CN117586952A - Separation reagent and separation method for separating plasma exosomes - Google Patents
Separation reagent and separation method for separating plasma exosomes Download PDFInfo
- Publication number
- CN117586952A CN117586952A CN202410083497.1A CN202410083497A CN117586952A CN 117586952 A CN117586952 A CN 117586952A CN 202410083497 A CN202410083497 A CN 202410083497A CN 117586952 A CN117586952 A CN 117586952A
- Authority
- CN
- China
- Prior art keywords
- separation
- plasma
- polyethylene glycol
- reagent
- separating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 85
- 238000000926 separation method Methods 0.000 title claims abstract description 72
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 46
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 31
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 22
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 22
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims description 21
- 238000005119 centrifugation Methods 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 11
- 108090000190 Thrombin Proteins 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 229960004072 thrombin Drugs 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 239000001166 ammonium sulphate Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- 230000001376 precipitating effect Effects 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 10
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 4
- 229920001477 hydrophilic polymer Polymers 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 70
- 230000000052 comparative effect Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 17
- 239000002245 particle Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 11
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biology, in particular to a separation reagent and a separation method for separating plasma exosomes. The separating agent comprises polyethylene glycol, ammonium sulfate and water. The separation reagent adopts the hydrophilic polymer polyethylene glycol to be matched with inorganic ammonium sulfate, so that exosomes are separated out from the plasma sample, the total exosomes in the purified plasma can be separated and separated in 1-2 hours, high-efficiency separation is realized, special centrifugal equipment is not needed, the separation reagent has the characteristics of high recovery rate, high purity and low price, can meet scientific research and clinical detection application, and has the same effect as the international common plasma exosome separation reagent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a separation reagent and a separation method for separating plasma exosomes.
Background
The exosomes are generally 50-150nm in diameter and have a double-layer membrane structure, and are widely used in various body fluid samples such as blood plasma, cerebrospinal fluid and urine. The exosome has small volume, stable structure and rich content, and has wide application prospect in clinical diagnosis, scientific research and other aspects.
In the related art, an ultracentrifugation or kit separation method is generally adopted to separate the plasma exosome sample, the method generally adopts an ultrahigh rotating speed of more than 100000g, the centrifugal equipment is expensive, the separation time is more than 10 hours, and the kit for separating the exosome of SBI company is expensive and cannot be widely applied.
At present, the technology for separating the plasma exosomes is not mature, and the common separation method has the problems of long time consumption, high reagent cost and the like, thus preventing the exosomes from being applied to scientific research and clinical diagnosis.
Disclosure of Invention
In order to solve the technical problems, the invention provides a separating reagent and a separating method for separating plasma exosomes, wherein the separating reagent adopts polyethylene glycol (PEG) to be matched with inorganic ammonium sulfate salt, so that exosomes are separated out from a plasma sample, the total exosomes in the purified plasma can be separated in 1-2h, and special centrifugal equipment is not needed.
According to a first aspect of the present invention there is provided a separation reagent for separating plasma exosomes comprising polyethylene glycol, ammonium sulphate and water.
In the scheme, the separating agent for separating the plasma exosomes comprises polyethylene glycol, ammonium sulfate and water, wherein the polyethylene glycol belongs to hydrophilic macromolecular substances, can compete with exosome surface proteins for water molecules, and can further separate the exosomes out by the rejection action of exosomes and the salting-out action generated by the ammonium sulfate with higher concentration, so that the total exosomes in the purified plasma can be separated and separated within 1-2 hours, high-efficiency separation is realized, special centrifugal equipment is not needed, and the separating agent has the characteristics of high recovery rate, high purity and low price, can meet scientific research and clinical detection application, and has the effect equivalent to that of the international common separating agent for the plasma exosomes.
Further, the separation reagent comprises 10% -18% polyethylene glycol, 1% -4% ammonium sulfate and 80% -88% water by weight percent.
In the scheme, the dosage of polyethylene glycol, ammonium sulfate and water in the separation reagent is limited within a reasonable range value, so that each component can play a better synergistic effect, and a better separation effect is achieved. Particularly, when the amount of ammonium sulfate in the separating agent is less than 1%, the separating effect is poor, and when the amount of ammonium sulfate in the separating agent is more than 4%, the ammonium sulfate is not well dissolved in a solution formed by polyethylene glycol and water.
To achieve a better separation effect, further, the separation reagent comprises 15% polyethylene glycol, 2% ammonium sulfate and 83% water by weight.
Further, the polyethylene glycol has a molecular weight of 5000-8000 daltons.
Further, the polyethylene glycol has a molecular weight of 6000 daltons.
Further, the separating agent is obtained by mixing polyethylene glycol, ammonium sulfate and water at room temperature for 10min-15min and filtering with a 0.22 μm filter membrane.
According to a second aspect of the present invention, there is also provided a method of separating plasma exosomes, comprising the steps of:
step S1: centrifuging the plasma sample for the first time, collecting supernatant, and discarding precipitate;
step S2: adding thrombin into the supernatant obtained in the step S1, flicking and uniformly mixing, performing secondary centrifugation after room temperature treatment for a period of time, collecting the supernatant, and discarding the precipitate;
step S3: adding the separating reagent into the supernatant obtained in the step S2, mixing the mixture upside down, precipitating the mixture on ice, centrifuging the mixture for the third time, and discarding the supernatant to retain the precipitate;
step S4: and (3) fully dissolving the precipitate obtained in the step (S3) by using PBS to obtain the total exosome solution of the plasma.
The room temperature is generally 10 to 30 ℃.
In the scheme, the separation method for separating the plasma exosome comprises the steps of firstly carrying out first centrifugation pretreatment on a plasma sample to obtain a plasma sample with cells and cell fragments removed, then adding thrombin to treat the plasma sample to remove fibrin in the plasma sample to obtain a treated plasma sample solution, then adding the separation reagent provided by the invention, mixing the mixture upside down, precipitating on ice, centrifuging, and removing other plasma components to obtain the plasma total exosome, wherein the total plasma exosome can be obtained in the whole process of 1-2 hours. The separation method provided by the invention does not need expensive treatment equipment, can efficiently extract the plasma exosome sample, has the characteristics of high recovery rate, high purity and low cost, can meet scientific research and clinical detection application, and is an ideal purification method.
Further, in the step S1, the first centrifugation is performed at 5000g-7000g for 15min-30min at 3-5 ℃.
In the scheme, the temperature, the centrifugal force and the centrifugal time of the first centrifugation are limited in a reasonable range value, so that cell fragments, apoptotic bodies and the like in the plasma sample can be separated more easily, and the separation effect can be improved.
Further, in the step S2, the volume ratio of the supernatant obtained in the step S1 to thrombin is (100-150): 1; preferably 125:1;
and/or thrombin at a concentration of 600U/mL-650U/mL; preferably 611U/mL;
and/or, the second centrifugation is performed for 3-8min at 10000g-15000g under the condition of 3-5 ℃.
In the above scheme, the volume ratio of the supernatant obtained in the step S1 to thrombin, the concentration of thrombin and the secondary centrifugation condition are limited in a reasonable range, so that the removal of fibrin in the blood plasma is facilitated, and the improvement of the separation effect is facilitated.
Further, in step S3, the volume ratio of the supernatant obtained in step S2 to the separation reagent is 1:1;
and/or the number of upside down is 5-10; preferably 8 times.
And/or the temperature of the ice precipitation is-4 ℃ to 4 ℃ and the time is 20min to 40min;
and/or, the third centrifugation is at 1000g-2000g for 3min-8min at 3-5 ℃.
In the above scheme, the volume ratio of the supernatant obtained in the step S2 to the separating reagent, the times of upside down, the temperature and time of ice precipitation and the third centrifugation condition are limited in a reasonable range, so that the removal of other plasma components and the improvement of the separation effect are facilitated.
The technical scheme provided by the invention has the following beneficial effects:
the separating agent for separating the plasma exosomes comprises polyethylene glycol, ammonium sulfate and water, and the hydrophilic polymer polyethylene glycol is matched with inorganic salt ammonium sulfate, so that the exosomes are separated out from a plasma sample, the total exosomes in the purified plasma can be separated and purified within 1-2 hours, high-efficiency separation is realized, special centrifugal equipment is not needed, and the separating agent has the characteristics of high recovery rate, high purity and low price, can meet scientific research and clinical detection application, and has the effect equivalent to that of the international common separating agent for the plasma exosomes.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of a separation method for separating plasma exosomes according to example 4 of the present invention;
FIG. 2 is a graph showing the particle size distribution of the total plasma exosomes obtained in example 4 of the present invention;
FIG. 3 is a graph showing the particle size distribution of the total plasma exosomes obtained in example 5 of the present invention;
FIG. 4 is a graph showing the particle size distribution of the total plasma exosomes obtained in example 6 of the present invention;
FIG. 5 is a graph showing the particle size distribution of the total plasma exosomes obtained in comparative example 1 of the present invention;
FIG. 6 is a graph showing the particle size distribution of the total plasma exosomes obtained in comparative example 2 of the present invention;
FIG. 7 is a graph showing the particle size distribution of the total plasma exosomes obtained in comparative example 3 of the present invention;
FIG. 8 is a graph showing the comparison of the surface marker test results of the total plasma exosomes obtained in examples 4, 5, 6 and comparative example 1 of the present invention;
FIG. 9 is a graph showing the comparison of the surface marker test results of the total plasma exosomes obtained in example 4 and comparative examples 1 and 2 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
Example 1
The present example provides a separation reagent for separating plasma exosomes, consisting of 15g polyethylene glycol, 2g ammonium sulphate and 83g deionized water, the molecular weight of the polyethylene glycol being 6000 daltons. The separating reagent is obtained by mixing polyethylene glycol, ammonium sulfate and water at room temperature for 15min and filtering with a 0.22 μm filter membrane.
Example 2
This example provides a separation reagent for separating plasma exosomes, which differs from example 1 in that it consists of 15g polyethylene glycol, 1g ammonium sulphate and 84g deionized water.
Example 3
This example provides a separation reagent for separating plasma exosomes, which differs from example 1 in that it consists of 15g polyethylene glycol, 4g ammonium sulphate and 81g deionized water.
Example 4
The embodiment provides a separation method for separating plasma exosomes, the flow chart of which is shown in fig. 1, comprising the following steps:
step S1: collecting peripheral blood samples of 1 volunteer by using EDTA anticoagulation blood collection tube, and preserving at 4 ℃; after removal, the human plasma sample was centrifuged at 6000g for 30min at 4℃to leave a supernatant, and the pellet was discarded.
Step S2: adding 2ul thrombin with concentration of 611U/mL into 250ul supernatant obtained in step S1, flicking, mixing, treating at room temperature for 5min, centrifuging at 12000g at 4deg.C for 5min, collecting supernatant, and discarding precipitate.
Step S3: adding the separating reagent of the example 1 into the supernatant obtained in the step S2 according to the volume ratio of 1:1, uniformly mixing the mixture upside down for 8 times, precipitating the mixture on ice at the temperature of 0 ℃ for 30min, centrifuging the mixture at 1500g for 5min at the temperature of 4 ℃, and discarding the supernatant to keep the precipitate.
Step S4: and (3) fully dissolving the precipitate obtained in the step (S3) by using 1mLPBS buffer solution to obtain the total exosome solution of the plasma.
Example 5
The present example provides a separation method for separating plasma exosomes, which differs from example 4 in that the separation reagent of example 2 is used in step S3.
Example 6
The present example provides a separation method for separating plasma exosomes, which differs from example 4 in that the separation reagent of example 3 is used in step S3.
Comparative example 1
This comparative example provides a method for separating plasma exosomes, which differs from example 4 in that in step S3 a separation reagent of the model Exoquick from SBI is used.
Comparative example 2
This comparative example provides a separation method for separating plasma exosomes, which is different from example 4 in that the separation reagent used in step S3, specifically, the separation reagent comprises 15g polyethylene glycol and 85g deionized water, and the molecular weight of the polyethylene glycol is 6000 daltons.
Comparative example 3
This comparative example provides a separation method for separating plasma exosomes, which is different from example 4 in that the salt ion in the solution is sodium chloride, and the separation reagent used in step S3 is different from that in the embodiment, specifically, the separation reagent includes 4g sodium chloride, 15g polyethylene glycol and 81g deionized water, and the molecular weight of the polyethylene glycol is 6000 daltons.
The total plasma exosome solutions obtained in examples 4 to 6 and comparative examples 1 to 3 were subjected to exosome particle size distribution testing using nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA).
As shown in FIG. 2, the results of the examination of the total plasma exosome solution obtained in example 4 showed that the exosome particle size range was between 60 and 300nm, with a peak at about 97nm at about 100 nm. As shown in FIG. 3, the results of the examination of the total plasma exosome solution obtained in example 5 showed that the exosome particle size ranges between 30 and 300nm, and that a plurality of peaks occurred at 90nm, 112nm, 166nm, etc. As shown in FIG. 4, the results of the examination of the total plasma exosome solution obtained in example 6 revealed that the exosome particle size ranges between 30 and 400nm, and that a plurality of peaks occurred at 31nm, 97nm, 140nm, 194nm, etc. As shown in FIG. 5, the results of the measurement of the total plasma exosome solution obtained in comparative example 1 showed that the exosome particle size range was between 60 and 320nm, and that a plurality of peaks appeared at 103nm, 186nm, etc., with the highest peak concentrated around 103 nm. As shown in FIG. 6, the results of the detection of the total plasma exosome solution obtained in comparative example 2 showed that the exosome particle size ranges between 29 and 250nm, a plurality of peaks at 86nm, 114nm, 158nm, etc., and the exosome concentration was low. As shown in FIG. 7, the results of the examination of the total plasma exosome solution obtained in comparative example 3 revealed that the exosome particle size range was between 60 and 450nm, and that a plurality of peaks appeared at 87nm, 135nm, 194nm, etc., with the highest peak concentrated around 87 nm.
As can be seen from the results of fig. 2 to 7, the particle size distribution of the total plasma exosomes obtained by combining the separation reagent according to the present invention with the separation method according to the present invention is uniform, concentrated, and highly uniform, and the purity of the exosomes obtained by separation is high, and especially the detection result of the total plasma exosomes solution according to example 4 of the present invention is close to the detection result of the total plasma exosomes solution according to comparative example 1, and the proportion of particles is high around 100nm, which indicates that the particle size distribution of the total plasma exosomes obtained by combining the separation reagent according to the present invention with the separation method according to the present invention is comparable to the international commonly used plasma exosomes separation reagent.
The total plasma exosome solutions obtained in examples 4 to 6 and comparative examples 1 to 2 were used for detecting the content of the exosome marker protein by Western blot. Table 1 shows the comparison of the gray scale analysis values of the surface markers of the total plasma exosomes obtained in examples 4, 5, 6 and comparative example 1, and Table 2 shows the comparison of the gray scale analysis values of the surface markers of the total plasma exosomes obtained in example 4 of the present invention and comparative examples 1, 2.
TABLE 1
TABLE 2
As can be seen from the results of FIG. 8 and Table 1, the expression level of the total plasma exosome markers obtained by the combination of the separation reagent of the present invention with the separation method of the present invention was similar to that of comparative example 1, indicating that the purity of the exosome obtained by the separation was high.
As can be seen from the results of FIG. 9 and Table 2, the expression level of the total plasma exosome markers obtained by the separation method of the present invention using the separation reagent of the present invention was close to that of comparative example 1 and higher than that of comparative example 2, indicating that the exosome obtained by the separation of the present invention was higher in purity.
The results from FIGS. 5, 6 and 2 show that the concentration of total plasma exosomes isolated by the separation reagent of the present invention in combination with the separation method of the present invention is higher than that of comparative examples 1 and 2, indicating that the separation effect of the present invention is better.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A separation reagent for separating plasma exosomes, comprising polyethylene glycol, ammonium sulphate and water.
2. The separation reagent of claim 1, wherein the separation reagent comprises, in weight percent, 10% -18% polyethylene glycol, 1% -4% ammonium sulfate, and 80% -88% water.
3. The separation reagent of claim 2, wherein the separation reagent comprises, in weight percent, 15% polyethylene glycol, 2% ammonium sulfate, and 83% water.
4. The separation reagent of claim 1, wherein the polyethylene glycol has a molecular weight of 5000-8000 daltons.
5. The separation reagent of claim 4, wherein the polyethylene glycol has a molecular weight of 6000 daltons.
6. The separating agent according to claim 1, wherein the separating agent is obtained by mixing polyethylene glycol, ammonium sulfate and water at room temperature for 10min to 15min and filtering with a 0.22 μm filter membrane.
7. A separation method for separating plasma exosomes, comprising the steps of:
step S1: centrifuging the plasma sample for the first time, collecting supernatant, and discarding precipitate;
step S2: adding thrombin into the supernatant obtained in the step S1, flicking and uniformly mixing, performing secondary centrifugation after room temperature treatment for a period of time, collecting the supernatant, and discarding the precipitate;
step S3: adding the separating reagent according to any one of claims 1-6 into the supernatant obtained in the step S2, mixing the mixture upside down, precipitating on ice, centrifuging for the third time, and discarding the supernatant to retain the precipitate;
step S4: and (3) fully dissolving the precipitate obtained in the step (S3) by using PBS to obtain the total exosome solution of the plasma.
8. The separation method according to claim 7, wherein in step S1, the first centrifugation is performed at 5000g to 7000g for 15min to 30min at 3℃to 5 ℃.
9. The method according to claim 7, wherein in step S2, the volume ratio of the supernatant obtained in step S1 to thrombin is (100-150): 1;
and/or thrombin at a concentration of 600U/mL-650U/mL;
and/or, the second centrifugation is performed for 3-8min at 10000g-15000g under the condition of 3-5 ℃.
10. The separation method according to claim 7, wherein in step S3, the volume ratio of the supernatant obtained in step S2 to the separation reagent is 1:1;
and/or the number of upside down is 5-10;
and/or the temperature of the ice precipitation is-4 ℃ to 4 ℃ and the time is 20min to 40min;
and/or, the third centrifugation is at 1000g-2000g for 3min-8min at 3-5 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410083497.1A CN117586952B (en) | 2024-01-19 | 2024-01-19 | Separation reagent and separation method for separating plasma exosomes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410083497.1A CN117586952B (en) | 2024-01-19 | 2024-01-19 | Separation reagent and separation method for separating plasma exosomes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117586952A true CN117586952A (en) | 2024-02-23 |
| CN117586952B CN117586952B (en) | 2024-04-26 |
Family
ID=89920632
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410083497.1A Active CN117586952B (en) | 2024-01-19 | 2024-01-19 | Separation reagent and separation method for separating plasma exosomes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117586952B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107532195A (en) * | 2015-03-31 | 2018-01-02 | 浦项工科大学校产学协力团 | The method that extracellular vesica is separated using double-aqueous phase system |
| CN109355258A (en) * | 2018-11-02 | 2019-02-19 | 北京恩泽康泰生物科技有限公司 | It is a kind of for extracting the extractant and its extracting method of extracellular vesica in body fluid |
| CN111148828A (en) * | 2017-07-26 | 2020-05-12 | 罗塞塔外排体株式会社 | Method for separating extracellular vesicles using cations |
| US20200188918A1 (en) * | 2018-12-18 | 2020-06-18 | Korea Institute Of Science And Technology | Novel method for isolating extracellular vesicles |
| CN115200969A (en) * | 2021-09-28 | 2022-10-18 | 武汉博越致和生物科技有限公司 | Method and kit for efficiently separating and purifying plasma exosomes |
| CN116590279A (en) * | 2023-04-26 | 2023-08-15 | 江苏康为世纪生物科技股份有限公司 | Kit for extracting plasma free DNA (deoxyribonucleic acid) based on hydroxyl magnetic beads and application method |
| CN116925163A (en) * | 2023-07-10 | 2023-10-24 | 海南玫瑰谷产业发展有限公司 | Method for obtaining rose glycoside by low-frequency ultrasound |
-
2024
- 2024-01-19 CN CN202410083497.1A patent/CN117586952B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107532195A (en) * | 2015-03-31 | 2018-01-02 | 浦项工科大学校产学协力团 | The method that extracellular vesica is separated using double-aqueous phase system |
| CN111148828A (en) * | 2017-07-26 | 2020-05-12 | 罗塞塔外排体株式会社 | Method for separating extracellular vesicles using cations |
| CN109355258A (en) * | 2018-11-02 | 2019-02-19 | 北京恩泽康泰生物科技有限公司 | It is a kind of for extracting the extractant and its extracting method of extracellular vesica in body fluid |
| US20200188918A1 (en) * | 2018-12-18 | 2020-06-18 | Korea Institute Of Science And Technology | Novel method for isolating extracellular vesicles |
| CN115200969A (en) * | 2021-09-28 | 2022-10-18 | 武汉博越致和生物科技有限公司 | Method and kit for efficiently separating and purifying plasma exosomes |
| CN116590279A (en) * | 2023-04-26 | 2023-08-15 | 江苏康为世纪生物科技股份有限公司 | Kit for extracting plasma free DNA (deoxyribonucleic acid) based on hydroxyl magnetic beads and application method |
| CN116925163A (en) * | 2023-07-10 | 2023-10-24 | 海南玫瑰谷产业发展有限公司 | Method for obtaining rose glycoside by low-frequency ultrasound |
Non-Patent Citations (1)
| Title |
|---|
| 高云龙等: ""硫酸铵-聚乙二醇双水相体系热力学研究"", 《高校化学工程学报》, vol. 4, no. 4, 31 December 1990 (1990-12-31), pages 291 - 301 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117586952B (en) | 2024-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022067774A1 (en) | Preparation method and application of sea cucumber polysaccharide | |
| CN112980765A (en) | Method for extracting high-purity centella asiatica exosome | |
| CN113214413A (en) | Sweet tea polysaccharide extract with high antioxidant activity and extraction method thereof | |
| CN117586952B (en) | Separation reagent and separation method for separating plasma exosomes | |
| CN112076207B (en) | High molecular weight cordyceps militaris polysaccharide, preparation method thereof and application of high molecular weight cordyceps militaris polysaccharide in preparation of anticomplement medicines | |
| CN110846295B (en) | Method for enriching potato acyl hydrolase based on functional nano magnetic beads | |
| CN113101737A (en) | Affinity tangential flow filtration system and construction method thereof, and exosome extraction method and application | |
| CN110964694A (en) | Method for extracting exosome based on density gradient centrifugation and ultracentrifugation | |
| CN108192892B (en) | Kit for extracting RNA by hydroxyl nano-magnetic bead method and extraction method | |
| CN115873786A (en) | Large-scale extraction method of exosomes in fresh milk | |
| CN107200774B (en) | Extraction and purification method of nostoc sphaeroides biliprotein and purified phycoerythrin | |
| Kirschner et al. | Modification of the 3H-leucine centrifugation method for determining bacterial protein synthesis in freshwater samples | |
| CN116376124A (en) | Method for rapidly separating and enriching exosomes based on chitosan with specific molecular weight | |
| CN114057907B (en) | Method for extracting, separating and purifying red ginseng polysaccharide | |
| CN113956338B (en) | Method for simultaneously extracting urease and Canavalia gladiata lectin from Canavalia gladiata | |
| CN112480283B (en) | Method for preparing neutral oligosaccharide from rhizoma polygonati | |
| CN114853919A (en) | Preparation method of black fungus superfine powder polysaccharide capable of preventing and inhibiting thrombus | |
| CN111617636B (en) | Plant extraction and purification process | |
| CN111887341A (en) | Method for extracting, separating and purifying miraculin | |
| CN105002153B (en) | A kind of preparation method of fibrin ferment | |
| CN107936133B (en) | Evening primrose leaf polysaccharide and preparation method thereof | |
| CN114397388A (en) | Urine exosome extraction kit based on combination of PEG precipitation method and SEC column method and application | |
| CN1093173C (en) | Papain blood group diagnose reagent prepn. method | |
| CN112724269B (en) | Preparation and application of bletilla striata polysaccharide | |
| CN114195908A (en) | Enzyme extraction and purification process of polysaccharide from leaves of litsea cubeba |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |