres is greater than 3.5 μm, the microspheres are magnetic microspheres, and the exosome marker antibody is an anti-CD63 antibody.

[0094] On the other hand, the present application also provides a method for preparing capture microspheres, which comprises the following steps: taking microspheres with an average particle size of 1 μm or more, adding exosome marker antibodies, mixing and incubating, and obtaining capture microspheres.

[0095] The microspheres in the method for preparing capture microspheres of the present application are any of the microspheres in the aforementioned first aspect.

[0096] In some embodiments, the exosome marker antibody is an anti-CD63 antibody, an anti-CD9 antibody, or an anti-CD81 antibody, preferably an anti-CD63 antibody.

[0097] In some embodiments, the microspheres are magnetic microspheres, and the exosome marker antibody is an anti-CD63 antibody. In some embodiments, the microspheres are carboxyl-modified magnetic microspheres, and the exosome marker antibody is an anti-CD63 antibody.

[0098] In some embodiments, the mass ratio of the exosome marker antibody to the microspheres is 0.005 to 0.2:1, for example, 0.005:1, 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, etc.

[0099] In some embodiments, the average particle size of the microspheres is greater than 3.5 μm, and the microspheres are magnetic microspheres.

[0100] In some embodiments, the average particle size of the microspheres is greater than 3.5 μm, the microspheres are magnetic microspheres, and the exosome marker antibody is an anti-CD63 antibody.

[0101] In some embodiments, the preparation method of the present application comprises the following steps:

[0102] The magnetic microspheres are magnetically adsorbed and the supernatant is removed;

[0103] The magnetic microspheres were then equilibrated and the supernatant was removed again;

[0104] Then add coupling agent for activation, magnetic adsorption, and remove the supernatant;

[0105] Wash the activated magnetic microspheres and resuspend them;

[0106] adding the exosome marker antibody and incubating;

[0107] Add blocking agent, incubate, and remove supernatant after magnetic adsorption;

[0108] Add the second buffer, magnetically adsorb, and remove the supernatant;

[0109] A second buffer is added and the mixture is resuspended to obtain the capture microspheres.

[0110] In some embodiments, the preparation method of the present application comprises the following steps:

[0111] Add magnetic microspheres into centrifuge tubes, magnetically adsorb, and remove supernatant;

[0112] Then, the first buffer or distilled water was added to equilibrate the magnetic microspheres, and the supernatant was removed again;

[0113] Add the first buffer or distilled water again, shake and mix, then add the coupling agent for activation, magnetic adsorption, and remove the supernatant;

[0114] Wash the activated magnetic microspheres with the first buffer and resuspend them with the first buffer or distilled water;

[0115] adding the exosome marker antibody and incubating;

[0116] Add blocking agent, incubate, and remove supernatant after magnetic adsorption;

[0117] Add the second buffer, shake and mix, magnetically adsorb, remove the supernatant, and repeat 3 to 8 times, for example, 3, 4, 5, 6, 7, 8 times;

[0118] A second buffer solution is added, the mixture is resuspended, and shaken to mix well to obtain the capture microspheres.

[0119] In some embodiments, the first buffer is MES buffer; and EDC and NHS are used as coupling agents.

[0120] In some embodiments, the activation temperature is 35-37°C, for example, it can be 35°C, 35.2°C, 35.5°C, 35.6°C, 35.8°C, 36°C, 36.2°C, 36.4°C, 36.5°C, 36.6°C, 36.8°C, 37°C, etc., and the activation time is 10-30min, for example, it can be 10min, 12min, 14min, 15min, 16min, 18min, 20min, 22min, 24min, 25min, 26min, 28min, 30min, etc.

[0121] In some embodiments, the activated magnetic microspheres are washed with the first buffer at least three times, for example, 3 times, 4 times, 5 times, 6 times, etc.

[0122] In some embodiments, after adding the exosome marker antibody, incubation is carried out at 30-37°C for 1-2 hours, for example, incubation can be carried out at 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, etc. for 1 hour, 1.1 hour, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2 hours etc.; after adding the blocking agent, incubate at 30-37°C for 1-2h, for example, incubate at 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, etc. for 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, etc.; the second buffer is PBS buffer.

[0123] In some embodiments, the first buffer is MES buffer; EDC and NHS are used as coupling agents; the activation temperature is 35-37°C, and the activation time is 10-30 min; the activated magnetic microspheres are washed at least 3 times with the first buffer; after adding the exosome marker antibody, incubate at 30-37°C for 1-2 h; after adding the blocking agent, incubate at 30-37°C for 1-2 h; the second buffer is PBS buffer.

[0124] In another aspect, the present application also provides a method for enriching high-purity exosomes, comprising the following steps:

[0125] Crude extraction: Take a body fluid sample, remove impurities by ultracentrifugation, and obtain crudely extracted exosomes;

[0126] Purification: adding any of the aforementioned capture microspheres or capture microspheres prepared by any of the aforementioned preparation methods to the crudely extracted exosomes, mixing and incubating, centrifuging, removing the supernatant, and resuspending to obtain high-purity exosomes.

[0127] In some embodiments, the body fluid sample is a plasma sample, a serum sample, a cerebrospinal fluid sample, or a urine sample.

[0128] In some embodiments, in the purification step, the volume ratio of the crudely extracted exosomes to the capture microspheres is 1:0.005-0.1, for example, 1:0.005, 1:0.006, 1:0.007, 1:0.008, 1:0.009, 1:0.01, 1:0.015, 1:0.02, 1:0.025, 1:0.03, 1:0.035, 1:0.04, 1:0.045, 1:0.05, 1:0.055, 1:0.06, 1:0.065, 1:0.07, 1:0.075, 1:0.08, 1:0.085, 1:0.09, 1:0.095, 1:0.1, etc., preferably 1:0.005-0.025.

[0129] In some embodiments, in the purification step, the incubation conditions are shaking incubation at 2-8°C for 12-16 hours. For example, the incubation temperature can be 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C, etc., and the incubation time can be 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, 16h, etc.

[0130] In some embodiments, in the purification step, the incubation conditions are 30-37°C for 1-2 h. For example, the incubation temperature may be 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, etc., and the incubation time may be 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, etc.

[0131] In some embodiments, in the polishing step, the incubation condition is 37° C. for 1 h.

[0132] In some embodiments, in the crude extraction step, the ultracentrifugation comprises:

[0133] Centrifuge at 5000-15000 g for 20-50 min at 2-5 °C and aspirate the supernatant;

[0134] Then filter using a 0.22 μm filter membrane;

[0135] Centrifuge again at 80,000 to 150,000 g for 1 to 3 h at 2 to 5 °C, remove the supernatant and resuspend the precipitate;

[0136] Then centrifuge again at 80,000 to 150,000 g at 2 to 5° C. for 1 to 3 h, remove the supernatant and resuspend the precipitate.

[0137] In some embodiments, in the crude extraction step, the ultracentrifugation comprises:

[0138] Centrifuge at 10,000 g for 30 min at 2-5 °C and aspirate the supernatant;

[0139] Then filter using a 0.22 μm filter membrane;

[0140] Centrifuge at 100,000 g for 1 h at 4 °C, remove the supernatant and resuspend the pellet;

[0141] The cells were then centrifuged again at 100,000 g for 1 h at 4 °C, the supernatant removed and the pellet resuspended.

[0142] In some embodiments, in the purification step, centrifugation is performed at 3000-5000 rpm for 5-10 min. For example, the centrifugation rate is 3000 rpm, 3200 rpm, 3500 rpm, 3800 rpm, 4000 rpm, 4300 rpm, 4500 rpm, 4700 rpm, 5000 rpm, etc., and the centrifugation time can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0143] Example

[0144] Example 1

[0145] (1) Crude extraction of exosomes

[0146] Use 4 mL of fresh plasma sample and centrifuge at 10,000 g for 30 min at 4°C. Carefully aspirate the supernatant. This step is to remove larger impurities such as cell debris in the sample.

[0147] It was then filtered using a 0.22 μm filter to remove impurities such as high-density lipoprotein and some cell debris;

[0148] Centrifuge at 100,000 g for 1 h at 4 °C. Carefully remove the supernatant and resuspend the pellet thoroughly with 4 mL PBS (pH 7.4). Then centrifuge again at 100,000 g for 1 h at 4 °C; carefully remove the supernatant and resuspend the pellet thoroughly with 0.4 mL PBS (pH 7.4). This step is intended to remove small-sized impurities or non-exosome vesicles with the help of ultracentrifugation technology.

[0149] (2) Preparation of magnetic capture microspheres

[0150] Place 10 mg of 1 μm magnetic beads (carrying carboxyl modification) that have been thoroughly shaken and mixed in a 1.5 mL centrifuge tube, and remove the supernatant after magnetic adsorption;

[0151] The magnetic beads were then fully equilibrated with 50 mM MES (pH 6.0), and the supernatant was removed again;

[0152] Add 800 μL 50 mM MES (pH 6.0) and shake to mix, then add 100 μL EDC (50 mM) solution and 100 μL NHS (50 mM) solution, shake to mix, invert and mix at 37°C for 30 min, and after the reaction is completed, magnetic adsorption is performed and the supernatant is removed with a pipette;

[0153] The activated magnetic beads were washed three times with 1 mL of 50 mM MES (pH 6.0) and resuspended with 900 μL of 50 mM MES (pH 6.0);

[0154] Add 100 μg of exosome marker antibody Anti-CD63 and mix at 37°C for 2 h;

[0155] After the incubation, add 300 μL of 0.4% BSA (diluted with PBS) and mix at 37°C for 1 h;

[0156] After the incubation, the supernatant was removed by pipetting after magnetic adsorption.

[0157] Add 1 mL of 20 mM PBS (pH 7.4) solution, shake and mix magnetically, remove the supernatant with a pipette, and repeat 5 times;

[0158] 1 mL of 20 mM PBS (pH 7.4) was added for resuspending, and the mixture was shaken to obtain carboxyl magnetic capture microspheres.

[0159] (3) Purified exosomes

[0160] Add 100 μL of crudely extracted exosome samples to 1.5 mL centrifuge tubes. If the sample is less than 100 μL, add 20 mM PBS (pH 7.4) to 100 μL.

[0161] Add 7 μL of carboxyl magnetic capture microspheres, mix well on a vortex mixer, and then place on a constant temperature mixer for shaking incubation. The incubation condition is 4°C overnight incubation for 18 h.

[0162] Centrifuge at 3000 rpm for 5 min and remove the supernatant with a pipette;

[0163] Add 100 μL of 20 mM PBS (pH 7.4) and resuspend.

[0164] Example 2

[0165] The difference between this embodiment and embodiment 1 is that the average particle size of the magnetic beads is 2.2 μm.

[0166] Example 3

[0167] The difference between this embodiment and embodiment 1 is that the average particle size of the magnetic beads is 3 μm.

[0168] Example 4

[0169] The difference between this embodiment and embodiment 1 is that the average particle size of the magnetic beads is 5 μm.

[0170] Example 5

[0171] The difference between this embodiment and embodiment 4 is that the exosome marker antibody is Anti-CD81.

[0172] Example 6

[0173] The difference between this embodiment and embodiment 4 is that the amount of capture microspheres used is 3 μL.

[0174] Example 7

[0175] The difference between this embodiment and embodiment 4 is that the amount of capture microspheres used is 1 μL.

[0176] Example 8

[0177] The difference between this embodiment and embodiment 1 is that the incubation condition in the step of purifying exosomes is incubation at 37° C. for 1 h.

[0178] Comparative Example 1

[0179] The difference between this comparative example and Example 1 is that the step of preparing magnetic capture microspheres and the step of purifying exosomes are not included.

[0180] Comparative Example 2

[0181] The difference between this comparative example and Example 8 is that the crude exosome extraction step is not included, that is, the plasma sample is directly used to incubate with the carboxyl magnetic capture microspheres.

[0182] The raw materials and experimental parameters of Examples 1-8 and Comparative Examples 1-2 are summarized in Table 1 below.

[0183] Table 1

[0184]

[0185] Example 9

[0186] (1) Crude extraction of exosomes

[0187] Use 4 mL of fresh plasma sample and centrifuge at 10,000 g for 30 min at 4°C. Carefully aspirate the supernatant. This step is to remove larger impurities such as cell debris in the sample.

[0188] It was then filtered using a 0.22 μm filter to remove impurities such as high-density lipoprotein and some cell debris;

[0189] Centrifuge at 100,000 g for 1 h at 4 °C. Carefully remove the supernatant and resuspend the pellet thoroughly with 4 mL PBS (pH 7.4). Then centrifuge again at 100,000 g for 1 h at 4 °C; carefully remove the supernatant and resuspend the pellet thoroughly with 0.4 mL PBS (pH 7.4). This step is intended to remove small-sized impurities or non-exosome vesicles with the help of ultracentrifugation technology.

[0190] (2) Preparation of latex capture microspheres

[0191] Take 10 mg of latex microspheres with an average particle size of 1 μm (carrying carboxyl modification) in a 1.5 mL centrifuge tube, add 900 μL of 50 mM MES (pH 6.0) and shake to mix, then add 100 μL of EDC (50 mM) solution and 100 μL of NHS (50 mM) solution, shake to mix, and invert and mix at 37 °C for 30 min;

[0192] After the reaction, 100 μg of exosome marker antibody Anti-CD63 was added and mixed at 37°C for 2 h;

[0193] After the incubation, add 100 μL of 1% BSA (diluted with PBS) and mix at 37°C for 1 h;

[0194] After incubation, place it in a centrifuge, centrifuge at 12000g for 20min, and discard the supernatant;

[0195] 1 mL of 20 mM PBS (pH 7.4) was added to resuspend, and the mixture was shaken to obtain carboxyl latex capture microspheres.

[0196] (3) Purified exosomes

[0197] Add 100 μL of crudely extracted exosome samples to 1.5 mL centrifuge tubes. If the sample is less than 100 μL, add 20 mM PBS (pH 7.4) to 100 μL.

[0198] Add 7 μL of carboxyl latex capture microspheres, mix well on a vortex mixer, and then place on a constant temperature mixer for shaking incubation. The incubation condition is 4°C overnight incubation for 18 h.

[0199] Centrifuge at 3000 rpm for 5 min and remove the supernatant with a pipette;

[0200] Add 100 μL of 20 mM PBS (pH 7.4) and resuspend.

[0201] Example 10

[0202] The difference between this embodiment and embodiment 9 is that the average particle size of the latex microspheres is 2 μm.

[0203] Embodiment 11

[0204] The difference between this embodiment and embodiment 9 is that the average particle size of the latex microspheres is 3 μm.

[0205] Example 12

[0206] The difference between this embodiment and embodiment 9 is that the average particle size of the latex microspheres is 5 μm.

[0207] Example 13

[0208] The difference between this embodiment and embodiment 12 is that the exosome marker antibody is Anti-CD81.

[0209] Embodiment 14

[0210] The difference between this embodiment and embodiment 12 is that the amount of capture microspheres used is 3 μL.

[0211] Embodiment 15

[0212] The difference between this embodiment and embodiment 12 is that the amount of capture microspheres used is 1 μL.

[0213] Example 16

[0214] The difference between this embodiment and embodiment 15 is that the incubation condition in the step of purifying exosomes is incubation at 37° C. for 1 h.

[0215] Comparative Example 3

[0216] The difference between this comparative example and Example 9 is that the step of preparing latex capture microspheres and the step of purifying exosomes are not included.

[0217] Comparative Example 4

[0218] The difference between this comparative example and Example 16 is that the crude exosome extraction step is not included, that is, the plasma sample is directly used to incubate with the carboxyl latex capture microspheres.

[0219] The raw materials and experimental parameters of Examples 9-16 and Comparative Examples 3-4 are summarized in Table 2 below.

[0220] Table 2

[0221]

[0222]

[0223] Test Example 1

[0224] The exosomes purified in each example and comparative example were subjected to exosome characterization and marker detection.

[0225] 1.1 Detection of fluorescence signal value of markers in exosomes

[0226] The analytical instrument used was Beckman DxFLEX, and the instrument was operated according to the instruction manual.

[0227] The testing process is as follows:

[0228] The protein concentration of the crudely extracted samples was determined according to the instructions of the commercial BCA protein concentration determination kit.

[0229] According to the measured protein concentration, add 0μg, 6.25μg, 12.5μg, 25μg, 50μg, and 100μg samples to a 1.5mL centrifuge tube. If the sample is less than 100μL, add 20mM PBS (pH7.4) to 100μL. For example, according to the instructions of the commercial BCA protein concentration determination kit, if the crude sample protein concentration is 1μg / μl, add 100μg of sample to the centrifuge tube, and the corresponding volume is 100μl; if the crude sample protein concentration is 2μg / μl, add 100μg of sample to the centrifuge tube, and the corresponding volume is 50μl, then add 50μl of 20mM PBS to 100μl. In addition, if the marker needs to be permeabilized, a commercial permeabilization agent should be used to permeabilize the membrane according to the instructions.

[0230] Add 1 μL of capture magnetic beads, mix well on a vortex mixer, and incubate at 37°C for 1 h.

[0231] Then centrifuge at 3000 g for 5 min and remove the supernatant with a pipette.

[0232] Add 100 μL 20 mM PBS (pH 7.4) to resuspend, add 0.1 μg CD9 / GPC1 / pTau217 / α-syn / ITGA3 and other fluorescent antibodies, mix well on a vortex mixer, and then incubate at 37°C in the dark for 1 hour.

[0233] Centrifuge at 3000 g for 5 min and remove the supernatant with a pipette.

[0234] 100 μL of 20 mM PBS (pH 7.4) was added for resuspending, mixed on a vortex mixer, and tested on the machine. Each tube of sample was mixed again before being tested on the machine. The test results of the exosomes prepared in Examples 1 to 8 are shown in Table 2, and the test results of the exosomes prepared in Examples 9 to 16 are shown in Table 4.

[0235] Table 3

[0236]

[0237] Table 4

[0238]

[0239] 1.2 BCA detection

[0240] Follow the instructions of the commercial BCA protein concentration assay kit. According to the BCA kit (BCA protein concentration assay kit, Biotech), the diluted standard (0.5 mg / ml) was added to the protein standard wells of the 96-well plate at 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 12 μL, 16 μL, and 20 μL in sequence. For wells with less than 20 μL, add PBS to 20 μL. Add 200 μL of BCA working solution to each well, place at 37 ° C, and incubate for 15 to 30 minutes. Use an enzyme reader to measure the absorbance at A562nm, and use Excel to organize the table based on the obtained data to obtain the BCA standard curve. Among them, the data at 0μL, 2μL, 4μL, 6μL, 8μL, 12μL, 16μL, and 20μL correspond to 0mg / ml, 0.05mg / ml, 0.1mg / ml, 0.15mg / ml, 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, and 0.5mg / ml respectively.

[0241] 1.3 Nanoparticle Tracking Analysis (NTA) Detection

[0242] The samples were processed as follows:

[0243] Take 10 μL of the extracted exosome sample and dilute it to 100 μL with 20 mM PBS (pH 7.4).

[0244] Exosomes were dissociated using 60 μL of a 1:1 mixture of 0.1% BSA PBS and 4% paraformaldehyde / 5% glutaraldehyde, and the supernatant was collected by centrifugation at 3000 g for 5 min.

[0245] The instrument used was Nanosight (model NS300), and the specific operation of the instrument was performed according to the instruction manual.

[0246] Click Start Camera according to the prompts, adjust the focal length, and select the appropriate Screen Gain and Camera Level. Select the required measurement method in SOP and adjust the various measurement parameters. Click Create and Run and follow the on-screen prompts to complete the test.

[0247] 1.4 Detection of the proportion of exosome membrane surface marker-positive particles / membrane particles

[0248] Dissociate the extracted exosome samples according to the sample processing method in 1.3.

[0249] NanoFCM (model U30) was used for detection and the instrument operation was performed according to the instruction manual.

[0250] a) Detection of the proportion of exosome membrane surface marker-positive particles

[0251] Take 100 μL of the dissociated sample, add 0.1 μg / Test of fluorescent antibodies such as CD9 / CD81, and incubate at 4°C overnight.

[0252] Ultracentrifuge at 100000g for 1h at 4°C, remove the supernatant, and resuspend in 100μL 20mM PBS (pH7.4). Unstained exosomes were used as blank control.

[0253] b) Detection of the proportion of particles with membrane

[0254] Follow the instructions of the commercial PKH67 staining kit.

[0255] 100 μL of the dissociated sample was diluted with 100 μL Diluent C, and 1 μL of PKH67 dye was diluted with 100 μL Diluent C.

[0256] 25 μL of diluted PKH67 dye was added to the diluted exosome sample and incubated at room temperature for 10 min.

[0257] Ultracentrifuge at 100000g for 1h at 4°C, remove the supernatant, and resuspend in 100 μL 20 mM PBS (pH 7.4). Unstained exosomes were used as blank controls. The test results of the exosomes prepared in Example 8, Comparative Example 1 and Comparative Example 2 are shown in Tables 5 and Figures 1 to 3 shown.

[0258] Table 5

[0259]

[0260] 1.5WB detection

[0261] The commercial WB detection kit was operated according to the instructions. The target proteins detected were CD63, CD9, TSG101 and Calnexin, among which Calnexin was used as a negative marker.

[0262] 1.6TEM detection

[0263] (1) Dissociate the extracted exosome samples according to the sample processing method in 1.3;

[0264] (2) resuspending the exosomes obtained in step (1) in 30 μL of 20 mM PBS (pH 7.4);

[0265] Pipette 10 μL of the sample and drop it on the copper grid to precipitate for 1 min, and remove the floating liquid with filter paper;

[0266] 10 μL of uranyl acetate (phosphotungstic acid) was added dropwise onto the copper grid and allowed to precipitate for 1 min, and the floating liquid was removed by filter paper.

[0267] Dry at room temperature for several minutes;

[0268] Thermo Scientific transmission electron microscope (model: Talos L120C) was used for imaging at 120 kV to obtain transmission electron microscope imaging results.

[0269] 1.7 Conclusion

[0270] Equal amounts of CD63 magnetic beads and CD63 latex microspheres were used to capture exosomes, and then detected with CD9 fluorescent antibody. The results showed that the fluorescence intensity detected after the exosomes were captured by magnetic beads was greater than that detected after the exosomes were captured by latex microspheres, and the low end had better discrimination.

[0271] Using the amount of exosomes added as the concentration gradient, carboxyl magnetic microspheres of different particle sizes were used as capture beads to capture exosomes, and there was good linearity, indicating that the number of exosomes captured by the magnetic beads increased with the increase in sample concentration. At the same time, it was found that carboxyl magnetic microspheres with larger particle sizes had better linearity, and magnetic microspheres of 3.5μm and above had better effects.

[0272] Equal amounts of CD63 magnetic beads and CD81 magnetic beads were used to capture exosomes, and then detected with CD9 fluorescent antibody. The results showed that the fluorescence intensity increased with the increase of sample volume, but the fluorescence intensity captured by CD81 magnetic beads was generally lower than that captured by CD63 magnetic beads, indicating that the exosome capture efficiency of CD81 magnetic beads was lower than that of CD63 magnetic beads.

[0273] After capturing exosomes with different amounts of CD63 magnetic beads and detecting them with CD9 fluorescent antibodies, it was found that the fluorescence signal value was the highest when the amount of magnetic beads was 1 μL. The current results suggest that the amount of magnetic beads above 3 μL (including 3 μL) exceeds the amount of magnetic beads required by the detection system, resulting in too few exosomes captured on a single magnetic bead, which in turn causes the average fluorescence signal of each particle (magnetic bead) detected to decrease. Based on this, this application ultimately uses 1 μL of magnetic beads as the capture magnetic bead sample amount. The current capture antibody dosage is 0.1 μg / T, which is significantly reduced compared to 10 μg / T and 0.5 μg / T in the embodiments of CN110133272A and CN111505264A, and therefore also plays a role in reducing costs.

[0274] The exosomes were captured with CD63 magnetic beads and incubated overnight at 4°C with shaking or at 37°C for 1 hour, and then detected with CD9 fluorescent antibody. The results showed that the fluorescence signal after incubation at 37°C for 1 hour was stronger than that after incubation at 4°C with shaking overnight, indicating that the ability of magnetic beads to capture exosomes was stronger under the condition of incubation at 37°C for 1 hour. Based on this, incubation at 37°C for 1 hour was finally selected as the condition for magnetic beads to capture exosomes. So far, the total duration of the improved magnetic bead capture method in this application is 3.5 hours, which is much shorter than the duration of not less than 12 hours in CN107893051A, CN111505264A and CN113652388A. Therefore, such adjustments in this application also serve to shorten the detection time.

[0275] The comparative example only used ultracentrifugation or only captured magnetic beads to enrich exosomes in plasma, and observed the proportion of membrane particles in exosomes obtained by different methods. The results showed that the proportion of membrane particles in the present application method was as high as 88.3%, which was much higher than the data in the comparative example. This shows that the exosomes obtained by the present application method are of higher purity, which is conducive to subsequent research.

[0276] Experimental Example 2: Further characterization and detection application of exosomes based on current methodology

[0277] 2.1 Experimental design

[0278] In this test example, the samples in Table 6 below were used to purify exosomes with reference to Example 8.

[0279] Table 6

[0280]

[0281] 2.2 Experimental Results

[0282] BCA standard curve Figure 4 As shown, according to Figure 4 The exosome concentration data of 10 random clinical plasma samples obtained using the BCA standard curve are shown in Table 7.

[0283] Table 7

[0284]

[0285] Example of NTA test data of clinical exosome sample 3 Figure 5 shown.

[0286] The percentage of CD9 positive particles in clinical exosome sample 3 is Figure 6 As shown, the proportion of CD81 positive particles in clinical sample exosome 3 is Figure 7 shown.

[0287] The WB results of clinical exosome sample 3 are as follows Figure 8 shown.

[0288] TEM image of clinical exosome sample 3 Fig. 9 shown.

[0289] Results of blood exosome pTau217 marker detection in patients with Alzheimer's disease and healthy controls Fig.10 shown.

[0290] Results of blood exosome GPC1 marker detection in pancreatic cancer patients and healthy controls Fig.11 shown.

[0291] Results of urine exosome ITGA3 marker detection in prostate cancer patients and healthy controls Fig.12 shown.

[0292] 2.3 Conclusion

[0293] The method of this application has a high concentration of enriched exosome particles, and plasma exosomes are 10 8 -10 9 The number of particles / mL was in the order of magnitude and the particle size was normally distributed evenly. The proportion of membrane particles exceeded 85%. The high proportion of positive markers on the surface of the exosome membrane also indicated that the purity of the exosome sample captured by the magnetic beads was high. At the same time, both the WB results and the TEM results showed that the method of this application had enriched the exosome particles. The above results collectively showed that the method of this application could enrich high-purity exosomes from plasma.

[0294] In terms of the detection effect of exosome-related disease markers, the magnetic bead capture and detection method of this application uses ptau217 as a single marker to distinguish Alzheimer's patients from normal people, and its AUC reached 0.910, which is better than the 0.877 reported in application CN 113637736A. The data of Xiao et al. in 2020 showed that the diagnostic AUC of GPC1 as a single marker for pancreatic cancer was 0.885, while the AUC of the method provided in this application can reach 0.936. The method provided in this application can achieve an AUC of 0.9 for the detection of ITGA3 in urine exosomes, P < 0.001, which is better than the data of Bijnsdorp et al.

[0295] In summary, the above data also show that the method of the present application is lower cost and more efficient than the currently disclosed methods, and has a high degree of clinical application. It can also cover a variety of body fluid samples and has good application prospects.

[0296] Although the embodiments of the present application are described above in conjunction with the accompanying drawings, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are merely illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present application, all of which belong to the protection of the present application.

Claims

1. A capture microsphere, wherein: It is a complex obtained by coupling microspheres with exosome marker antibodies, and the average particle size of the microspheres is greater than 1 μm.

2. The capture microsphere according to claim 1, wherein: The average particle size of the microspheres is greater than 3.5 μm.

3. The capture microsphere according to claim 1 or 2, wherein: The microspheres are magnetic microspheres, latex microspheres or silica microspheres, preferably magnetic microspheres.

4. The capture microsphere according to any one of claims 1 to 3, wherein The exosome marker antibody is an anti-CD63 antibody, an anti-CD9 antibody or an anti-CD81 antibody, preferably an anti-CD63 antibody; Preferably, the mass ratio of the exosome marker antibody to the microspheres is 0.005-0.2:

1.

5. A method for preparing capture microspheres, wherein: The following steps are involved: Microspheres with an average particle size of more than 1 μm were taken, and exosome marker antibodies were added and mixed and incubated to obtain capture microspheres.

6. The preparation method according to claim 5, wherein: The average particle size of the microspheres is greater than 3.5 μm.

7. The preparation method according to claim 5 or 6, wherein: The microspheres are magnetic microspheres, latex microspheres or silica microspheres, preferably magnetic microspheres.

8. The preparation method according to any one of claims 5 to 7, wherein The exosome marker antibody is an anti-CD63 antibody, an anti-CD9 antibody or an anti-CD81 antibody, preferably an anti-CD63 antibody; Preferably, the mass ratio of the exosome marker antibody to the microspheres is 0.005-0.2:

1.

9. The preparation method according to any one of claims 5 to 8, wherein The preparation method comprises the following steps: The magnetic microspheres are magnetically adsorbed and the supernatant is removed; The magnetic microspheres were then equilibrated and the supernatant was removed again; Then add coupling agent for activation, magnetic adsorption, and remove the supernatant; Wash the activated magnetic microspheres and resuspend them; adding the exosome marker antibody and incubating; Add blocking agent, incubate, and remove supernatant after magnetic adsorption; Add the second buffer, magnetically adsorb, and remove the supernatant; Add a second buffer and resuspend to obtain the capture microspheres; Preferably, the preparation method comprises the following steps: Add magnetic microspheres into centrifuge tubes, magnetically adsorb, and remove supernatant; Then, the first buffer or distilled water was added to equilibrate the magnetic microspheres, and the supernatant was removed again; Add the first buffer or distilled water again, shake and mix, then add the coupling agent for activation, magnetic adsorption, and remove the supernatant; Wash the activated magnetic microspheres with the first buffer and resuspend them with the first buffer or distilled water; adding the exosome marker antibody and incubating; Add blocking agent, incubate, and remove supernatant after magnetic adsorption; Add the second buffer, shake and mix, magnetically adsorb, remove the supernatant, and repeat 3 to 8 times; Add a second buffer, resuspend, and shake to mix, to obtain the capture microspheres; Preferably, the first buffer is MES buffer; Preferably, EDC and NHS are used as coupling agents; Preferably, the activation temperature is 35-37°C and the activation time is 10-30 min; Preferably, the activated magnetic microspheres are washed at least 3 times with the first buffer; Preferably, after adding the exosome marker antibody, incubate at 30-37° C. for 1-2 h; Preferably, after adding the blocking agent, incubate at 30-37°C for 1-2h; Preferably, the second buffer is PBS buffer.

10. A method for enriching high-purity exosomes, wherein: The following steps are involved: Crude extraction: Take a body fluid sample, remove impurities by ultracentrifugation, and obtain crudely extracted exosomes; Purification: Add the capture microspheres according to any one of claims 1 to 4 or the capture microspheres prepared by the preparation method according to any one of claims 5 to 9 to the crudely extracted exosomes, mix and incubate, centrifuge, remove the supernatant, and resuspend to obtain high-purity exosomes.

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