Human plasma neurogenic exosome and total exosome and separation method thereof

Abstract

The invention discloses a human plasma neurogenic exosome and a total exosome and a separation method thereof, the separation method of the human plasma neurogenic exosome comprises the following steps: step A, taking streptavidin magnetic beads, cleaning with 0.005%-0.05% Tween 20-DPBS, and resuspending with BSA-DPBS to obtain a resuspension liquid I; step B, adding a biotinylated NCAM antibody into the resuspension I, incubating, cleaning and resuspending to obtain a resuspension II; step C, mixing the resuspension II with the plasma total exosome, and incubating at 4 DEG C; and step D, removing the supernatant, washing with DPBS, eluting, and neutralizing to obtain the human plasma neurogenic exosome. According to the separation method of the human plasma neurogenic exosome, the neurogenic exosome with fewer impurities and more complete morphological structure can be obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to human plasma neurogenic exosomes and total exosomes and a method for separating the same. Background Art

[0002] Exosomes are extracellular vesicles with a complete double-layer membrane structure, ranging from 30 to 200 nm in diameter, that can carry proteins, RNA, and lipids released by various cell types. Exosomes are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk.

[0003] In the brain, exosomes participate in processes such as synaptic plasticity, neuronal stress responses, intercellular communication, and neurogenesis. Exosomes can cross the blood-cerebrospinal fluid barrier, and their content varies depending on the secreting and recipient cells, making them biomarkers of neurological dysfunction. Intercellular communication in the central nervous system plays an important role in brain growth, development, and maintenance of homeostasis. Studying neurogenic exosomes can provide information for disease diagnosis and therapeutic intervention.

[0004] The prerequisite for studying the regulation of human physiological functions by exosomes is to isolate and extract high-quality exosomes. Currently, methods for isolating exosomes include ultracentrifugation, polymer precipitation, and immunoaffinity.

[0005] Currently, the isolation of neurogenic exosomes from human plasma is often contaminated with exosomes from other cell sources due to nonspecific binding of the magnetic beads used in immunocapture. Incomplete elution and neutralization of the exosomes after immunocapture also result in incomplete morphology and structure. Furthermore, similar processes in the industry for extracting total exosomes from human plasma have resulted in low purity and high levels of impurities. Summary of the Invention

[0006] To address the above technical problems, the first objective of the present invention is to provide a method for isolating neurogenic exosomes from human plasma, thereby isolating neurogenic exosomes with high purity and relatively intact morphological structure. A second objective of the present invention is to provide neurogenic exosomes from human plasma. A third objective of the present invention is to provide a method for isolating total exosomes from human plasma. A fourth objective of the present invention is to provide total exosomes from human plasma.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] As a first aspect of the present invention, a method for isolating neurogenic exosomes from human plasma comprises the following steps:

[0009] Step A: Take streptavidin magnetic beads, wash them with 0.005%-0.05% Tween 20-DPBS, and then resuspend them with BSA-DPBS to obtain resuspension solution I;

[0010] Step B, adding biotinylated NCAM antibody to resuspension I, incubating, washing, and resuspending to obtain resuspension II;

[0011] Step C, resuspension II is mixed with total plasma exosomes and then incubated;

[0012] Step D: removing the supernatant, washing, eluting, and neutralizing to obtain human plasma neurogenic exosomes.

[0013] Preferably, the mass fraction of Tween 20-DPBS used for washing the streptavidin magnetic beads in step A is 0.01%.

[0014] Specifically, the elution in step D was incubated at room temperature for 10 minutes.

[0015] Preferably, in the step D, elution is performed with Glycine-HCl and neutralization is performed with Tris-HCl.

[0016] Preferably, the concentration of Glycine-HCl is 0.5 M and the pH value is 3.0; and / or the concentration of Tris-HCl is 1 M and the pH value is 8.0.

[0017] Specifically, the step of eluting with Glycine-HCl comprises:

[0018] After adding Glycine-HCl, vortex on a vortex shaker for 30 seconds, incubate at room temperature for 10 minutes, centrifuge, and collect the supernatant.

[0019] Specifically, in step B, biotinylated NCAM antibody was added and incubated at room temperature for 1 h.

[0020] Specifically, the resuspension II of step C is mixed with the total exosomes from plasma and incubated at 4° C. for 16-18 h.

[0021] According to the present invention, the total plasma exosomes in step C are prepared by the following method:

[0022] Exosome precipitant was added to the plasma, mixed and incubated, centrifuged and the supernatant was removed, centrifuged again and the supernatant was removed, and resuspended with DPBS to obtain total plasma exosomes.

[0023] Preferably, the exosome precipitant comprises PEG and NaCl, and when the exosome precipitant is added to plasma, the volume ratio of the plasma to the exosome precipitant is 5:1.

[0024] Specifically, the PEG in the exosome precipitant is PEG6000 or PEG8000.

[0025] Preferably, the mass fraction of PEG6000 or PEG8000 is 30%.

[0026] Preferably, after adding the exosome precipitant to the plasma, the NaCl concentration in the plasma is 0.3M-0.5M.

[0027] Preferably, after adding the exosome precipitant to the plasma, the NaCl concentration in the plasma is 0.3 M.

[0028] Preferably, the method for isolating neurogenic exosomes from human plasma comprises the following steps:

[0029] (1) Magnetic bead pretreatment: Take 100 μL of streptavidin magnetic beads, add 500 μL of Tween 20-DPBS reagent to wash, place on a magnetic stand for 1 min, remove the supernatant, repeat washing twice, and add 500 μL of 3% BSA-DPBS to resuspend;

[0030] (2) Binding of magnetic beads to biotinylated antibody reagent: 3 μg of biotinylated NCAM antibody was added to streptavidin magnetic beads resuspended in 500 μL 3% BSA-DPBS, and the beads were incubated on a rotary mixer at room temperature for 1 h. The beads were then placed on a magnetic stand for 1 min, the supernatant was removed, and the beads were washed with 500 μL DPBS. The washing was repeated three times and the beads were resuspended in 100 μL DPBS.

[0031] (3) Immunocapture of neurogenic exosomes by magnetic bead-antibody complex: 100 μL of the resuspension obtained in step (2) (containing the magnetic bead-antibody complex) was mixed with 500 μL of total plasma exosomes and incubated overnight (16-18 h) at 4°C on a rotary mixer.

[0032] (4) Washing and elution of neurogenic exosomes: After incubation overnight, place on a magnetic stand for 1 min, remove the supernatant, add 500 μL DPBS for washing, and repeat washing for a total of 3 times. The exosomes bound to the magnetic beads are neurogenic exosomes;

[0033] (5) Washing and elution of plasma neurogenic exosomes: add 100 μL of 0.5 M Glycine-HCl, pH 3.0, resuspend, place on a vortex shaker, vortex for 30 seconds, incubate at room temperature for 10 minutes, centrifuge briefly using a desktop centrifuge, place on a magnetic stand for 1 minute, and then aspirate all the supernatant;

[0034] (6) Neutralization of plasma neurogenic exosomes: Take a new 1.5 mL centrifuge tube, add 22.5 μL 1 M Tris-HCl, pH 8.0, transfer all the supernatant in step (5) to the 1.5 mL centrifuge tube, and mix thoroughly to obtain 122.5 μL neurogenic exosomes.

[0035] According to the present invention, the amino acid sequence of the antigenic determinant of the biotinylated NCAM antibody is shown in Seq ID NO:4.

[0036] As a second aspect of the present invention, a human plasma neurogenic exosome is prepared by any of the methods described above.

[0037] As a third aspect of the present invention, a method for isolating total exosomes from human plasma comprises: adding an exosome precipitant to the plasma, mixing, incubating, centrifuging, removing the supernatant, centrifuging again, removing the supernatant, and resuspending with DPBS to obtain total exosomes from the plasma; the exosome precipitant comprises PEG and NaCl, and when the exosome precipitant is added to the plasma, the volume ratio of the plasma to the exosome precipitant is 5:1.

[0038] Preferably, the PEG in the exosome precipitant is PEG6000 or PEG8000, the mass fraction of the PEG6000 or PEG8000 is 30%, and after the exosome precipitant is added, the NaCl concentration in the plasma is 0.3M-0.5M.

[0039] As a fourth aspect of the present invention, a human plasma total exosome is prepared by any of the above-mentioned methods for isolating human plasma total exosomes.

[0040] The method for isolating human plasma neurogenic exosomes of the present invention has the following beneficial effects:

[0041] Streptavidin magnetic beads are washed with Tween 20-DPBS and then immunocaptured with biotinylated NCAM antibodies to obtain neurogenic exosomes of high purity, free of nonspecific binding to exosomes from other sources. Following immunocapture, elution with Glycine-HCl and neutralization with Tris-HCl can also yield neurogenic exosomes with more complete morphology. Furthermore, when obtaining total plasma exosomes, using an exosome precipitant containing 30% PEG6000 or PEG8000 and 0.3-0.5M NaCl can yield higher purity and fewer impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the characterization result diagram of experimental group 1;

[0043] Figure 2 This is the characterization result diagram of experimental group 2;

[0044] Figure 3 The NTA and BCA results of experimental group 3 are shown;

[0045] Figure 4 This is the exosome purity analysis diagram of experimental group 3;

[0046] Figure 5 and Figure 6 This is the WB result of experimental group 4;

[0047] Figure 7 Different homemade antibodies were used for protein marker analysis after exosome isolation in Experiment 5;

[0048] Figure 8 For Experiment 5, homemade antibodies and commercial antibodies were used to analyze protein markers after exosome isolation;

[0049] Figure 9 and Figure 10 This is the WB result of experimental group 6;

[0050] Figure 11 This is the transmission electron microscopy result of experimental group seven. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to more fully understand the technical solutions of the present invention, exemplary embodiments of the present invention will be described in more comprehensive and detailed manner below with reference to the accompanying drawings.

[0052] Unless otherwise specified, the reagents and materials used in the following examples were all commercially available. The experimental methods used in the following examples and comparative examples were all conventional methods unless otherwise specified.

[0053] It should be noted that the following protease / protein phosphatase inhibitor was purchased from Thermo Fisher Scientific as Halt Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-Free (100×), which was diluted 100-fold before use, namely the 1* protease / protein phosphatase inhibitor described in the examples.

[0054] 1. Isolation of Total Exosomes from Plasma

[0055] 1. Method for isolating total exosomes from plasma:

[0056] (1) Preparation of plasma:

[0057] Take 10 mL of blood sample placed in an EDTA-2k anticoagulant tube and centrifuge it directly at 2500 g and 4°C for 15 min. Pipette the supernatant and transfer it to a 15 mL centrifuge tube and centrifuge it again at 2500 g and 4°C for 15 min. Pipette the supernatant as plasma.

[0058] (2) Plasma freezing and thawing:

[0059] If total exosome isolation is performed directly, transfer 3 mL of plasma to a 15 mL centrifuge tube for direct separation;

[0060] If total exosome isolation is not performed directly, 3 mL of plasma is transferred into 5 mL cryovials and stored vertically in a -80°C refrigerator. Before separation, the plasma frozen at -80°C is placed in a 37°C constant temperature water bath, quickly thawed, and then 3 mL of plasma is transferred to a 15 mL centrifuge tube.

[0061] (3) Isolation of total exosomes from plasma:

[0062] To 3 mL of plasma, 600 μL of exosome precipitant was added (the volume ratio of plasma to exosome precipitant was 5:1), the mixture was gently inverted and incubated in a 4°C refrigerator for 1 h, followed by centrifugation at 4500 g for 10 min at 4°C. After removing the supernatant, the centrifugation was repeated at 4500 g for 1 min at 4°C to remove the residual supernatant. Subsequently, 750 μL of DPBS (containing 1* protease / protein phosphatase inhibitor) was added to resuspend the exosome pellet and transfer it to a 1.5 mL centrifuge tube to obtain total plasma exosomes.

[0063] 2. Examples and Comparative Examples of Exosome Precipitants

[0064] (1) Experimental Group 1: Effects of different PEG types and concentrations on the separation of total exosomes from plasma

[0065] The exosome precipitant contains PEG and NaCl. To explore the effects of different PEG types and concentrations on the separation of total exosomes from plasma, two PEGs of different lengths, PEG6000 and PEG8000, and two PEG concentrations, 30% and 50% (both by mass fraction), were used. Two sets of examples (Example 1 and Example 2) and two sets of comparative examples (Comparative Example 1 and Comparative Example 2) were set up:

[0066] The exosome precipitant formulation in Example 1 is 30% PEG6000 + 0.3M NaCl (0.3M is the NaCl concentration in the plasma after the exosome precipitant is mixed with the plasma, i.e., the final concentration of NaCl);

[0067] The exosome precipitant formulation in Example 2 is 30% PEG8000 + 0.3M NaCl;

[0068] The exosome precipitant formulation in Comparative Example 1 was 50% PEG6000 + 0.3 M NaCl;

[0069] The exosome precipitant formulation in Comparative Example 2 was 50% PEG8000+0.3M NaCl.

[0070] During the experiment, each group of examples or comparative examples was repeated twice, and each repetition used 500 μL of frozen plasma and 100 μL of exosome precipitant (the volume ratio of thawed plasma to exosome precipitant was 5:1), and the total exosomes in plasma were separated according to the above-mentioned method for separating total exosomes in plasma.

[0071] (2) Experimental Group 2: Effects of different NaCl concentrations on the isolation of total exosomes from plasma

[0072] To investigate the effect of different NaCl concentrations on the separation of total exosomes from plasma, three exosome precipitants with different NaCl concentrations were used: 0 M, 1.8 M, and 3 M. The final NaCl concentrations after mixing the exosome precipitants with plasma were 0 M, 0.3 M, and 0.5 M, respectively. Four examples (Examples 1, 2, 3, and 4) and two comparative examples (Comparative Example 3 and Comparative Example 4) were set up:

[0073] The exosome precipitant formulation in Example 1 is 30% PEG6000 + 0.3M NaCl;

[0074] The exosome precipitant formulation in Example 2 is 30% PEG8000 + 0.3M NaCl;

[0075] The exosome precipitant formulation in Example 3 is 30% PEG6000 + 0.5M NaCl;

[0076] The exosome precipitant formulation in Example 4 is 30% PEG8000 + 0.5M NaCl;

[0077] The exosome precipitant formulation in Comparative Example 3 was 30% PEG6000;

[0078] The exosome precipitant formulation in Comparative Example 4 was 30% PEG8000.

[0079] (The above NaCl concentrations refer to the final NaCl concentration after the exosome precipitant and plasma are mixed.)

[0080] During the experiment, each group of examples or comparative examples was repeated twice, and each repetition used 500 μL of frozen plasma and 100 μL of exosome precipitant (the volume ratio of thawed plasma to exosome precipitant was 5:1). The total exosomes in plasma were separated according to the above-mentioned method for separating total exosomes in plasma.

[0081] The exosome precipitation agent formulas of Examples 1-4 and Comparative Examples 1-4 in the above two groups of experiments are shown in Table 1.

[0082] Table 1 Formulations of exosome precipitants in Examples 1-4 and Comparative Examples 1-4

[0083] Example PEG Final NaCl concentration Example 1 30% PEG6000 0.3M Example 2 30% PEG8000 0.3M Example 3 30% PEG6000 0.5M Example 4 30% PEG8000 0.5M Comparative Example 1 50% PEG6000 0.3M Comparative Example 2 50% PEG8000 0.3M Comparative Example 3 30% PEG6000 - Comparative Example 4 30% PEG8000 -

[0084] (3) Experimental Group 3: Comparison of the isolation effect of total exosomes from plasma with that of SBI brand commercial exosome isolation reagent

[0085] Comparative Example 5 was set up, using System Biosciences (SBI) brand ExoQuick exosome precipitation solution (referred to as SBI precipitant) for comparison with the exosome precipitant formula of Example 1 (30% PEG6000 + 0.3M NaCl). Each group was repeated twice, and each repeat used 500 μL of frozen plasma.

[0086] In Comparative Example 5, the volume ratio of thawed plasma to SBI precipitant was 4:1, and in Example 1, the volume ratio of thawed plasma to exosome precipitant was 5:1. The total exosomes in plasma were separated according to the above-mentioned method for separating total exosomes in plasma.

[0087] 3. Effect embodiment

[0088] The total plasma exosomes isolated from the above three groups of experiments were characterized by the following methods:

[0089] Nanoparticle tracking analysis (NTA) was performed using a Malvern Panalytical NanoSight NS300 to determine the number of exosomes. The protein concentration of the exosomes was determined using the BCA assay. The purity of the exosomes was determined by the number of exosomes per unit of exosomal protein (i.e., exosome number / protein concentration). The expression of exosome markers was analyzed using Western blot (WB). CD81, TSG101, and Alix are positive exosome markers, while ApoA-1 and Calnexin are negative exosome markers.

[0090] (1) Effect Example 1

[0091] The results of the two groups of examples (Example 1 and Example 2) and the two groups of comparative examples (Comparative Example 1 and Comparative Example 2) in the experimental group 1 were characterized according to the above method.

[0092] Figure 1 is the characterization result of experimental group 1; Figure 1 A is the exosome purity diagram of each group of examples and comparative examples in experimental group 1; Figure 1 B is the protein immunoblotting (WB) image of each group of examples and comparative examples in experimental group 1. Figure 1 A and Figure 1 B from left to right: Example 1, Comparative Example 1, Example 2, Comparative Example 2.

[0093] Figure 1 The results of A showed that the purity of exosomes in Examples 1 and 2 was better than that in Comparative Examples 1 and 2;

[0094] Figure 1 The results of B show that Examples 1 and 2, and Comparative Examples 1 and 2 all expressed the positive exosome markers CD81, TSG101, and Alix, and did not express the negative marker Calnexin. However, the expression of the negative marker ApoA-1 in Examples 1 and 2 was lower than that in Comparative Examples 1 and 2.

[0095] Therefore, the purity of the total plasma exosomes separated by the exosome precipitants of Examples 1 and 2 was higher than that of Comparative Examples 1 and 2, and the expression of exosome markers was also better than that of Comparative Examples 1 and 2. That is, the exosome precipitant containing 30% PEG6000 or 30% PEG8000 had a better separation effect on total plasma exosomes than the exosome precipitant containing 50% PEG6000 or 50% PEG8000.

[0096] (2) Effect Example 2

[0097] The results of the four groups of examples (Examples 1, 2, 3, and 4) and two groups of comparative examples (Comparative Examples 1 and 2) in Experimental Group 2 were characterized according to the above method.

[0098] Figure 2 is the characterization result of experimental group 2; Figure 2 A is the exosome purity diagram of each group of examples and comparative examples in experimental group 2; Figure 2 B is the protein immunoblotting (WB) image of each group of examples and comparative examples in experimental group 2. Figure 1 A and Figure 1 B from left to right: Comparative Example 3, Example 1, Example 3, Comparative Example 4, Example 2, Example 4.

[0099] Figure 2The results of A showed that the purity of the exosomes in Examples 1, 2, 3, and 4 was better than that in Comparative Examples 3 and 4; the purity of the exosomes in Examples 1 and 2 was better than that in Examples 3 and 4, but the difference was not significant.

[0100] Figure 2 The results of B showed that all six groups of examples and comparative examples expressed the positive exosome markers CD81, TSG101, and Alix, and did not express the negative marker Calnexin, but at the same time all expressed the negative marker ApoA-1.

[0101] Therefore, the purity of the total plasma exosomes separated by the exosome precipitants of Examples 1, 2, 3, and 4 was higher than that of Comparative Examples 3 and 4, that is, the exosome precipitant containing a final concentration of 0.3M-0.5M NaCl had a better separation effect on the total plasma exosomes than the exosome precipitant without NaCl.

[0102] (3) Effect Example 3

[0103] The results of Example 1 and Comparative Example 5 (SBI precipitant) in Experimental Group 3 were characterized according to the above method. Figure 3 and Figure 4 shown. Figure 3 The NTA and BCA results of experimental group 3 are shown; Figure 4 This is the purity analysis chart of exosomes in experimental group 3. Figure 3 and Figure 4 The horizontal axis in the figure is Nyuen, and the comparative example 5 is Figure 3 and Figure 4 The horizontal axis is represented as SBI.

[0104] Figure 3 The results showed that the exosome protein concentration separated by Nyuen precipitant (Example 1) was lower; the particle concentration and particle size were similar to those of SBI precipitant (Comparative Example 5);

[0105] Figure 4 The results showed that the purity of exosomes separated by Nyuen precipitant (Example 1) was significantly higher than that of SBI precipitant (Comparative Example 5).

[0106] 2. Isolation of Neurogenic Exosomes

[0107] 1. Experimental Group 4:

[0108] In order to explore the effect of whether the magnetic beads were washed during pretreatment and the type and concentration of the washing reagent on whether the magnetic beads non-specifically bound to exosomes from other sources in plasma, five groups of comparative examples were set up, specifically comparative examples 6-10.

[0109] It should be noted that the total plasma exosomes used in the examples and comparative examples of experimental groups 4, 5, and 6 for isolating neurogenic exosomes were all plasma total exosomes prepared by the method of Example 1, 2, 3, or 4, or commercially available plasma total exosomes.

[0110] Comparative Example 6:

[0111] (1) Take 100 μL of streptavidin magnetic beads and resuspend them in 500 μL of 3% BSA-DPBS without washing;

[0112] (2) Without binding to the antibody, the resuspension in step (1) was placed directly on a rotary mixer and incubated at room temperature for 1 h. Then, the supernatant was removed after being placed on a magnetic stand for 1 min. The cells were washed with 500 μL DPBS, and the washing was repeated 3 times before being resuspended in 100 μL DPBS.

[0113] (3) The resuspension obtained in step (2) was mixed with the total plasma exosomes and placed on a rotary mixer. Incubate at 4°C overnight (16-18 hours). The beads were then placed on a magnetic stand for 1 minute, the supernatant removed, and washed with 500 μL of DPBS. The washing was repeated three times. The neurogenic exosomes were bound to the magnetic beads.

[0114] Comparative Example 7:

[0115] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of DBPS to wash, place on a magnetic stand for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and then add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are the same as in Comparative Example 6.

[0116] Comparative Example 8:

[0117] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.01% Tween20-DPBS to wash, place on a magnetic rack for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are the same as in Comparative Example 6.

[0118] Comparative Example 9:

[0119] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.05% Tween20-DPBS to wash, place on a magnetic rack for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are the same as in Comparative Example 6.

[0120] Comparative Example 10:

[0121] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.005% Tween20-DPBS to wash, place on a magnetic rack for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are the same as in Comparative Example 6.

[0122] Whether washing was performed during magnetic bead pretreatment in Comparative Examples 6-10, as well as the washing reagents and types, are shown in Table 2.

[0123] Table 2 Washing reagents for magnetic bead pretreatment in Comparative Examples 6-10

[0124] Comparative Example Washing reagents Comparative Example 6 - Comparative Example 7 DBPS Comparative Example 8 0.01% Tween 20-DPBS Comparative Example 9 0.05% Tween 20-DPBS Comparative Example 10 0.005% Tween 20-DPBS

[0125] Effect embodiment 4:

[0126] 100 μL 1× SDS Loading Buffer was added to the product obtained in Comparative Example 6-10 to prepare WB samples. Western blot (WB) was used to analyze the expression of exosome positive markers (CD81, TSG101 and Alix) to determine whether the magnetic beads were bound to exosomes. Figure 5 and Figure 6 shown.

[0127] (1) Figure 5 To compare the effects of different types of washing reagents on the nonspecific binding of magnetic beads to exosomes, from left to right are the WB results of Comparative Examples 6, 7, and 8, i.e., no washing, DPBS washing, and 0.01% Tween20-DPBS washing.

[0128] Figure 5 The results showed that non-specific binding of exosomes was present after immunocapture of magnetic beads without washing (Comparative Example 6) or washing with DPBS (Comparative Example 7), while non-specific binding of exosomes was not present after immunocapture of magnetic beads washed with 0.01% Tween 20-DPBS (Comparative Example 8).

[0129] (2) Figure 6 To compare the effects of different concentrations of Tween 20-DPBS on the nonspecific binding of magnetic beads to exosomes, from left to right are the WB results of Comparative Examples 6, 9, 8, and 10: no wash, 0.05% Tween 20-DPBS wash, 0.01% Tween 20-DPBS wash, and 0.005% Tween 20-DPBS wash.

[0130] Figure 6The results showed that: after immune capture, there was no nonspecific binding of exosomes to the magnetic beads washed with 0.05% Tween20-DPBS (Comparative Example 9), 0.01% Tween20-DPBS (Comparative Example 8) and 0.005% Tween20-DPBS (Comparative Example 10); while nonspecific binding of exosomes was found after immune capture with the magnetic beads not washed (Comparative Example 6).

[0131] 2. Experimental Group 5:

[0132] To explore the plasma neurogenic exosome separation effects of different antibodies, this example prepared antibodies for immunocapture of neurogenic exosomes and compared their plasma neurogenic exosome separation effects with those of commercial antibodies.

[0133] 2.1 Preparation of antibodies for immunocapture of neurogenic exosomes and verification of their functional activity

[0134] Two antibodies were prepared against the neuronal markers L1CAM and NCAM respectively for immunocapture of neurogenic exosomes.

[0135] The two epitopes of L1CAM (Ensembl ID: ENSP00000359077.1) correspond to positions 140-330aa and 718-907aa, and the amino acid sequences are:

[0136] KETVKPVEVEEGESVVLPCNPPPSAEPLRIYWMNSKILHIKQDERVTMGQNGNLYFAN

[0137] VLTSDNHSDYICHAHFPGTRTIIQKEPIDLRVKATNSMIDRKPRLLFPTNSSSHLVALQG

[0138] QPLVLECIAEGFPTPTIKWLRPSGPMPADRVTYQNHNKTLQLLKVGEEDDGEYRCLAENSLGSARHAYYVTVE(Seq ID NO:1) and

[0139] PVDVKGEGNETTNMVITWKPLRWMDWNAPQVQYRVQWRPQGTRGPWQEQIVSDP

[0140] FLVVSNTSTFVPYEIKVQAVNSQGKGPEPQVTIGYSGEDYPQAIPELEGIEILNSSAVLV

[0141] KWRPVDLAQVKGHLRGYNVTYWREGSQRKHSKRHIHKDHVVVPANTTSVILSGLRPYSSYHLEVQAFNGRGGSGPAS (Seq ID NO: 2).

[0142] The two epitopes of NCAM (Ensembl ID: ENSP00000318472.8) correspond to positions 124-307aa and 511-694aa, and the amino acid sequences are:

[0143] PTPQEFREGEDAVIVCDVVSSLPPTIIWKHKGRDVILKKDVRFIVLSNNYLQIRGIKKT

[0144] DEGTYRCEGRILARGEINFKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFP

[0145] EPTMSWTKDGEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAGEQDATIHLKVFAK(Seq ID NO:3) and

[0146] SSPSIDQVEPYSSTAQVQFDEPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASM

[0147] EGIVTIVGLKPETTYAVRLAALNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDG

[0148] NSIKVNLIKQDDGGSPIRHYLVRYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEYEVYVVAENQQGKSK (Seq ID NO: 4). The polypeptide fragment corresponding to the antigen epitope was directly obtained by synthesis.

[0149] Healthy 4-5 week old Balb / c mice were immunized with NCAM or L1CAM antigen at a dose of 50 μg per mouse. The antigen was diluted to 25 μL with saline, mixed with an equal volume of Freund's complete adjuvant, and emulsified by ultrasonic phacoemulsification before subcutaneous injection at multiple sites. Three weeks later, an equal amount of L1CAM (amino acid sequences shown in Seq ID NO:1 and Seq ID NO:2) or NCAM (amino acid sequences shown in Seq ID NO:3 and Seq ID NO:4) was diluted to 25 μL, mixed with an equal volume of Freund's incomplete adjuvant, and emulsified by ultrasonic phacoemulsification before subcutaneous injection at multiple sites. This immunization cycle was repeated three weeks later.

[0150] One week after the third immunization, all mice underwent eye bleeding and serum was isolated. Mouse serum titers were assessed using an ELISA plate coated with NCAM or L1CAM antigens. The procedure was as follows: ELISA plates were coated with NCAM or L1CAM antigen at a concentration of 1 μg / ml. 50 μL of the solution was added to each well of a 96-well microplate. The plate was incubated at 37°C for 2 hours and then incubated at 4°C for 16-36 hours, or at 4°C for 16-36 hours and then at 37°C for 2 hours. The plate was washed twice with PBST (PBS containing 0.5% Tween-20). Then, 200 μL of blocking solution containing 1% BSA was added to each well. The plate was incubated at 37°C for 2 hours or at 4°C overnight, then patted dry and stored at -20°C until further use. For testing, 100 μL of mouse serum at various concentrations was added to each well of the ELISA plate (set up in duplicate) and incubated at room temperature for 2 hours. After washing three times with PBST, 100 μL of diluted HRP-labeled rabbit anti-mouse Ig antibody was added and incubated at room temperature for 1 hour. After washing three more times with PBST, 100 μL of color development solution was added to each well for color development. The reaction was then terminated by adding 100 μL of stop solution (2M H2SO4) to each well. The OD value of each well at a wavelength of 450 nm was immediately measured using a microplate reader. For mice with serum antibody titers >10,000, a surge immunization was performed one week after eyeball blood collection. Each mouse was injected with 100 μL of antigen protein (10 μg) via tail vein injection.

[0151] Three days after the mice were immunized, spleen cells were harvested for fusion. Hybridoma SP2 / 0 cells were cultured in an incubator at 37°C and 5% CO to ensure healthy growth. The medium was changed the day before fusion. The cell fusion process was as follows: the spleen of the mouse was harvested, ground, washed, and counted. The splenocytes and SP2 / 0 cells were mixed at a ratio of 6:1. The electrofusion solution was preheated in a 37°C water bath. The supernatant of the mixed cells was discarded, and preheated electrofusion solution at 37°C was added. The mixture was centrifuged at 1200 rpm for 3 minutes, washed twice, and the supernatant discarded. The cells were then resuspended in electrofusion solution and incubated at 37°C. The cell suspension was added to an electrofusion dish, which was placed under an inverted microscope with the power on to observe whether the cells moved and formed neat rows. After fusion was complete, the cells were allowed to rest for 5 minutes. The cells in the electrofusion dish were harvested and centrifuged at 1200 rpm for 3 minutes, and the supernatant discarded. Two million sp2 / 0 cells were evenly seeded into 96-well plates, with 150 μL per well. Screening was initially performed using HAT medium (containing hypoxanthine, methotrexate, and thymidine), with the medium replaced every 3-4 days. After 10 days, HT medium was switched. When the hybridoma cell density in the 96-well plate exceeded 10%, the supernatant was removed and analyzed by ELISA using an NCAM or L1CAM antigen-coated plate (ELISA method as described above). Selected positive hybridoma clones were passaged to 24-well plates for expansion and subcloning by limiting dilution. Three rounds of subcloning were performed to obtain hybridoma cell lines stably expressing the target antibody, which were then preserved and banked.

[0152] At the same time, the hybridoma cell lines after the third round of subcloning were expanded in serum-free culture, and the cell supernatant was collected and purified using a Protein G affinity column to obtain four antibodies, which were named L1CAM-1 (amino acid sequence shown in Seq ID NO: 1), L1CAM-2 (amino acid sequence shown in Seq ID NO: 2), NACM-1 (amino acid sequence shown in Seq ID NO: 3), and NACM-2 (amino acid sequence shown in Seq ID NO: 4). After biotinylation, the functional activity of these four antibodies was verified to determine whether they could effectively isolate neurogenic exosomes (L1CAM-1 group, L1CAM-2 group, NACM-1 group and NACM-2 group). No antibody was added (no antibody group) as the control group, and a total of 5 groups were divided; after using 3 ml of plasma to obtain total exosomes, 100 μL of streptavidin magnetic beads were taken, 500 μL of 0.01% Tween20-DPBS were added for washing, and the beads were placed on a magnetic stand for 1 minute, after which the supernatant was removed. After repeating the washing for a total of 3 times, 500 μL of 3% BSA-DPBS was added for resuspending, and 3 μg of biotinylated L1CAM-1, L1CAM-2, NACM-1 and NACM-2 antibodies were added respectively. The beads were placed on a rotary mixer and incubated at room temperature for 1 hour. Subsequently, the beads were placed on a magnetic stand for 1 minute, after which the supernatant was removed, and 500 μL of The beads were washed with DPBS, repeated three times, and resuspended in 100 μL of DPBS. The beads in the no-antibody group were placed directly on a rotary mixer and incubated at room temperature for 1 hour. The beads were then placed on a magnetic rack for 1 minute, the supernatant removed, and washed with 500 μL of DPBS, repeated three times, and resuspended in 100 μL of DPBS. The beads in the L1CAM-1, L1CAM-2, NACM-1, NACM-2, and no-antibody groups were then mixed with total exosomes and placed on a rotary mixer for overnight incubation at 4°C. The beads were then placed on a magnetic rack for 1 minute, the supernatant removed, and washed with 500 μL of DPBS, repeated three times. After that, 100 μL of 1× SDS loading buffer was added to prepare the beads for Western blotting (WB). Western blotting was performed to analyze the expression of exosome markers (TSG101 and Alix) and neuronal markers (L1CAM and NCAM) to determine which antibody groups bound the magnetic beads to neuronal exosomes.

[0153] The experimental results are as follows Figure 7 The results showed that the NACM-2 antibody group could isolate and obtain neurogenic exosomes, while the other antibody groups could not effectively isolate and obtain neurogenic exosomes.

[0154] Comparison of the isolation effects of plasma neurogenic exosomes between NCAM-2 antibody and commercial antibodies

[0155] Comparative effect of the NACM-2 antibody obtained in Experimental Group 5, Section 2.1, and commercial antibodies on the isolation of plasma neurogenic exosomes. The commercial antibodies used in this example include Abcam's recombinant Anti-L1CAM antibody [EPR18750], recombinant Anti-L1CAM antibody [EPR23241-224], and recombinant Anti-NCAM1 antibody [EP2567Y], as well as Thermo Fisher's CD171 monoclonal antibody (eBio5G3).

[0156] After biotinylation, NACM-2 antibody and four commercial antibodies were used to isolate neurogenic exosomes. The isolation effects were compared (EPR18750 group, EPR23241-224 group, EP2567Y group, eBio5G3 group and NACM-2 group). No antibody was added (no antibody group) as the control group, and a total of 6 groups were divided. After obtaining total exosomes using 3 ml of plasma, 100 μL of streptavidin magnetic beads were taken, washed with 500 μL of 0.01% Tween20-DPBS, placed on a magnetic rack for 1 minute, and the supernatant was removed. The washing was repeated 3 times and then 500 μL of 3% The cells were resuspended in BSA-DPBS and then added with 3 μg of biotinylated EPR18750, EPR23241-224, EP2567Y, eBio5G3, and NACM-2 antibodies, respectively. The cells were incubated on a rotary mixer for 1 hour at room temperature, then placed on a magnetic rack for 1 minute, the supernatant removed, and washed with 500 μL of DPBS, repeated three times, and resuspended in 100 μL of DPBS. The cells in the no-antibody group were directly placed on a rotary mixer and incubated at room temperature for 1 hour. The cells were then placed on a magnetic rack for 1 minute, the supernatant removed, washed with 500 μL of DPBS, repeated three times, and resuspended in 100 μL of DPBS. The EPR18750, EPR23241-224, EP2567Y, eBio5G3, NACM-2, and no-antibody groups were then mixed with total exosomes and incubated on a rotary mixer overnight at 4°C with rotation. The supernatant was then removed after being placed on a magnetic stand for 1 minute. The beads were washed with 500 μL of DPBS and washed three times. 100 μL of 1×SDS loading buffer was added to prepare WB samples. The expression of exosome positive markers (TSG101 and Alix) and neuronal markers (L1CAM and NCAM) was analyzed by Western blot (WB) to determine which antibody group magnetic beads bound to neuronal exosomes.

[0157] The experimental results are as follows Figure 8 The results showed that the NACM-2 antibody group could isolate and obtain neurogenic exosomes, while the other antibody groups could not effectively isolate and obtain neurogenic exosomes.

[0158] Therefore, subsequent experiments were performed using the NACM-2 antibody obtained in Example 1.

[0159] 3. Experimental Group 6:

[0160] In order to explore the effects of the type and concentration of washing reagents on the specific binding of magnetic beads to neurogenic exosomes, five groups of comparative examples and three groups of examples were set up.

[0161] The five groups of comparative examples are comparative examples 7, 8, 9, 10 and comparative example 11, and the three groups of embodiments are embodiments 5, 6 and 7.

[0162] Example 5:

[0163] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.01% Tween20-DPBS to wash, place on a magnetic stand for 1 min, remove the supernatant, repeat washing for a total of 3 times, and then add 500 μL of 3% BSA-DPBS to resuspend;

[0164] (2) Take the streptavidin magnetic beads resuspended in 500 μL 3% BSA-DPBS in step (1), add 3 μg of biotinylated NCAM-2 antibody, place on a rotary mixer and incubate at room temperature for 1 hour, then place on a magnetic stand for 1 minute, remove the supernatant, add 500 μL DPBS to wash, repeat washing for a total of 3 times, and then add 100 μL DPBS to resuspend;

[0165] (3) The resuspension obtained in step (2) was mixed with the total plasma exosomes and placed on a rotary mixer, and incubated overnight at 4°C; then, the mixture was placed on a magnetic stand for 1 minute, the supernatant was removed, and 500 μL DPBS was added for washing. The washing was repeated 3 times. The exosomes bound to the magnetic beads were neurogenic exosomes.

[0166] Example 6:

[0167] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.05% Tween20-DPBS to wash, place on a magnetic rack for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are consistent with Example 5.

[0168] Example 7:

[0169] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.005% Tween20-DPBS to wash, place on a magnetic rack for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and then add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are consistent with Example 5.

[0170] Comparative Example 11:

[0171] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of DPBS to wash, place on a magnetic rack for 1 minute, remove the supernatant, repeat washing for a total of 3 times, and then add 500 μL of 3% BSA-DPBS to resuspend; the subsequent steps are consistent with Example 5.

[0172] The differences between Examples 5-7 and Comparative Examples 7-11 are the type and concentration of the washing reagent during the magnetic bead pretreatment, and whether the magnetic beads bind to the biotinylated NCAM-2 antibody, as shown in Table 3.

[0173] Table 3 Differences between Examples 5-7 and Comparative Examples 7-11

[0174] Example Type and concentration of washing reagents Whether it binds to NCAM antibody Example 5 0.01% Tween 20-DPBS yes Example 6 0.05% Tween 20-DPBS yes Example 7 0.005% Tween 20-DPBS yes Comparative Example 7 DBPS no Comparative Example 8 0.01% Tween 20-DPBS no Comparative Example 9 0.05% Tween 20-DPBS no Comparative Example 10 0.005% Tween 20-DPBS no Comparative Example 11 DBPS yes

[0175] Effect embodiment 5:

[0176] 100 μL of 1×SDS Loading Buffer was added to the products obtained in Examples 5-7 and Comparative Examples 7-11 to prepare WB samples. Western blot (WB) was used to analyze the expression of exosome positive markers (TSG101 and Alix) and neurogenic markers (L1CAM) to determine whether neurogenic exosomes were bound to the magnetic beads bound to the antibody and whether exosomes were bound to the magnetic beads not bound to the antibody. The results are shown in Figure 2. Figure 9 and Figure 10 shown.

[0177] (1) Figure 9 Comparison of Western Blot results with and without antibodies when using DPBS and 0.01% Tween 20-DPBS as wash reagents. From left to right are Example 5, Comparative Example 8, Comparative Example 11, and Comparative Example 7, i.e., the 0.01% Tween 20-DPBS-with antibodies group, the 0.01% Tween 20-DPBS-without antibodies group, the DPBS-with antibodies group, and the DPBS-without antibodies group, respectively.

[0178] Figure 9 The results showed that after immunocapture with magnetic beads washed with DPBS and 0.01% Tween20-DPBS, the antibody group could isolate and obtain neurogenic exosomes; and there was no nonspecific binding of exosomes after immunocapture with magnetic beads washed with 0.01% Tween20-DPBS; there was nonspecific binding of exosomes after immunocapture with magnetic beads washed with DPBS.

[0179] (2) Figure 10Comparison of Western blotting results with and without antibodies when washing magnetic beads at different Tween 20-DPBS concentrations. From left to right are Examples 6, 5, and 7, and Comparative Examples 9, 8, and 10, i.e., the 0.05% Tween 20-DPBS-with antibodies group, the 0.01% Tween 20-DPBS-with antibodies group, and the 0.005% Tween 20-DPBS-with antibodies group, as well as the 0.05% Tween 20-DPBS-without antibodies group, the 0.01% Tween 20-DPBS-without antibodies group, and the 0.005% Tween 20-DPBS-without antibodies group.

[0180] Figure 8 The results showed that: 0.005% Tween20-DPBS, 0.01% Tween20-DPBS and 0.05%

[0181] After immunocapture with magnetic beads washed with Tween20-DPBS, the antibody group was able to isolate and obtain neurogenic exosomes, and the corresponding non-antibody group showed no non-specific binding of exosomes. In the antibody group, 0.01% Tween20-DPBS had a better washing effect, and the expression of exosome positive markers (TSG101 and Alix) and neurogenic marker (L1CAM) was better than that of 0.005% Tween20-DPBS and 0.05% Tween20-DPBS.

[0182] 3. Elution and Neutralization of Exosomes after Immunocapture with Magnetic Beads

[0183] Experimental Group 7:

[0184] In order to explore the effect of different incubation times during elution on the morphology of neurogenic exosomes, a group of examples (Example 8) and two groups of comparative examples (Comparative Examples 12 and 13) were set up.

[0185] Example 8:

[0186] (1) Take 100 μL of streptavidin magnetic beads, add 500 μL of 0.01% Tween20-DPBS to wash, place on a magnetic stand for 1 min, remove the supernatant, repeat washing for a total of 3 times, and then add 500 μL of 3% BSA-DPBS to resuspend;

[0187] (2) Take the streptavidin magnetic beads resuspended in 500 μL 3% BSA-DPBS in step (1), add 3 μg of biotinylated NCAM-2 antibody, place on a rotary mixer and incubate at room temperature for 1 hour, then place on a magnetic stand for 1 minute, remove the supernatant, add 500 μL DPBS to wash, repeat washing for a total of 3 times, and then add 100 μL DPBS to resuspend;

[0188] (3) The resuspension obtained in step (2) was mixed with the total exosomes from plasma and placed on a rotary mixer. Incubated overnight at 4°C. The mixture was then placed on a magnetic stand for 1 min, the supernatant was removed, and 500 μL of DPBS was added for washing. The washing was repeated 3 times.

[0189] (4) Add 100 μL of 0.5 M Glycine-HCl, pH 3.0, resuspend, place on a vortex shaker, vortex for 30 seconds, incubate at room temperature for 10 minutes, centrifuge briefly using a desktop centrifuge, place on a magnetic stand for 1 minute, and then aspirate all the supernatant;

[0190] (5) Transfer the supernatant to a 1.5 mL centrifuge tube containing 22.5 μL of 1 M Tris-HCl, pH 8.0 and mix well to obtain 122.5 μL of neurogenic exosomes.

[0191] Comparative Example 12:

[0192] Compared with Example 8, only Glycine-HCl was added in step (4) and the incubation time at room temperature was 0 min after vortexing on a vortex shaker for 30 seconds. The remaining steps were consistent with Example 8.

[0193] Comparative Example 13:

[0194] Compared with Example 8, only Glycine-HCl was added in step (4) and the incubation time at room temperature was 20 minutes after vortexing on a vortex shaker for 30 seconds. The other steps were consistent with Example 8.

[0195] Effect Example 6:

[0196] Transmission electron microscopy (TEM) was used to observe whether the neurogenic exosomes obtained in Example 8 and Comparative Examples 12 and 13 had intact morphology. Figure 11 shown. Figure 11 From left to right in the figure are Comparative Example 12, Example 8 and Example 13, which are transmission electron microscopy results at elution time of 0 min, 10 min and 20 min, respectively.

[0197] Figure 11 The results showed that incubation for 10 minutes during elution can ensure the sufficient elution of neurogenic exosomes without damaging the morphology of neurogenic exosomes by the acidic environment.

[0198] The above description is merely an example of the implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for isolating neurogenic exosomes from human plasma, characterized in that: The steps include: Step A: Take streptavidin magnetic beads, wash them with 0.005%-0.05% Tween 20-DPBS, and resuspend them with BSA-DPBS to obtain resuspension solution I; Step B, adding biotinylated NCAM antibody to resuspension I, incubating, washing, and resuspending to obtain resuspension II; Step C, resuspension II is mixed with total plasma exosomes and then incubated; Step D: remove the supernatant, wash, elute, and neutralize to obtain human plasma neurogenic exosomes.

2. The method for isolating neurogenic exosomes from human plasma according to claim 1, wherein: The mass fraction of Tween 20-DPBS used for washing the streptavidin magnetic beads in step A is 0.01%.

3. The method for isolating neurogenic exosomes from human plasma according to claim 1 or 2, wherein: Incubate at room temperature for 10 min during elution in step D.

4. The method for isolating neurogenic exosomes from human plasma according to claim 1 or 2, wherein: In step D, the solution was eluted with Glycine-HCl and neutralized with Tris-HCl.

5. The method for isolating neurogenic exosomes from human plasma according to claim 4, wherein: The concentration of the Glycine-HCl is 0.5 M and the pH value is 3.0, and / or the concentration of the Tris-HCl is 1 M and the pH value is 8.

0.

6. The method for isolating neurogenic exosomes from human plasma according to claim 1, wherein: In step B, biotinylated NCAM antibody was added and incubated at room temperature for 1 h.

7. The method for isolating neurogenic exosomes from human plasma according to claim 1, wherein: The resuspension II from step C was mixed with the total plasma exosomes and incubated at 4°C for 16-18 hours.

8. The method for isolating neurogenic exosomes from human plasma according to claim 1, wherein The total plasma exosomes in step C are prepared by the following method: Exosome precipitant was added to the plasma, and the mixture was mixed and incubated; centrifuged and the supernatant was removed; centrifuged again and the supernatant was removed; and the total exosomes in the plasma were obtained by resuspending with DPBS.

9. The method for isolating neurogenic exosomes from human plasma according to claim 8, wherein: The exosome precipitant contains PEG and NaCl. When the exosome precipitant is added to the plasma, the volume ratio of the plasma to the exosome precipitant is 5:

1.

10. The method for isolating neurogenic exosomes from human plasma according to claim 9, wherein: The PEG in the exosome precipitant is PEG6000 or PEG8000, and the mass fraction of the PEG6000 or PEG8000 is 30%. After the exosome precipitant is added, the NaCl concentration in the plasma is 0.3M-0.5M.

11. The method for isolating neurogenic exosomes from human plasma according to any one of claims 1-2 and 5-10, wherein: The amino acid sequence of the antigenic determinant of the biotinylated NCAM antibody is shown in Seq ID NO:

4.

12. Human plasma neurogenic exosomes, prepared by the method for isolating human plasma neurogenic exosomes according to any one of claims 1 to 11.

13. A method for isolating total exosomes from human plasma, characterized in that: An exosome precipitant is added to the plasma, followed by mixing and incubation; centrifugation, removal of the supernatant; re-centrifugation, removal of the supernatant; and resuspending with DPBS to obtain total plasma exosomes. The exosome precipitant contains PEG and NaCl. When the exosome precipitant is added to the plasma, the volume ratio of the plasma to the exosome precipitant is 5:

1.

14. The method for isolating total exosomes from human plasma according to claim 13, wherein: The PEG in the exosome precipitant is PEG6000 or PEG8000, and the mass fraction of the PEG6000 or PEG8000 is 30%. After the exosome precipitant is added, the NaCl concentration in the plasma is 0.3M-0.5M.

15. A human plasma total exosome, characterized in that It is prepared by the method for isolating total exosomes from human plasma according to any one of claims 13 or 14.

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