CN112831457A

CN112831457A - Method for separating and concentrating exosome

Info

Publication number: CN112831457A
Application number: CN202110178197.8A
Authority: CN
Inventors: 李萍; 肖木亮; 高秀春; 王绍成
Original assignee: Liaoning Rengen Biosciences Co ltd
Current assignee: Liaoning Rengen Biosciences Co ltd
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2021-05-25

Abstract

The invention belongs to the technical field of biological detection, and relates to a method for quickly separating and concentrating exosomes. And separating and obtaining the exosome particles from the sample by combining the anion exchange resin with the exosomes in the sample. The method disclosed by the invention is simple to operate, short in time, low in cost and good in repeatability, can be used for quickly concentrating exosomes in a sample into a small volume, can be generally concentrated by 20-40 times, and hardly loses exosome particles. The method is convenient for extracting exosomes in a large-volume low-concentration sample, and is suitable for particle size analysis, electron microscope detection, Western blot, exosome DNA and RNA extraction and other related downstream experiments.

Description

Method for separating and concentrating exosome

Technical Field

The invention belongs to the technical field of biological detection, and relates to a method for quickly separating and concentrating exosomes.

Background

The exosome is a micro vesicle which is secreted by a living cell and contains abundant proteins, nucleic acids and lipid bilayer structures, and the diameter of the exosome is 30-150 nm. Exosomes are found in a wide range of body fluids, such as urine, ascites, blood, cell culture fluid, cerebrospinal fluid, and the like. Exosomes are capable of delivering functional molecules, including proteins, lipids and nucleic acids, to recipient cells, participate in intercellular communication, and affect various physiological and pathological functions of the cells, so that attention of many researchers is paid, and it is important to separate exosomes of a certain concentration before intensive exosome research.

The ultracentrifugation method for separating exosomes in the prior art is the most common method at present, is widely applied to separation of samples such as serum, plasma, cell culture fluid, urine and the like, is also the gold standard in the current separation method, can be used for extraction of exosomes in large-volume and low-concentration samples, but wastes time and labor, is easy to destroy the structure and function of exosomes, and has low recovery rate. The density gradient centrifugation method is a method for obtaining exosomes according to the density of exosomes by utilizing the continuous distribution of media with different densities from low to high to form a density gradient layer under the condition of long-time ultracentrifugal force, but the operation is complicated, the repeatability is poor, and the method needs to be provided with various expensive ultrahigh-speed centrifuges and is not suitable for extracting exosomes in large batch. The ultrafiltration method is to separate exosomes in a sample by using ultrafiltration membranes with different pore diameters to obtain exosomes, and the method is high in cost, suitable for concentrating exosomes in a small-volume sample, and not beneficial to extraction of exosomes in a large-volume low-concentration sample. The polymer precipitation method is characterized in that a high molecular polymer is used for competing for water around an exosome, so that the exosome is aggregated, the exosome concentration of a large-volume and low-concentration sample can be carried out, the operation is simple and convenient, the cost is low, but the impurity-containing protein is more, the polymer which is difficult to remove is generated, and the analysis of a downstream experiment is influenced. The immune affinity method is to realize the enrichment of exosomes by utilizing the specific interaction between corresponding antibodies and exosome membrane proteins, but the magnetic beads and the antibodies in the method are expensive and have harsh storage conditions, and the method is not beneficial to the extraction of exosomes in a large-volume and low-concentration sample. The size exclusion method is to separate the exosomes by using the particle sizes, can remove a part of protein components with small particles, has good separation and purification effects, and is not suitable for extracting the exosomes in a large-volume sample. The ion exchange chromatography uses ion exchange resin as filler to fill a chromatographic column, protein is separated according to the difference of charges carried by the protein, after the sample is added, the protein adsorbed on the column is eluted by adjusting different salt concentrations of a mobile phase, and fractions of the eluent are collected to play a good purification role.

Therefore, there is an urgent need to develop a method for separating and concentrating exosomes with simple operation, low cost, stability and high efficiency aiming at cell culture supernatant or organism liquid sample.

Disclosure of Invention

The invention aims to solve the technical problems of low exosome content, large sample volume, complex sample components and difficult exosome extraction of the existing samples such as cell culture supernatant or biological body fluid, and provides a novel method for quickly separating and concentrating exosomes, namely a method for extracting exosomes by using anion exchange resin without filling chromatographic columns.

In order to achieve the purpose, the technical scheme implemented by the invention is as follows:

a method of isolating and concentrating exosomes:

s1, adding a binding buffer solution into a sample containing an exosome;

s2, adding a binding resin into the system to enable the binding resin to be bound with exosomes in a sample;

s3, washing the bonding resin by using a washing solution to remove impure proteins;

and S4, eluting the combined resin by using an eluent containing a high-concentration salt solution to obtain exosomes in the sample.

The sample is cell culture supernatant or biological fluid; wherein, when the sample is cell culture supernatant, the cell culture supernatant needs 1500g, centrifugates for 15min at 4 ℃, abandons the precipitate, and the supernatant passes through a 0.22 μm filter membrane for standby;

when the sample is biological fluid, the biological fluid is needed to be centrifuged for 15min at 4 ℃ at 3000g, the precipitate is discarded, and the supernatant is filtered through a 0.22 mu m filter membrane for later use.

The volume ratio of the binding buffer solution to the sample is 1/25-1/5; wherein the binding buffer solution can be Tris buffer solution or phosphate buffer solution or MOPES buffer solution with the pH value of 7.0-8.5.

When the binding buffer solution is a Tris buffer solution, the concentration of Tris is 50-500 mM, and the pH value can be adjusted to 7.0-8.5 by HCl;

when the binding buffer solution is phosphate buffer solution, the concentration of NaCl is 20-100 mM, the concentration of KCl is 0.5-6 mM, and Na₂HPO₄The concentration of KH is 1-10 mM₂PO₄The concentration is 0.5-20 mM, and the pH is 7.0-8.5;

when the binding buffer solution is an MOPES buffer solution, the MOPES concentration is 10-100 mM, the sodium acetate concentration is 5-100 mM, the EDTA concentration is 5-100 mM, and the pH value is 7.0-8.5.

And the binding buffer solution and the sample are uniformly mixed and then incubated for 5-20 min.

The volume ratio of the binding resin to the sample is 1/50-1/10; wherein the binding resin is a weak anion exchange resin or a strong anion exchange resin, so that the binding resin is combined with the exosome in the sample.

The resin takes polyacrylate or agarose or cellulose or polystyrene as a matrix, and the matrix is prepared by reacting- (CH)₂)₂N(CH₃)₂Modifying to obtain weak anion exchange resin; or matrix through- (CH)₂)₂N⁺(CH₃)₃Modifying to obtain the strong anion exchange resin.

And (S2) after the binding resin and the sample system are uniformly mixed, incubating at room temperature for 10-60 min, and centrifuging at 1500-3000 g for 2-5 min.

The washing solution is HEPES buffer solution or phosphate buffer solution or Tris buffer solution with the pH value of 6.8-8.3.

When the washing solution is HEPES buffer solution, the concentration of HEPES is 50-500 mM, and the pH value is 6.8-8.3;

when the washing solution is phosphate buffer solution, the concentration of NaCl is 20-100 mM, the concentration of KCl is 0.5-6 mM, and Na₂HPO₄The concentration of KH is 1-10 mM₂PO₄The concentration is 0.5-10 mM, and the pH is 6.8-8.3;

when the washing solution is Tris buffer solution, the concentration of Tris is 50-500 mM, and the pH value can be adjusted to 6.8-8.3 by HCl.

And in the step S3, adding the washing solution into the purification column, incubating for 1-5 min, centrifuging for 2-5 min at 1500-3000 g, and discarding the filtrate.

The eluent is HEPES buffer solution or phosphate buffer solution or Tris buffer solution with the pH value of 6.3-7.4 and containing high-concentration NaCl.

When the eluent is HEPES buffer solution with high-concentration NaCl, the concentration of NaCl is 100-700 mM, the concentration of HEPES is 50-500 mM, and the pH value is 6.3-7.4;

when the eluent is phosphate buffer solution of high-concentration NaCl, the concentration of NaCl is 100-700 mM, the concentration of KCl is 0.5-10 mM, and Na is added₂HPO₄The concentration of KH is 1-20 mM₂PO₄The concentration is 0.5-10 mM, and the pH is 6.3-7.4;

when the eluent is Tris buffer solution with high concentration of NaCl, the concentration of NaCl is 100-700 mM, the concentration of Tris is 50-500 mM, and the pH value can be adjusted to 6.3-7.4 by HCl.

And S4, adding the eluent into a purification column, incubating for 3-7 min, centrifuging for 2-5 min at 200-500 g, adding the filtrate into the purification column again, incubating for 3-7 min, and centrifuging for 2-5 min at 1500-3000 g.

The invention can have the following beneficial effects:

the method can concentrate the exosomes in the cell culture supernatant or the biological body fluid with very low exosome content into small volume, has simple operation, low cost, short time consumption and stable result, and the obtained exosome concentrated solution contains salt ions, has no pollution of polymers, and is time-saving and labor-saving.

Drawings

FIG. 1 is a graph showing a comparison of ELISA tests for each component in the isolation and concentration of exosomes from cell culture according to the present invention.

FIG. 2 is a Western Blot identification of exosomes isolated and concentrated from cell culture fluid according to the present invention.

FIG. 3 is a transmission electron microscopy identification of exosomes isolated and concentrated from cell culture fluid according to the present invention; 3-1HEK293T, 3-2 Lncap; FIGS. 3-3 BALL.

FIG. 4 is a NTA identification chart of the isolation and concentration of exosomes from cell culture fluid according to the present invention; FIG. 4-1HEK293T, FIG. 4-2Lncap, FIG. 4-3 BALL.

FIG. 5 is a comparison of ELISA tests for each component in the isolation and concentration of exosomes from urine samples according to the present invention.

FIG. 6 is a Western Blot identification of exosomes isolated and concentrated from urine samples according to the present invention.

FIG. 7 is a transmission electron microscopy identification of exosomes isolated and concentrated from urine samples according to the present invention; urine A in FIG. 7-1, and urine B in FIG. 7-2.

FIG. 8 is a graph of NTA identification of exosomes isolated and concentrated from a urine sample according to the present invention; urine A in FIG. 8-1 and urine B in FIG. 8-2.

Detailed description of the preferred embodiment

The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The ion exchange resin used in the invention is anion exchange resin, which takes polymer (agarose and cellulose) as a matrix, the matrix is bonded with ion exchange groups after surface hydrophilic modification, and simultaneously has good compatibility to bioactive macromolecules and excellent physical and chemical stability, thereby greatly improving the purification efficiency. The ion exchanger is mainly composed of a high molecular polymer matrix, a charge group and a balance ion. The charged group is covalently bonded to the high molecular weight polymer to form a charged ion-exchangeable group. Counterions are counterions bound to charged groups that can undergo reversible exchange reactions with other ionic groups in solution. The action of anion exchangers is that the ion exchanger which balances the negative charge of the ion is able to exchange with the negatively charged ionic groups.

The invention utilizes the ion exchange function of anion exchange resin, namely, the exosome in the sample is regulated by the binding buffer solution to make the protein on the outer membrane of the exosome carry negative charges and be combined with the anion exchange resin with positive charges, and because the outer membrane of the exosome contains abundant proteins, the combination sites of the exosome and the anion exchange resin are more than that of a single free protein, and the combination is firmer. During washing, the salt concentration and pH of the washing solution are adjusted to remove most of free protein, the free protein is easier to remove because the binding sites of the free protein and the anion exchange resin are few, and then the elution is carried out by the eluent containing high-concentration salt solution, so that a large amount of exosomes are obtained from the sample.

The reagent and equipment adopted by the invention are conventional reagents and equipment in the technical field, and the ELISA detection kit is a human total exosome capture and quantification kit (cell culture supernatant) and a human total exosome capture and quantification kit (urine) purchased from Liaoning Runzi Biotechnology Limited. The reagents, materials and equipment used in the following examples are commercially available.

Example 1

Method for separating and concentrating exosomes from cell culture solution

The culture supernatants of three cell lines HEK293T (DMEM)/Lncap (RPM1640)/BALL (RPM1640) were subjected to exosome extraction and identified.

(1) 10ml of different cell culture solutions (HEK293T/Lncap/BALL) were centrifuged at 1500g for 10min at 4 ℃ to remove suspended cells and cell debris, the supernatant was filtered through a 0.22 μm pore size filter to remove larger microvesicles, and 5ml of the cell culture solutions were pipetted into different 10ml centrifuge tubes (noted as HEK293T, Lncap, BALL, respectively).

(2) Pipette 500. mu.l of binding buffer into the centrifuge tube containing the culture medium in (1), cover tightly, and mix by inversion. Wherein the binding buffer is 200mM Tris-HCl buffer (pH8.0).

(3) Adding 300 μ l of the binding resin into the centrifuge tube in (2), covering the cap tightly, mixing by continuous inversion at room temperature, incubating for 15min, centrifuging at 1500g for 2min, taking out the centrifuge tube gently, taking 500 μ l of supernatant (without discarding) with a pipette, carefully pouring off the remaining supernatant,the resin was gently blown up with liquid (500. mu.l) in a pipette, transferred all to a purification column (collection tube already placed), left to stand for 2min, centrifuged at 2,000g at room temperature for 2min, and the filtrate and collection tube were discarded. Wherein the binding resin is agarose as matrix and has a chemical structure of- (CH)₂)₂N(CH₃)₂) The anion exchange resin of (a) may be prepared as described in the prior art or may be obtained from sigma.

(4) Sucking 500 μ l of washing solution, adding into a purification column, standing for 5min, centrifuging at 2000g room temperature for 2min, and discarding the filtrate and collection tube. Wherein the washing solution is 68mM HEPES buffer solution, and the pH value is 7.4.

(5) Transferring the purification column into a 1.5ml centrifuge tube with low protein adsorption, adding 200 mul of eluent, incubating for 5min, centrifuging at 350g for 2min at room temperature, transferring the eluent into the purification column again, incubating for 5min, centrifuging at 2500g at room temperature for 3min, and obtaining the liquid in the centrifuge tube, namely the concentrated exosome solution. Wherein the eluent is 450mM NaCl, 5.6mM KCl, 5.2mM Na₂HPO₄，6mM KH₂PO₄Phosphate buffer, pH 6.5.

(6) Exosome ELISA quantitative detection

The presence of exosomes in each sample was verified by ELISA (total exosome capture and quantification kit for human, product number RGEXOC96-1) (reading see table 1, results figure 1) with PBS as negative control.

TABLE 1

	HEK293T	Lncap	BALL
				Stock solution (OD)₄₅₀Value)	0.625	0.745	0.543
Filtrate (OD)₄₅₀Value)	0.063	0.065	0.059
				Cleaning solution (OD)₄₅₀Value)	0.078	0.065	0.064
Eluent (OD)₄₅₀Value)	1.542	1.643	1.401

Negative control OD detected by ELISA experiment₄₅₀0.057, as can be seen from Table 1, the OD of the filtrate of the cell culture fluid sample HEK293T/Lncap/BALL₄₅₀0.063, 0.065, 0.059, respectively, which is substantially identical to the negative control. That is, adding 300. mu.l of the binding resin to a 5ml sample of the cell culture solution can capture almost all of the exosomes in the sample. Wash OD of sample HEK293T/Lncap/BALL₄₅₀0.078, 0.065 and 0.064, respectively, indicating that the exosomes were hardly lost during washing. As can be seen from FIG. 1, the values of the eluates for separating and concentrating the three cell culture solutions are significantly higher than those of the stock solutions, indicating that the eluates contain a large amount of exosomes, which can be concentrated about 25-fold with respect to the volume of the stock solutions.

(7) BCA protein quantitative determination

The protein content in each wash and eluate was determined by BCA protein quantification assay (see table 2) with PBS as negative control.

TABLE 2

	HEK293T	Lncap	BALL
				Stock solution (mg/ml)	2.145	2.512	2.758
Filtrate (mg/ml)	1.825	2.143	2.300
				Washing liquid (mg/ml)	0.212	0.220	0.312
Eluent (mg/ml)	1.848	1.924	2.056

As shown in Table 2, the binding resin was rarely bound to the free protein in each cell culture fluid sample, and 300. mu.l of the binding resin was bound to about 0.3-0.5 mg/ml of the protein in 5ml of the cell culture fluid sample, indicating that the positively charged protein in the sample was not bound to the binding resin, i.e., the free protein in the sample was selectively bound to the binding resin.

As is clear from tables 1 and 2, the protein contained in the outer membrane of the exosome was more strongly bound to the bound resin than the free protein, and the partially free protein on the resin was removed by washing with the washing solution, but the exosome was hardly lost.

(8) Western Blot detection

The exosome solution obtained after elution in step (5) was validated by Western Blot experiment (see fig. 2).

Electrophoresis was run with 10% SDS-PAGE gel:

preparation of 10% SDS-PAGE separation gel:

composition (I)	Volume of	Unit of
			H₂O	4	ml
30％Acr/Bise	3.3	ml
			1.5M Tris-HCL pH8.8	2.5	ml
10％SDS	100	μl
			10％AP	100	μl
TEMED
		10	μl

Preparation of 10% SDS-PAGE concentrated gel:

composition (I)	Volume of	Unit of
			H₂O	1.53	ml
30％Acr/Bise	0.33	ml
			0.5M Tris-HCL pH6.8	0.63	ml
10％SDS	25	μl
			10％AP	30	μl
TEMED	3	μl

Sample loading pretreatment:

protein quantification was performed separately on each eluate from step (4) using the BCA protein assay: detecting to obtain the protein concentration of the BALL eluent as 2.4 mg/ml; the protein concentration of the HEK293T eluate was 3.1 mg/ml; the Lncap eluate had a protein concentration of 2.9 mg/ml.

And (3) respectively sucking 20 mu l of each eluent in the step (4) into a 0.5mL centrifuge tube, adding 5 mu l of 5 Xloading buffer solution to a final concentration of 1X, wherein 25 mu l of the buffer solution is used totally, before loading, heating the sample at 99 ℃ for 10min to denature the protein, and after cooling to room temperature, respectively taking 20 mu l of the sample for electrophoresis loading.

The electrophoresis time is about 1.5h generally, the voltage of the concentrated gel is 80V and the constant voltage is 30min, and the voltage of the separation gel is 120V and the constant voltage is 40-50 min. The electrophoresis was terminated as soon as bromophenol blue (approx. 20kDa) came out.

Film transfer: wet transfer was performed using PVDF membrane, 250mM constant current transfer for 1.5 h.

And (3) sealing: PVDF membrane was soaked in 4% skimmed milk powder (TBST lysis) and incubated on a destaining shaker for 1h at room temperature or overnight at 4 ℃.

Washing: the PVDF membrane was washed 3 times with TBST, 8 min/time.

Primary antibody incubation: primary anti-antibody CD9 was solubilized with 4% BSA (TBST) at 1: 500(v/v) dilution; CD63 was diluted 1:1000(v/v) with 4% BSA (TBST lysis); ALIX was diluted 1:400(v/v) with 4% BSA (TBST lysis). Incubation was performed at room temperature for 2h or at 4 ℃ overnight.

Washing: the PVDF membrane was washed 3 times with TBST, 8 min/time.

And (3) secondary antibody incubation: the secondary HRP-labeled goat anti-mouse was diluted 1:3000(v/v) with 4% skim milk powder (TBST solubilized) and incubated at room temperature for 1 h.

Washing: the PVDF membrane was washed 3 times with TBST, 8 min/time.

Color development: ECL luminescence color development, with gel imaging system analysis of target band molecular weight.

As shown in FIG. 2, the exosomes isolated and concentrated from the HEK293T/Lncap/BALL three cell culture solutions were verified by Western Blot, and ALIX/CD9/CD63 bands were clearly visible and deep.

(9) Transmission Electron Microscopy (TEM) detection

Exosomes were removed in 5 μ L and diluted to 10 μ L. Sucking 10 mu L of a sample, dropwise adding the sample on a copper net for incubation for 3min, sucking floating liquid by using filter paper, dropwise adding 10 mu L of uranyl acetate on the copper net for incubation for 1min, sucking the floating liquid by using the filter paper, drying for several minutes at normal temperature, and performing electron microscope detection imaging at 100 kv. Exosomes obtained from the three culture fluids (HEK293T/Lncap/BALL) by the method are detected by an electron microscope (see figure 3), and the exosomes of each sample are complete in structure and are like a saucer as can be seen from figure 3-1HEK293T, figure 3-2Lncap, and figure 3-3 BALL.

(10) Nano particle size detection

Nanoparticle Tracking Analyzer (NTA) detection method: diluting the sample with water to obtain a particle concentration of 1 × 10⁷Perml and 1X 10⁹In the/mL range. The number and size of particles in the sample were then measured using a Zeta View PMX110 instrument at 405nm laser and photographs were taken at 30/sec for 1 minute. Finally the movement of the particles was analyzed using NTA software (zetaview8.02.28).

The exosomes obtained by this method were tested by NTA (see fig. 4), and the number of particles with a particle size of 109.2nm was 98.7% obtained from HEK293T of fig. 4-1, Lncap of fig. 4-2, and BALL of cell culture fluid HEK293T, and the result was 9.6E +10 particles/ml. The number of particles with an exosome particle size of 108.3nm obtained from the cell culture fluid Lncap was 98.4%, and the detection result was 9.4E +10 particles/ml. The number of particles with an exosome particle size of 123nm obtained from the cell culture fluid BALL was 99%, and the detection result was 2.6E +10 particles/ml.

The experiments prove that the invention is feasible and effective for separating and concentrating exosomes in various cell lines cultured by two common culture media of DMEM and RPM1640, and keeps the integrity and biological functions of the exosomes.

Example 2

The method for separating and concentrating the exosome is established, and the exosome is further extracted and identified from a human urine sample.

(1) 15ml of human urine A and urine B are respectively centrifuged at 4 ℃ and 3000g for 10min to remove cell debris and impurities, the supernatant is retained and filtered by a filter membrane with the aperture of 0.22 mu m to remove larger micro vesicles, and 5ml of each of the urine A and the urine B (respectively marked as samples: urine A and urine B) is respectively added into 2 10ml centrifuge tubes.

(2) Pipette 500. mu.l of binding buffer into the centrifuge tube containing the culture medium in (1), cover tightly, and mix by inversion. Wherein, when the binding buffer is MOPES buffer, the MOPES concentration is 15mM, the sodium acetate concentration is 20mM, the EDTA concentration is 15mM, and the pH value is 8.3.

(3) And (3) sucking 200 mu l of binding resin, adding the binding resin into the centrifuge tube in the step (2), covering the cover tightly, continuously reversing and mixing the binding resin evenly at room temperature, incubating for 15min, centrifuging for 2min at 1500g, taking the centrifuge tube out of the centrifuge lightly, taking 500 mu l of supernatant (not to be discarded) by using a pipette, carefully pouring out the residual supernatant, slightly blowing the resin by using liquid (500 mu l) in the pipette, completely transferring the resin into a purification column (placed into a collection tube), standing for 2min, centrifuging for 2min at 2,000g at room temperature, and discarding filtrate and the collection tube. Wherein the bonding resin is prepared from cellulose as matrix and has a chemical structure of- (CH)₂)₂N⁺(CH₃)₃The anion exchange resin of (a) may be prepared as described in the prior art or may be obtained from GE, USA.

(4) The 500. mu.l of washing solution was pipetted into a purification column, allowed to stand for 5min, centrifuged at 2,000g at room temperature for 2min, and the filtrate and collection tube were discarded together. Wherein the washing solution is 45mM Tris-HCl buffer solution, and the pH value is 7.02.

(5) Transferring the purification column into a 1.5ml centrifuge tube with low protein adsorption, adding 150 mu l of eluent, incubating for 5min, centrifuging at 350g for 2min at room temperature, transferring the eluent into the purification column again, incubating for 5min, centrifuging at 2500g at room temperature for 3min, and obtaining the liquid in the centrifuge tube, namely the concentrated exosome solution. Wherein the eluent is 375mM NaCl, 2.1mM KCl, 6.8mM Na₂HPO₄，3.1mMKH₂PO₄The pH of the phosphate buffer of (1) was 7.0.

(6) Exosome ELISA quantitative detection

The presence of exosomes in each sample was verified by ELISA (total exosome capture and quantification kit for human, product number RGEXOU96-1) (reading see table 3, results figure 5) with PBS as negative control.

TABLE 3

	Urine A	Urine B
			Stock solution (OD)₄₅₀Value)	0.645	0.723
Filtrate (OD)₄₅₀Value)	0.072	0.065
			Cleaning solution (OD)₄₅₀Value)	0.102	0.090
Eluent (OD)₄₅₀Value)	1.442	1.603

Negative control OD detected by ELISA experiment₄₅₀0.056, as can be seen from Table 3, the OD of the filtrate in urine samples A and B₄₅₀0.072 and 0.065, respectively, which is substantially consistent with the negative control, i.e., adding 200. mu.l of binding resin to a 5ml urine sample allowed almost complete capture of exosomes in the sample. Wash OD of urine samples A and B₄₅₀0.102 and 0.090, respectively, indicating that exosomes were hardly lost upon washing. As can be seen from fig. 5, the elution values of the urine samples a and B are significantly higher than the original solution, which indicates that the elution solution contains a large amount of exosomes, and the exosomes can be concentrated by about 25-30 times relative to the original solution volume.

(7) BCA protein quantitative determination

The protein content in each wash and eluate was determined by BCA protein quantification assay (see table 4) with PBS as negative control.

TABLE 4

	Urine A	Urine B
			Stock solution (protein mg/ml)	9.432	8.789
Filtrate (protein mg/ml)	8.661	8.056
			Washing liquid (protein mg/ml)	0.421	0.526
Eluent (protein mg/ml)	2.089	2.125

As shown in Table 4, the binding resin was rarely bound to the free protein in each sample of urine, and 200ul of the binding resin was bound to about 0.5-0.8 mg/ml of the protein in 5ml of the urine, indicating that the positively charged protein in the sample was not bound to the binding resin, i.e., the free protein in the sample was selectively bound to the binding resin.

As can be seen from tables 3 and 4, washing with the wash solution removed some of the free protein on the resin, but almost no exosomes were lost.

(8) Western Blot detection

The exosome solution obtained after elution in step (5) was validated by Western Blot experiment (see fig. 6).

The Western Blot experiment method and the operation steps are the same as above.

As shown in FIG. 6, the ALIX/CD9/CD63 bands of urine A and urine B were clearly visible and the bands were deep as confirmed by Western Blot.

(9) Transmission Electron Microscopy (TEM) detection

The structure of the exosomes obtained from urine a and urine B by this method was examined by Transmission Electron Microscopy (TEM) (see fig. 7), the operation method is the same as above, and the exosomes from fig. 7-1, urine a and urine B from fig. 7-2 were structurally complete and in the form of a saucer.

(10) Nano particle size detection

The exosomes obtained from urine a and urine B by this method were detected by a nanoparticle particle tracking analyzer (NTA) (see fig. 8), the operation was the same as above, and in fig. 8-1, urine a and urine B of fig. 8-2, it was found that the exosomes obtained from urine a had a particle size of 107.4nm, which accounted for 97.1% and a concentration of 1.3E +10particles/ml, respectively. Urine B obtained 96.5% of exosomes having a particle size of 107.5nm at a concentration of 1.3E +10 particles/ml.

The experiments prove that the method is feasible and effective for separating and concentrating exosomes in urine, and keeps the integrity and biological functions of exosomes.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method of isolating and concentrating exosomes, characterized by:

s1, adding a binding buffer solution into a sample containing an exosome;

s3, washing the bonding resin by using a washing liquid;

2. The method of claim 1, wherein: the sample is cell culture supernatant or biological fluid;

wherein, when the sample is cell culture supernatant, the cell culture supernatant needs 1500g, centrifugates for 15min at 4 ℃, abandons the precipitate, and the supernatant passes through a 0.22 μm filter membrane for standby;

3. The method of claim 1, wherein: the volume ratio of the binding buffer solution to the sample is 1/25-1/5; wherein the binding buffer solution is Tris buffer solution or phosphate buffer solution or MOPES buffer solution with the pH value of 7.0-8.5; wherein, when the binding buffer solution is a Tris buffer solution, the concentration of Tris is 50-500 mM, and the pH value can be adjusted to 7.0-8.5 by HCl; when the binding buffer solution is phosphate buffer solution, the concentration of NaCl is 20-100 mM, the concentration of KCl is 0.5-6 mM, and Na₂HPO₄The concentration of KH is 1-10 mM₂PO₄The concentration is 0.5-20 mM, and the pH is 7.0-8.5; when the binding buffer solution is an MOPES buffer solution, the MOPES concentration is 10-100 mM, the sodium acetate concentration is 5-100 mM, the EDTA concentration is 5-100 mM, and the pH value is 7.0-8.5.

4. A method according to claim 1 or 3, characterized in that: and the binding buffer solution and the sample are uniformly mixed and then incubated for 5-20 min.

5. The method of claim 1, wherein: the volume ratio of the binding resin to the sample is 1/50-1/10; wherein the binding resin is a weak anion exchange resin or a strong anion exchange resin; the resin takes polyacrylate or agarose or cellulose or polystyrene as a matrix, and the matrix is prepared by reacting- (CH)₂)₂N(CH₃)₂Or- (CH)₂)₂N⁺(CH₃)₃The modification results in a weak anion exchange resin or a strong anion exchange resin.

6. The method of claim 1 or 5, wherein: and (S2) after the binding resin and the sample system are uniformly mixed, incubating at room temperature for 10-60 min, and centrifuging at 1500-3000 g for 2-5 min.

7. The method of claim 1, wherein: the washing solution is HEPES buffer solution or phosphate buffer solution or Tris buffer solution with the pH value of 6.8-8.3; when the washing solution is an HEPES buffer solution, the concentration of the HEPES is 50-500 mM, and the pH value is 6.8-8.3; when the washing solution is phosphate buffer solution, the concentration of NaCl is 20-100 mM, the concentration of KCl is 0.5-6 mM, and Na₂HPO₄The concentration of KH is 1-10 mM₂PO₄The concentration is 0.5-10 mM, and the pH is 6.8-8.3; when the washing solution is Tris buffer solution, the concentration of Tris is 50-500 mM, and the pH value can be adjusted to 6.8-8.3 by HCl.

8. The method of claim 1 or 7, wherein: and in the step S3, adding the washing solution into the purification column, incubating for 1-5 min, centrifuging for 2-5 min at 1500-3000 g, and discarding the filtrate.

9. The method of claim 1, wherein: the eluent is HEPES buffer solution or phosphate buffer solution or Tris buffer solution with the pH value of 6.3-7.4 and containing high-concentration NaCl; when the eluent is HEPES buffer solution with high concentration of NaCl, the concentration of NaCl is 100-700 mM, the concentration of HEPES is 50-500 mM, and the pH value is 6.3-7.4;

when the eluent is phosphate buffer solution of high-concentration NaCl, the concentration of NaCl is 100-700 mM, the concentration of KCl is 0.5-10 mM, and Na is added₂HPO₄The concentration of KH is 1-20 mM₂PO₄The concentration is 0.5-10 mM, and the pH is 6.3-7.4; when the eluent is Tris buffer solution with high concentration of NaCl, the concentration of NaCl is 100-700 mM, the concentration of Tris is 50-500 mM, and the pH value can be adjusted to 6.3-7.4 by HCl.

10. The method of claim 1 or 9, wherein: and S4, adding the eluent into a purification column, incubating for 3-7 min, centrifuging for 2-5 min at 200-500 g, adding the filtrate into the purification column again, incubating for 3-7 min, and centrifuging for 2-5 min at 1500-3000 g.