Separation method of plasma or serum exosome

Abstract

The invention discloses a plasma or serum exosome separation method which comprises the following steps: firstly, coupling an antibody for resisting four transmembrane protein CD81 on the surface of an exosome by using a magnetic bead to form an immunomagnetic bead; pretreating a serum / plasma sample, enriching exosomes, co-incubating with the immunomagnetic beads, washing the magnetic beads after incubation capture is completed, and washing off uncombined impurities; after being captured, the exosome can be directly cracked, the magnetic beads are removed for downstream experiments, or the magnetic beads are eluted to obtain the complete exosome. The exosome affinity antibody is matched with the magnetic beads, the plasma / serum sample exosome is separated, the unique affinity separation technology not only can remove high-abundance protein in the serum / plasma sample, but also can eliminate other vesicles with similar membrane structures with the exosome, so that the purity of the separated exosome is far higher than that of the exosome obtained by other separation methods.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular vesicles, and particularly relates to a method for separating exosomes from plasma or serum. Background Art

[0002] Exosomes are small vesicles with a lipid bilayer structure, 30-150 nm in diameter, formed by cellular invagination. They are present in nearly all biological fluids and serve as crucial mediators of intercellular communication. Exosomes contain a diverse composition, including nucleic acids, proteins, lipids, amino acids, and cellular metabolites. Exosomes reflect their cell of origin and can transfer cell-specific components from the source cell to the recipient cell. In multicellular organisms, research on the production and functional mechanisms of exosomes spans the spectrum from physiological tissue regulation to pathogenic injury and organ remodeling. Exosomes have sparked a surge in research as potential diagnostic and therapeutic tools for a variety of diseases, including neurodegenerative disorders, cardiovascular dysfunction, and cancer. A growing body of research demonstrates that exosomes play an irreplaceable role in disease prognosis, diagnosis, and even treatment.

[0003] Unique tetraspanins (CD9, CD63, and CD81) on the surface of exosomes are highly expressed as exosome markers, affecting antigen presentation and adhesion, and inhibiting the migration and invasion of tumor cells. In addition, they can also be used as exosome surface-specific proteins to isolate and purify exosomes.

[0004] As an important medium for intercellular communication, exosomes play an increasingly important role in biological research and clinical applications. However, efficient, economical purification methods that maintain the integrity of exosomes have always been the main challenge facing this field. Especially for serum / plasma samples, the separation and purification of exosomes is particularly difficult. Serum / plasma samples are complex in composition. In addition to exosomes, there are also a large number of highly abundant proteins such as albumin and immunoglobulins. The huge concentration difference makes the separation of exosomes like looking for a needle in a haystack. In addition to albumin and immunoglobulins, serum / plasma also contains thousands of other proteins, including lipoproteins, complement proteins, coagulation factors, cytokines, etc. Their size, density, and charge may overlap with exosomes, further increasing the difficulty of exosome separation.

[0005] Ultracentrifugation (UC) is currently the most commonly used exosome isolation technology and the gold standard for exosome separation and enrichment. The UC method is based on the density and size differences between exosomes and impurities in the sample for separation. First, the sample is centrifuged at a low speed to remove larger cells, cell debris, and dead cells. Then, the supernatant is ultracentrifuged twice at a centrifugal force exceeding 100,000g to obtain exosomes. Ultracentrifugation has the advantages of mature technology and standardized operation, making it suitable for most sample separations. However, for research laboratories and clinical environments, this method is time-consuming, labor-intensive, and heavily instrument-dependent. In addition, the exosomes enriched by ultracentrifugation are still mixed with protein aggregates, subcellular organelles, etc. with density and size similar to exosomes, which is particularly unfriendly to serum / plasma samples and can easily cause contamination by foreign proteins.

[0006] Density gradient centrifugation is an optimized ultracentrifugation method that enhances purification by adding a density gradient medium to the sample. While this method can yield higher-purity exosomes, it is more complex and time-consuming (possibly requiring over 24 hours). It also requires an ultracentrifuge, and the purified sample, in a highly concentrated medium, requires replacement through methods such as ultrafiltration.

[0007] Polymer precipitation is a simple method that works by adding a polymer to a sample, altering the dispersion of exosomes and causing them to precipitate, thereby achieving isolation and enrichment. PEG is the most common polymer reagent used for exosome isolation. This method is simple to use, requiring no expensive equipment or complex procedures, and can be used to process large sample volumes. Despite its ease of use, its major drawbacks are low purity and the susceptibility to the introduction of interfering substances (such as protein aggregates). This is particularly true for high-throughput and omics studies, where the presence of large amounts of protein and nucleic acid can impair subsequent analysis and research.

[0008] Ultrafiltration is a separation method based on molecular weight. Samples are passed through filter membranes of varying pore sizes to remove impurities and obtain exosomes. Contaminants larger than the membrane pore size (or molecular weight cut-off) are removed, while components smaller than the molecular weight cut-off are collected through the membrane structure. Ultrafiltration is one of the simplest methods for isolating exosomes, requiring no expensive specialized equipment or hazardous chemicals and allowing the separation of exosomes from large volumes of liquid. However, clogging of the filter membrane surface can result in low exosome yields.

[0009] Size exclusion chromatography (SEC), similar in principle to ultrafiltration, is a separation technique based on molecular size. The sample is loaded into a porous gel column (such as dextran gel or agarose gel). Larger particles are unable to enter the gel pores, resulting in a faster elution rate. Conversely, smaller particles can readily enter the gel pores and elute more slowly, achieving the desired separation. While traditional SEC techniques can effectively separate exosomes, they typically require a large chromatography system. This high cost of chromatography equipment limits SEC to industrial-scale applications.

[0010] There are two main exosome analysis kits available on the market for serum / plasma samples. CellGuidance Systems' EX02-50-Exospin Blood Exosome Isolation Kit utilizes a three-step process: initial enrichment by PEG precipitation, fine purification by SEC size exclusion, and concentration by ultrafiltration centrifugation to isolate highly purified exosomes from complex blood samples. However, this kit is relatively cumbersome to use and cannot process a large number of samples simultaneously. Wako's MagCapture™ Exosome Extraction Kit utilizes Tim4 (T-cell immunoglobulin domain and mucindomain-containing protein 4), which specifically binds to phosphatidylserine (PS) on the EV membrane surface. The highly specific PS-Tim4 interaction, combined with gentle elution using a chelating agent, allows for the isolation of intact EVs with high purity. However, because this method primarily relies on the lipid bilayer structure, the isolated exosomes are susceptible to contamination by impurities other than the exosome membrane.

[0011] Patent announcement number CN117586952B provides a separation reagent and separation method for separating plasma exosomes. The scheme uses separation reagents polyethylene glycol and ammonium sulfate to separate exosomes. The principle is consistent with the polymer precipitation method, which is easy to introduce protein-like interfering substances. Summary of the Invention

[0012] The present invention aims to provide a method for isolating exosomes from plasma or serum, which uses an immunoaffinity method to coat highly specific antibodies on magnetic beads to capture exosomes, thereby achieving the effect of specific isolation of exosomes.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for separating exosomes from plasma or serum, the method comprising: First, magnetic beads are coupled with antibodies against the tetraspanin CD81 on the surface of exosomes to form immunomagnetic beads. Then, the serum / plasma sample is pretreated to enrich the exosomes and then incubated with the immunomagnetic beads. After incubation and capture, the magnetic beads are washed to remove unbound impurities. After exosome capture, the exosomes can be directly lysed and the magnetic beads removed for downstream experiments, or the magnetic beads can be eluted to obtain intact exosomes.

[0014] Furthermore, the CD81 antibody includes a framework region and a complementary determining region (CDR), wherein the complementary determining region (CDR) includes CDR1, CDR2, and CDR3, wherein the CDR1 sequence is shown in SEQ ID NO: 2, the CDR2 sequence is shown in SEQ ID NO: 4, and the CDR3 sequence is shown in SEQ ID NO: 6; the complementary determining region (CDR) is separated by four framework regions (FR1, FR2, FR3, and FR4), wherein FR1 is shown in SEQ ID NO: 1, FR2 is shown in SEQ ID NO: 3, FR3 is shown in SEQ ID NO: 5, and FR4 is shown in SEQ ID NO: 7; The sequence listing is as follows: .

[0015] This antibody, independently developed and screened, is highly selective for the extracellular domain of CD81 and is specifically designed for exosome detection, isolation, and purification. This anti-CD81 nanobody is a fusion of a recombinant VHH with the Fc region of rabbit IgG. As a nanobody, it offers minimal steric hindrance, making it more likely to bind to hidden antigen sites on the membrane surface. Furthermore, it exhibits minimal batch-to-batch variability and a stable structure. The recombinant Fc region of rabbit IgG effectively increases the number of amino sites available for carboxyl coupling, preventing the concentration of antibody-to-bead coupling sites in the heavy chain variable region, which could disrupt the antigen-binding domain. This maximizes the integrity of the antigen-binding region and improves the binding efficiency of the nanobody to exosome surface proteins.

[0016] Furthermore, the preparation method of immunomagnetic beads is as follows: 10 mg / ml carboxyl magnetic microspheres are washed with MEST buffer, and then half the volume of 20 mg / mL EDC in MEST and 20 mg / mL Sulfo-NHS in MEST are added for activation for 15 minutes, followed by addition of 0.1-1.0 mg / ml antibody solution for two hours, followed by blocking with 1% BSA solution for two hours, and finally washing and storage to form immunomagnetic beads.

[0017] Furthermore, pretreatment of serum / plasma samples included centrifugation to remove cell debris or filtering through a 0.22 μm filter to remove cell debris and other aggregates.

[0018] Furthermore, the enrichment process is to mix and incubate the immunomagnetic beads with the serum sample, allowing the Anti-CD81 antibody on the magnetic beads to fully contact and bind with the CD81 protein on the surface of the exosomes in the sample. After the incubation is completed, the magnetic beads are washed 3-5 times to wash away the unbound sample residues.

[0019] Furthermore, after washing, the exosome-carrying magnetic beads can be directly lysed with a lysis buffer, and then the magnetic beads can be removed with a magnetic stand, and the supernatant can be taken for Western Blot detection.

[0020] Furthermore, if complete exosomes need to be obtained, the magnetic beads need to be eluted.

[0021] Further, the specific method is as follows: 1. Pass the plasma sample through a 0.22 μm filter membrane; 2. Take 300 μl of 10 mg / ml antibody-coupled magnetic beads and wash them three times with 1 ml of PBS each time. During the last wash, divide the magnetic beads evenly into 6 EP tubes and remove the supernatant. 3. Mark two of the sample tubes as positive tubes, two as test tubes, and two as negative tubes; 4. Add the corresponding sample to the corresponding EP tube, 1 ml per tube; 5. Vortex the magnetic beads and place them on a rotary mixer and incubate at room temperature for 2 hours. 6. After the incubation time is up, briefly centrifuge the magnetic beads and place them on the magnetic stand. Aspirate the reaction solution and collect the flow-through using an EP tube. 7. Wash the magnetic beads 3 times with PBS, 1 ml each time; 8. Wash once more with 1 ml of pure water. After washing, discard the washing solution. 9. Add 100 μl of eluent and elute at room temperature for 1 min. Collect the eluate and immediately neutralize it with 10 μl of pre-cooled neutralization buffer. 10. Repeat step 8. After repeated elution, combine the eluates for subsequent detection.

[0022] The above technical solution can achieve the following beneficial effects: The present invention utilizes exosome affinity antibodies in combination with magnetic beads to separate exosomes from plasma / serum samples. The unique affinity separation technology can not only remove high-abundance proteins in serum / plasma samples, but also exclude other vesicles with similar membrane structures to exosomes, making the purity of the separated exosomes much higher than other separation methods. Because the separation method of the present invention does not damage the exosomes by external forces, the structure of the exosomes is more complete, which is beneficial for downstream observation and other applications. The exosome separation method of the present invention is simple to operate, takes a short time, and can also be used with fully automatic extraction instruments for high-throughput operations, which is in line with the future application trend of exosomes in the diagnosis and medical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a picture of the sample stock solution (particle size: 58.9nm, concentration: 3.69E+11 / ml).

[0024] Figure 2 This is a picture of the sample after enrichment by precipitation method (particle size: 86.5 nm, concentration: 2.23E+10 / ml).

[0025] Figure 3 This is a sample image after enrichment using the method of the present invention (particle size: 82.6 nm, concentration: 1.45E+11 / ml).

[0026] Figure 4 This is a comparison of electron microscopic photos of exosomes (a. sample solution, b. exosomes obtained by precipitation method, c. exosomes obtained by the method of the present invention). DETAILED DESCRIPTION

[0027] The following is combined with Figure 1-4 The present invention will be further described: A method for separating exosomes from plasma or serum, the method comprising: First, magnetic beads are coupled with antibodies against the tetraspanin CD81 on the surface of exosomes to form immunomagnetic beads. Then, the serum / plasma sample is pretreated to enrich the exosomes and then incubated with the immunomagnetic beads. After incubation and capture, the magnetic beads are washed to remove unbound impurities. After exosome capture, the exosomes can be directly lysed and the magnetic beads removed for downstream experiments, or the magnetic beads can be eluted to obtain intact exosomes.

[0028] The CD81 antibody comprises a framework region and a complementary determining region (CDR), wherein the complementary determining region (CDR) comprises CDR1, CDR2, and CDR3, wherein the CDR1 sequence is as shown in SEQ ID NO:2, the CDR2 sequence is as shown in SEQ ID NO:4, and the CDR3 sequence is as shown in SEQ ID NO:6; the complementary determining region (CDR) is separated by four framework regions (FR1, FR2, FR3, and FR4), wherein FR1 is as shown in SEQ ID NO:1, FR2 is as shown in SEQ ID NO:3, FR3 is as shown in SEQ ID NO:5, and FR4 is as shown in SEQ ID NO:7; The sequence listing is as follows: .

[0029] The preparation method of immunomagnetic beads is as follows: 10mg / ml carboxyl magnetic microspheres are washed with MEST buffer, and then half the volume of 20mg / mL EDC in MEST and 20mg / mL Sulfo-NHS in MEST is added for activation for 15 minutes. Then, 0.1-1.0mg / ml antibody solution is added for two hours, and then blocked with 1% BSA solution for two hours. Finally, the beads are washed and stored to form immunomagnetic beads.

[0030] Serum / plasma samples were pretreated by centrifugation to remove cell debris or by filtering through a 0.22 μm filter to remove cell debris and other aggregates.

[0031] The enrichment process is to mix and incubate the immunomagnetic beads with the serum sample, allowing the Anti-CD81 antibody on the magnetic beads to fully contact and bind with the CD81 protein on the surface of the exosomes in the sample. After incubation, the magnetic beads are washed 3-5 times to wash away the unbound sample residues.

[0032] After washing, the exosome magnetic beads can be directly lysed with lysis buffer, and then the magnetic beads can be removed with a magnetic stand, and the supernatant can be taken for Western Blot detection.

[0033] If intact exosomes are to be obtained, the magnetic beads need to be eluted.

[0034] The kit in the embodiment mainly comprises the following components: immunomagnetic beads, sample binding solution, magnetic bead washing solution, exosome elution solution, and exosome neutralization solution.

[0035] First, magnetic beads are coupled to antibodies against the tetraspanin CD81 on the surface of exosomes to form immunomagnetic beads. Pretreated serum / plasma samples are then incubated with the immunomagnetic beads. After capture, the beads are washed to remove unbound impurities. After capture, the exosomes can be directly lysed for downstream experiments or eluted for further use.

[0036] The anti-CD81 antibody described in this invention is an independently developed nanobody that is highly selective for the extracellular domain 2 (Phe113-Lys201) ​​of exosomal CD81. The method first uses CD81 recombinant protein to immunize alpacas to obtain a single-domain antibody gene and construct a single-domain antibody expression library. Phage display screening is then used to screen for high-affinity anti-CD81 single-domain antibodies from this library. After sequencing the antibody gene sequence, genetic engineering methods are used to generate anti-CD81 single-domain antibodies, which are then tested for affinity. The results demonstrate that the anti-CD81 nanobody obtained through alpaca immunization and screening has a half-maximal effective concentration (EC50) of only 14.04 nM for human CD81-His protein, demonstrating high affinity for the CD81 protein. The present Nanobody comprises a framework region and a complementary determining region (CDR), wherein the complementary determining region (CDR) comprises CDR1, CDR2, and CDR3, wherein the CDR1 sequence is shown in SEQ ID NO: 2, the CDR2 sequence is shown in SEQ ID NO: 4, and the CDR3 sequence is shown in SEQ ID NO: 6. The complementary determining region (CDR) is separated by four framework regions (FR1, FR2, FR3, and FR4), wherein FR1 is shown in SEQ ID NO: 1, FR2 is shown in SEQ ID NO: 3, FR3 is shown in SEQ ID NO: 5, and FR4 is shown in SEQ ID NO: 7.

[0037] The sequence listing is as follows:

[0038] Table 1 Anti-CD81 nanobody sequence information.

[0039] The magnetic beads used in this invention can have a particle size range of 200-3000 nm and can be carboxyl magnetic beads, epoxy magnetic beads, or tosyl (tosyl)-activated magnetic beads. Epoxy and tosyl magnetic beads do not require activation, while carboxyl magnetic beads require activation with EDC / NHS. Carboxyl magnetic beads can be activated with either NHS or Sulfo-NHS, with Sulfo-NHS being more effective. The preparation method involves washing 10 mg / ml carboxyl magnetic microspheres with MEST buffer (pH 5.0-6.0), adding half the volume of 20 mg / mL EDC in MEST and 20 mg / mL Sulfo-NHS in MEST, and activating for 15 minutes. Then, adding 0.1-1.0 mg / ml of antibody solution for two hours, blocking with 1% BSA solution for two hours, and finally washing and storing to form immunomagnetic beads.

[0040] Plasma / serum samples require pre-centrifugation to remove cell debris or pass through a 0.22μm filter to remove cell debris and other aggregates. After sample pretreatment, exosome enrichment begins. This involves incubating immunomagnetic beads with the serum sample, allowing the anti-CD81 antibody on the beads to fully contact and bind to the CD81 protein on the surface of exosomes in the sample. After incubation, the beads are washed 3-5 times to remove any unbound sample residue. After washing, the exosome-laden beads can be directly lysed using a lysis buffer. After lysis, the beads are removed using a magnetic rack, and the supernatant is collected for Western blotting (WB).

[0041] To obtain intact exosomes, the magnetic beads must be eluted. Antigen-antibody binding occurs through non-covalent interactions: electrostatic attraction, van der Waals attraction, hydrogen bonding, and hydrophobic interactions. The principle behind the antigen-antibody elution buffer is to utilize a strong acidic environment to protonate negatively charged groups, disrupting electrostatic attraction and interfering with hydrogen bonds at the antigen-antibody interface, thereby reducing the antigen-antibody complex to monomeric form. After CD81 exosomes are captured by immunomagnetic beads, a magnetic bead-antibody-exosome complex is formed. Because the antibody forms a stable amide bond with the magnetic microspheres, a strongly acidic buffer is used to elute the antigen-containing exosomes. The elution buffer used is 0.1 M glycine-HCl, pH 2.5-3.1. Alternative elution buffers include 0.1 M citric acid, pH 2.5-3.1, or 2.5% acetic acid. After elution, exosomes must be neutralized immediately to prevent the acidic environment from damaging the exosome structure. Neutralization buffer can be 1M Tris-HCl, pH 7.5-9.0, added at a ratio of 1:10.

[0042] The eluted exosomes can be characterized by the following methods: Western blot analysis to test the characteristic proteins on the surface of exosomes (CD9, CD63, CD81); nanoparticle tracking analysis (NTA) to test the particle concentration and size distribution; electron microscopy to observe the morphology of exosomes and the particle-protein ratio to assess the purity.

[0043] Anti-CD81 nanobody expression The nanobody gene of the selected competitively positive clones was linked to the IgG1 Fc vector and then ligated into the pcDNA3.1 vector for transient expression in HEK293F cells. The plasmid was extracted using an endotoxin-free plasmid extraction kit (Biomiga). The plasmid was mixed with the transfection reagent PEI (Polysciences, Inc.) in a 1:3 ratio and allowed to stand for 30 minutes. The mixture was then added to HEK293F cells and cultured in a shaking incubator at 37°C with 5% CO2 for 7 days. The supernatant was then centrifuged and collected. The supernatant was adjusted to pH 7.0 and loaded onto a Protein A affinity chromatography column (Borgron Biotechnology Co., Ltd.) and eluted with 100% 0.1M Gly-HCl (pH 3.0); the eluate was pre-added with 10% 1M Tris-HCl (pH 8.5).

[0044] Carboxyl magnetic beads activated and coupled to Anti-CD81 antibody step: 1) Place 100 mg of carboxyl magnetic beads in a 15 mL centrifuge tube and wash three times with MEST buffer. 2) Prepare 10 mL each of 20 mg / mL EDC in MEST and 20 mg / mL Sulfo-NHS in MEST (prepare immediately for use); 3) Add EDC and Sulfo-NHS to the magnetic microsphere centrifuge tube, mix well, and place in a vertical mixer for reaction activation for 15 minutes; 4) Prepare 10 ml of 0.2 mg / mL Anti-CD81 antibody solution; 5) After activation of the magnetic microspheres, remove the supernatant by magnetic aspiration, add the antibody solution, mix well, and place on a vertical mixer for 2 hours; 6) Prepare 10 ml of 10 mg / mL BSA blocking buffer and mix thoroughly. 7) After the SA grafting reaction of magnetic microspheres is completed, the supernatant is removed by magnetic aspiration, BSA blocking solution is added, mixed, and placed on a vertical mixer for mixing reaction and blocking for 1 hour; 8) After the blocking reaction is completed, remove the supernatant by magnetic aspiration and repeatedly wash the magnetic microspheres with TBST buffer; 9) Wash three times with Storage buffer; 10) Remove the supernatant by magnetic aspiration, accurately add 10 mL of Storage buffer to the volume, and maintain the concentration at 10 mg / mL to obtain the immunomagnetic beads.

[0045] Immunomagnetic beads capture exosomes in serum / plasma 1. Filter the plasma sample through a 0.22 μm filter.

[0046] 2. Take 300 μl of 10 mg / ml antibody-coupled magnetic beads and wash them three times with 1 ml of PBS each time. During the last wash, divide the magnetic beads evenly into 6 EP tubes and remove the supernatant.

[0047] 3. Label two of the sample tubes as positive tubes, two as test tubes, and two as negative tubes.

[0048] 4. Add the corresponding samples to the corresponding EP tubes, 1 ml per tube.

[0049] 5. Vortex the beads and incubate them on a rotary mixer at room temperature for 2 hours.

[0050] 6. After the incubation time is up, briefly centrifuge the magnetic beads and place them on the magnetic rack. Aspirate the reaction solution and collect the flow-through using an EP tube.

[0051] 7. Wash the magnetic beads three times with PBS, 1 ml each time.

[0052] 8. Wash once more with 1 ml of pure water. After washing, discard the washing solution.

[0053] 9. Add 100 μl of eluent and elute at room temperature for 1 min. Collect the eluate and immediately neutralize with 10 μl of pre-cooled neutralization buffer.

[0054] 10. Repeat step 8. After repeated elution, combine the eluates for subsequent detection.

[0055] Comparison of detection after purification of exosomes by different methods Figure 1 Displays the sample stock solution image (particle size: 58.9nm, concentration: 3.69E+11 / ml).

[0056] Figure 2 The image shows the sample after precipitation enrichment (particle size: 86.5 nm, concentration: 2.23E+10 / ml).

[0057] Figure 3 The image of the sample after enrichment using the method of the present invention is shown (particle size: 82.6 nm, concentration: 1.45E+11 / ml).

[0058] Figure 4 Comparison of electron micrographs of exosomes (a. Sample stock solution, b. Exosomes obtained by precipitation method, c. Exosomes obtained by the method of the present invention).

[0059]

[0060] Table 2 Protein ratio of exosome particles after isolation.

[0061] Analysis of Results: After extracting exosomes using the PEG precipitation method and the affinity magnetic bead method of the present invention, NTA was used to measure particle concentration and size distribution. Exosome morphology was observed using electron microscopy, and purity was assessed using the granule-to-protein ratio. NTA results showed that the exosomes obtained using the present method had a narrower particle size distribution and a higher particle concentration. Electron microscopy results showed that the exosomes obtained using the present method were more intact, without fragmentation or aggregation. Granule-to-protein ratio results showed that the exosomes obtained using the present method had a higher granule-to-protein ratio, and the exosome purity was 5.8 times higher than that obtained using the precipitation method.

[0062] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention are within the scope of protection of the claims attached to this application.

Claims

1. A method for isolating exosomes from plasma or serum, characterized by: The method is as follows: First, magnetic beads are coupled with antibodies against the tetraspanin CD81 on the surface of exosomes to form immunomagnetic beads. Then, the serum / plasma sample is pretreated to enrich the exosomes and then incubated with the immunomagnetic beads. After incubation and capture, the magnetic beads are washed to remove unbound impurities. After exosome capture, the exosomes can be directly lysed and the magnetic beads removed for downstream experiments, or the magnetic beads can be eluted to obtain intact exosomes.

2. The method for isolating exosomes from plasma or serum according to claim 1, wherein: The CD81 antibody comprises a framework region and a complementary determining region (CDR), wherein the complementary determining region (CDR) comprises CDR1, CDR2, and CDR3, wherein the CDR1 sequence is shown in SEQ ID NO:2, the CDR2 sequence is shown in SEQ ID NO:4, and the CDR3 sequence is shown in SEQ ID NO:6; the complementary determining region (CDR) is separated by four framework regions (FR1, FR2, FR3, and FR4), wherein FR1 is shown in SEQ ID NO:1, FR2 is shown in SEQ ID NO:3, FR3 is shown in SEQ ID NO:5, and FR4 is shown in SEQ ID NO:7; The sequence listing is as follows: 。 3. The method for isolating exosomes from plasma or serum according to claim 1, wherein: The preparation method of immunomagnetic beads is as follows: 10 mg / ml carboxyl magnetic microspheres are washed with MEST buffer, and then half the volume of 20 mg / mL EDC in MEST and 20 mg / mL Sulfo-NHS in MEST are added for activation for 15 minutes. Then, 0.1-1.0 mg / ml antibody solution is added for two hours, and then blocked with 1% BSA solution for two hours. Finally, the beads are washed and stored to form immunomagnetic beads.

4. The method for isolating exosomes from plasma or serum according to claim 1, wherein: Serum / plasma samples were pretreated by centrifugation to remove cell debris or by filtering through a 0.22 μm filter to remove cell debris and other aggregates.

5. The method for isolating exosomes from plasma or serum according to claim 1, wherein: The enrichment process is to mix and incubate the immunomagnetic beads with the serum sample, allowing the Anti-CD81 antibody on the magnetic beads to fully contact and bind with the CD81 protein on the surface of the exosomes in the sample. After incubation, the magnetic beads are washed 3-5 times to wash away the unbound sample residues.

6. The method for isolating exosomes from plasma or serum according to claim 1 or 5, characterized in that: After washing, the exosome magnetic beads can be directly lysed with lysis buffer, and then the magnetic beads can be removed with a magnetic stand, and the supernatant can be taken for Western Blot detection.

7. The method for isolating exosomes from plasma or serum according to claim 1, wherein: If intact exosomes are to be obtained, the magnetic beads need to be eluted.

8. The method for isolating exosomes from plasma or serum according to claim 1, wherein: The specific method is as follows: The plasma samples were filtered through a 0.22 μm filter; Take 300 μl of 10 mg / ml antibody-coupled magnetic beads and wash them three times with 1 ml of PBS each time. During the last wash, divide the magnetic beads evenly into 6 EP tubes and remove the supernatant. Mark two of the sample tubes as positive tubes, two as test tubes, and two as negative tubes; Add the corresponding sample to the corresponding EP tube, 1 ml per tube; Vortex the magnetic beads and incubate them at room temperature for 2 h. After the incubation time is up, briefly centrifuge the beads and place them on a magnetic stand. Aspirate the reaction solution and collect the flow-through using an EP tube. The magnetic beads were washed three times with PBS, 1 ml each time; Wash once more with 1 ml of pure water. After washing is complete, discard the washing solution; Add 100 μl of eluent and elute at room temperature for 1 min. Collect the eluate and immediately neutralize it with 10 μl of pre-cooled neutralization buffer. Repeat step 8, and after repeated elution, combine the eluates for subsequent detection.

Images