CN114323851A - Method for separating exosomes in serum and plasma based on ultrafiltration and affinity chromatography technology - Google Patents
Method for separating exosomes in serum and plasma based on ultrafiltration and affinity chromatography technology Download PDFInfo
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Abstract
The invention relates to the technical field of exosome extraction, and discloses a method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies, which comprises the following steps: 1) sample treatment: centrifuging serum or plasma at 5000g for 20min, and collecting supernatant; 2) and (3) ultrafiltration: filtering the supernatant with 0.45um filter membrane, and collecting the filtered liquid; 3) affinity chromatography: performing affinity chromatography by using an affinity chromatography medium simultaneously containing three antibodies consisting of CD9\ CD63\ CD 81; 4) collecting exosomes; 5) and (3) carrying out various detections, wherein the various detections comprise NTA detection, exosome sample transmission electron microscope detection and Wb detection. The method for separating exosomes from serum and plasma based on the ultrafiltration and affinity chromatography technology adopts the combination of the combined ultrafiltration and affinity chromatography, and compared with the affinity chromatography, the yield is improved from 8.9E +8Particles/mL to 1.2E +10Particles/mL, and compared with the ultrafiltration method, the NTA detection result is changed from a hetero peak to a single peak, so that the yield and purity of exosomes can be obviously improved, and the method can be better applied to downstream NTA, electron microscopy and wb detection.
Description
Technical Field
The invention relates to the technical field of exosome extraction, in particular to a method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies.
Background
Exosome refers to a small membrane vesicle (30-150nm) containing complex RNA and protein, which is a disc vesicle with the diameter of 40-100nm, and is firstly found in sheep reticulocytes in 1983, and is named as "exosome" by Johnstone in 1987, so that various cells can secrete exosome under normal and pathological states, and the exosome mainly comes from a multivesicular body formed by invagination of intracellular lysosomal microparticles and is released into extracellular matrix after the multivesicular body outer membrane is fused with cell membrane.
All cultured cell types secrete exosomes and exosomes are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid and milk, and the precise molecular mechanisms for their secretion and uptake and their composition, "cargo" and corresponding functions have just been studied, and exosomes are regarded as specifically secreted membrane vesicles, involved in cell-cell communication, and are of increasing interest for the study of exosomes, whether for their function or for how they are known to be used in the development of minimally invasive diagnostics.
The existing method for separating exosomes from serum and plasma mainly comprises an ultracentrifugation method, an ultrafiltration method, a size exclusion chromatography method, an affinity chromatography method and a microfluidic chip method, but all the methods have the defects, wherein the ultracentrifugation method depends on a high-speed centrifuge which is a valuable device, the cost is high, the purity of the ultrafiltration method and the size exclusion chromatography method is insufficient, the yield of the affinity chromatography method is low, the existing technology of the microfluidic chip method is immature, and the method for separating exosomes from serum and plasma based on the ultrafiltration and affinity chromatography technologies is provided to solve the problems.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies, the method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies combines ultrafiltration and affinity chromatography, the yield is improved to 1.2E +10Particles/mL from 8.9E +8Particles/mL compared with the affinity chromatography, compared with the ultrafiltration method, the NTA detection result is changed from a foreign peak to a single peak, the yield and purity of exosomes can be obviously improved, and the method can be better applied to downstream NTA, electron microscopy and wb detection.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies comprises the following steps:
1) sample treatment: centrifuging serum or plasma at 5000g for 20min, and collecting supernatant;
2) and (3) ultrafiltration: filtering the supernatant with 0.45um filter membrane, and collecting the filtered liquid;
3) affinity chromatography: performing affinity chromatography by using an affinity chromatography medium simultaneously containing three antibodies consisting of CD9\ CD63\ CD 81;
4) collecting exosomes;
5) and (5) carrying out various detections.
Preferably, the assays in step 5) include NTA assay, transmission electron microscopy assay for exosome sample and Wb assay.
Preferably, the NTA detection comprises the following steps:
(1) pumping pure water into the sample cell through an injector to clean the sample cell for 3 times;
(2) after cleaning, diluting the exosome resuspended in PBS to a proper concentration, pumping the exosome into a sample pool through a syringe, and then pushing the whole sample pool into a zetaview host;
(3) and observing real-time dynamic images of the exosome particles through a computer display screen.
Preferably, the transmission electron microscopy detection of the exosome sample comprises the following steps:
(1) taking out 10 mu L of exosome;
(2) sucking 10 μ L of sample, dripping on copper net, precipitating for 1min, and sucking away the supernatant with filter paper;
(3) dripping 10 μ L uranyl acetate on copper net for precipitation for 1min, and removing the floating liquid with filter paper;
(4) drying for several minutes at normal temperature;
(5) detecting and imaging by an electron microscope at 100 kv;
(6) and obtaining the imaging result of the transmission electron microscope.
Preferably, the Wb detection includes the steps of:
(1) extracting protein;
(2) protein concentration quantification: determining the sample protein concentration using a BCA protein concentration assay kit;
(3) SDS-PAGE electrophoresis:
1. sample treatment:
determining the sample loading amount according to the concentration of the sample, and ensuring that the total protein loading amount of each sample is 50 mu g;
adding proper amount of 5 times protein loading buffer solution into the protein sample, and boiling water bath at 95-100 ℃ for 5 min;
2. preparing glue and loading sample:
firstly, preparing separation glue, adding TEMED, immediately shaking up and pouring glue, slowly adding a proper amount of water on a glue surface to flatten the glue surface, pouring off water on the upper layer of the glue after about 45min, and sucking the rest water with absorbent paper;
preparing concentrated glue, adding TEMED, shaking up and filling glue, filling the residual space with the concentrated glue, and inserting a comb into the concentrated glue;
thirdly, pulling out the comb, putting the electrophoresis frame into an electrophoresis tank, adding electrophoresis buffer solution, and adding the sample into the spot sample hole;
performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until the bromophenol blue reaches the lower edge of the gel plate;
3. film transfer:
firstly, preparing membrane-transfer filter paper and a PVDF membrane, wherein the PVDF membrane is activated by methanol before being used, and the activation time is 3 min;
secondly, placing a 'sandwich' structure of the rotary film according to the directions of the anode and the cathode, sequentially arranging rotary film sponge, 3 layers of filter paper, a PVDF film, glue, 3 layers of filter paper and rotary film sponge from the anode to the cathode, and removing bubbles in each layer in the placing process;
thirdly, the membrane is rotated according to the constant current of 300mA, and the membrane rotating time is adjusted according to the molecular weight of the target protein;
4. antibody incubation:
firstly, adding the transferred membrane into a closed liquid chamber for temperature sealing for 1 h;
② removing the sealing liquid, adding the primary antibody diluted by the primary antibody diluent at 4 ℃ overnight (the primary antibody concentration range is 0.5-2 ug/ml);
thirdly, recovering the diluted primary antibody, and washing the primary antibody for three times by TBST, 5min each time;
adding the diluted secondary antibody of the secondary antibody diluent, incubating for 30min at room temperature, washing for four times with TBST on a shaking bed at room temperature, 5min for each time (the dilution ratio of the secondary antibody is 1: 10000);
5. and (3) chemiluminescence detection:
dripping a freshly prepared ECL mixed solution (A: B is 1:1) to the protein surface side of the membrane, and exposing in a dark room;
adjusting exposure conditions according to different light intensities, developing and fixing.
(III) advantageous effects
Compared with the prior art, the invention provides a method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies, which has the following beneficial effects:
the method for separating exosomes from serum and plasma based on the ultrafiltration and affinity chromatography technology adopts the combination of the combined ultrafiltration and affinity chromatography, and compared with the affinity chromatography, the yield is improved from 8.9E +8Particles/mL to 1.2E +10Particles/mL, and compared with the ultrafiltration method, the NTA detection result is changed from a hetero peak to a single peak, so that the yield and purity of exosomes can be obviously improved, and the method can be better applied to downstream NTA, electron microscopy and wb detection.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a schematic diagram showing the result of NTA detection of exosomes extracted by the method;
FIG. 3 is a schematic diagram showing the result of transmission electron microscope detection of exosomes extracted by the method;
FIG. 4 is a schematic diagram of the Wb detection result of the exosomes extracted by the method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The method adopts the combination of ultrafiltration and affinity chromatography, improves the yield from 8.9E +8Particles/mL to 1.2E +10Particles/mL compared with the affinity chromatography, obviously improves the purity compared with the ultrafiltration, and changes the NTA detection result from a hybrid peak to a single peak, so the method provided by the invention can obviously improve the yield and the purity of exosome and can be better applied to downstream NTA, electron microscope and wb detection.
The method for separating exosomes from serum and plasma based on the ultrafiltration and affinity chromatography technology mainly comprises the following materials and reagents:
0.45um filter membrane, CD9\ CD63\ CD81 antibody, affinity chromatography medium and centrifuge.
The extraction steps are as follows:
1) sample treatment: centrifuging 5000g of serum or plasma for 20min by using a centrifuge, and taking supernatant;
2) and (3) ultrafiltration: filtering the supernatant with 0.45um filter membrane, and collecting the filtered liquid;
3) affinity chromatography: affinity chromatography is carried out by using an affinity chromatography medium simultaneously containing three antibodies of CD9\ CD63\ CD81, so as to ensure that an exosome with higher purity is obtained;
4) collecting exosomes;
5) and (5) carrying out various detections.
Wherein, each detection comprises NTA detection, exosome sample transmission electron microscope detection and Wb detection.
The following describes the procedure and results of the various tests in detail:
A. NTA detection:
the method comprises the following specific steps:
(1) pumping pure water into the sample cell through an injector to clean the sample cell for 3 times;
(2) after cleaning, diluting the exosome resuspended in PBS to a proper concentration, pumping the exosome into a sample pool through a syringe, and then pushing the whole sample pool into a zetaview host;
(3) observing real-time dynamic images of the exosome particles through a computer display screen;
the detection result is shown in figure 2, through NTA detection, compared with an affinity chromatography, the yield is improved from 8.9E +8Particles/mL to 1.2E +10Particles/mL, the purity is also obviously improved compared with an ultrafiltration method, and the NTA detection result is changed from a hybrid peak to a single peak;
B. carrying out transmission electron microscopy detection on the exosome sample:
the specific detection steps are as follows:
(1) taking out 10 mu L of exosome;
(2) sucking 10 μ L of sample, dripping on copper net, precipitating for 1min, and sucking away the supernatant with filter paper;
(3) dripping 10 μ L uranyl acetate on copper net for precipitation for 1min, and removing the floating liquid with filter paper;
(4) drying for several minutes at normal temperature;
(5)100kv is subjected to electron microscope (Hitachi, HT-7700) detection imaging;
(6) obtaining a transmission electron microscope imaging result;
the detection result is shown in fig. 3, and a remarkable tray-shaped exosome structure is obtained.
C. And (5) detecting Wb:
(1) extracting protein;
(2) protein concentration quantification: determining the sample protein concentration using a BCA protein concentration assay kit;
(3) SDS-PAGE electrophoresis:
1. sample treatment:
determining the sample loading amount according to the concentration of the sample, and ensuring that the total protein loading amount of each sample is 50 mu g;
adding proper amount of 5 times protein loading buffer solution into the protein sample, and boiling water bath at 95-100 ℃ for 5 min;
2. preparing glue and loading sample:
firstly, preparing separation glue, adding TEMED, immediately shaking up and pouring glue, slowly adding a proper amount of water on a glue surface to flatten the glue surface, pouring off water on the upper layer of the glue after about 45min, and sucking the rest water with absorbent paper;
preparing concentrated glue, adding TEMED, shaking up and filling glue, filling the residual space with the concentrated glue, and inserting a comb into the concentrated glue;
thirdly, pulling out the comb, putting the electrophoresis frame into an electrophoresis tank, adding electrophoresis buffer solution, and adding the sample into the spot sample hole;
performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until the bromophenol blue reaches the lower edge of the gel plate;
3. film transfer:
firstly, preparing membrane-transfer filter paper and a PVDF membrane, wherein the PVDF membrane is activated by methanol before being used, and the activation time is 3 min;
secondly, placing a 'sandwich' structure of the rotary film according to the directions of the anode and the cathode, sequentially arranging rotary film sponge, 3 layers of filter paper, a PVDF film, glue, 3 layers of filter paper and rotary film sponge from the anode to the cathode, and removing bubbles in each layer in the placing process;
thirdly, the membrane is rotated according to the constant current of 300mA, and the membrane rotating time is adjusted according to the molecular weight of the target protein;
4. antibody incubation:
firstly, adding the transferred membrane into a closed liquid chamber for temperature sealing for 1 h;
② removing the sealing liquid, adding the primary antibody diluted by the primary antibody diluent at 4 ℃ overnight (the primary antibody concentration range is 0.5-2 ug/ml);
thirdly, recovering the diluted primary antibody, and washing the primary antibody for three times by TBST, 5min each time;
adding the diluted secondary antibody of the secondary antibody diluent, incubating for 30min at room temperature, washing for four times with TBST on a shaking bed at room temperature, 5min for each time (the dilution ratio of the secondary antibody is 1: 10000);
5. and (3) chemiluminescence detection:
dripping a freshly prepared ECL mixed solution (A: B is 1:1) to the protein surface side of the membrane, and exposing in a dark room;
adjusting exposure conditions according to different light intensities, developing and fixing;
the detection result is shown in FIG. 4, in which the exosomes in the serum plasma all detected the expression of the surface marker CD9\ CD63\ CD81 of the exosomes.
The invention has the beneficial effects that:
the method combines combined ultrafiltration and affinity chromatography, and compared with affinity chromatography, the yield is improved from 8.9E +8Particles/mL to 1.2E +10Particles/mL, compared with the ultrafiltration method, the NTA detection result is changed from a hybrid peak to a single peak, the yield and purity of exosome can be obviously improved, and the method can be better applied to downstream NTA, electron microscopy and wb detection.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technologies is characterized by comprising the following steps:
1) sample treatment: centrifuging serum or plasma at 5000g for 20min, and collecting supernatant;
2) and (3) ultrafiltration: filtering the supernatant with 0.45um filter membrane, and collecting the filtered liquid;
3) affinity chromatography: performing affinity chromatography by using an affinity chromatography medium simultaneously containing three antibodies consisting of CD9\ CD63\ CD 81;
4) collecting exosomes;
5) and (5) carrying out various detections.
2. The method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technique according to claim 1, wherein each test in step 5) comprises NTA test, exosome sample transmission electron microscope test and Wb test.
3. A method for separating exosomes from serum plasma based on ultrafiltration and affinity chromatography technique according to claim 2, characterized in that the NTA detection comprises the following steps:
(1) pumping pure water into the sample cell through an injector to clean the sample cell for 3 times;
(2) after cleaning, diluting the exosome resuspended in PBS to a proper concentration, pumping the exosome into a sample pool through a syringe, and then pushing the whole sample pool into a zetaview host;
(3) and observing real-time dynamic images of the exosome particles through a computer display screen.
4. The method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technology according to claim 2, wherein the exosome sample transmission electron microscopy detection comprises the following steps:
(1) taking out 10 mu L of exosome;
(2) sucking 10 μ L of sample, dripping on copper net, precipitating for 1min, and sucking away the supernatant with filter paper;
(3) dripping 10 μ L uranyl acetate on copper net for precipitation for 1min, and removing the floating liquid with filter paper;
(4) drying for several minutes at normal temperature;
(5) detecting and imaging by an electron microscope at 100 kv;
(6) and obtaining the imaging result of the transmission electron microscope.
5. The method for separating exosomes from serum and plasma based on ultrafiltration and affinity chromatography technique according to claim 2, wherein said Wb detection comprises the following steps:
(1) extracting protein;
(2) protein concentration quantification: determining the sample protein concentration using a BCA protein concentration assay kit;
(3) SDS-PAGE electrophoresis:
1. sample treatment:
determining the sample loading amount according to the concentration of the sample, and ensuring that the total protein loading amount of each sample is 50 mu g;
adding proper amount of 5 times protein loading buffer solution into the protein sample, and boiling water bath at 95-100 ℃ for 5 min;
2. preparing glue and loading sample:
firstly, preparing separation glue, adding TEMED, immediately shaking up and pouring glue, slowly adding a proper amount of water on a glue surface to flatten the glue surface, pouring off water on the upper layer of the glue after about 45min, and sucking the rest water with absorbent paper;
preparing concentrated glue, adding TEMED, shaking up and filling glue, filling the residual space with the concentrated glue, and inserting a comb into the concentrated glue;
thirdly, pulling out the comb, putting the electrophoresis frame into an electrophoresis tank, adding electrophoresis buffer solution, and adding the sample into the spot sample hole;
performing constant-pressure electrophoresis according to the concentration glue of 80V and the separation glue of 120V until the bromophenol blue reaches the lower edge of the gel plate;
3. film transfer:
firstly, preparing membrane-transfer filter paper and a PVDF membrane, wherein the PVDF membrane is activated by methanol before being used, and the activation time is 3 min;
secondly, placing a 'sandwich' structure of the rotary film according to the directions of the anode and the cathode, sequentially arranging rotary film sponge, 3 layers of filter paper, a PVDF film, glue, 3 layers of filter paper and rotary film sponge from the anode to the cathode, and removing bubbles in each layer in the placing process;
thirdly, the membrane is rotated according to the constant current of 300mA, and the membrane rotating time is adjusted according to the molecular weight of the target protein;
4. antibody incubation:
firstly, adding the transferred membrane into a closed liquid chamber for temperature sealing for 1 h;
② removing the sealing liquid, adding the primary antibody diluted by the primary antibody diluent at 4 ℃ overnight (the primary antibody concentration range is 0.5-2 ug/ml);
thirdly, recovering the diluted primary antibody, and washing the primary antibody for three times by TBST, 5min each time;
adding the diluted secondary antibody of the secondary antibody diluent, incubating for 30min at room temperature, washing for four times with TBST on a shaking bed at room temperature, 5min for each time (the dilution ratio of the secondary antibody is 1: 10000);
5. and (3) chemiluminescence detection:
dripping a freshly prepared ECL mixed solution, wherein A: B is 1:1, onto the protein surface side of the membrane, and exposing in a dark room;
adjusting exposure conditions according to different light intensities, developing and fixing.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540307A (en) * | 2022-02-16 | 2022-05-27 | 多莱泌生物科技(武汉)有限公司 | Preparation method of liver-targeting engineered exosome |
| CN115678840A (en) * | 2022-11-15 | 2023-02-03 | 陕西慧康生物科技有限责任公司 | Separation and extraction method of exosome |
| CN118961345A (en) * | 2024-10-12 | 2024-11-15 | 北京众驰伟业科技发展有限公司 | Multi-sample processing method for coagulation tester |
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