CN111575228A - Immunomagnetic bead separation method capable of obtaining complete exosomes - Google Patents
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Abstract
The invention relates to an immunomagnetic bead separation method capable of obtaining complete exosomes, which is characterized in that trypsin with a specific concentration is used as a main body to prepare a separation solution to digest antibody protein which plays a role in connection between immunomagnetic beads and exosomes and separate the immunomagnetic beads from the exosomes, so that the exosomes are separated from the immunomagnetic beads, the integrity and the bioactivity of the exosomes are kept, and the contained contents such as protein, nucleic acid and the like are not damaged, so that the immunomagnetic bead separation method can be further used for research on exosome morphology, bioactivity and the like and characterization detection methods such as TEM, NTA and the like.
Description
Technical Field
The invention relates to the technical field of biological experiments, in particular to an immunomagnetic bead separation method capable of obtaining complete exosomes.
Background
Exosomes (exosomes): the vesicle is a disc vesicle with the diameter of 30-150nm secreted by most cells, contains various nucleic acids, proteins and other substances, and can exchange information among cells. When the cells exocytosis to form exosomes, molecules such as various proteins, lipids, RNA and some fusion genes in the cells selectively enter the vesicles, and therefore the exosomes carry a lot of biological information derived from the mother cells. On the other hand, exosomes have the same membrane structure as cells, namely phospholipid bilayers, and can well protect proteins and nucleic acids carried by the exosomes from external influences and keep the original properties of the exosomes.
At present, more and more researches show that substances in exosomes have close relation with the occurrence and development of various human diseases. The gold standard method for exosome extraction is a gradient ultracentrifugation method, but the method is long in time consumption, labor-consuming, expensive in experimental equipment and not beneficial to research development. The immunomagnetic bead method can specifically and rapidly capture exosomes in a body fluid sample, and can complete the full-automatic extraction of the exosomes by combining with automatic equipment.
The final exosome-magnetic bead complex obtained by the method for capturing exosomes by immunomagnetic beads is, for example, domestic patent: CN107893051A, CN109738625A, CN106289926A, etc., all of which use magnetic beads to capture exosomes to obtain exosome-magnetic bead complexes, which cannot be well characterized and detected by TEM, NTA, etc., and thus it cannot be fully proved that exosomes but other vesicles are captured by magnetic beads. At present, no proper method is available for separating exosomes from immunomagnetic beads on the premise of ensuring the integrity of exosomes, and in the prior art, high-concentration salt solution, extremely-low-pH salt solution or strong protein denaturant directly lyse exosomes on the magnetic beads, so that separation is achieved, and structurally-complete exosomes cannot be obtained.
Therefore, there is a need in China for a method for separating exosomes from immunomagnetic beads, which is simple, convenient and rapid and can ensure the integrity of exosomes. The existing exosome-magnetic bead separation method has space for improvement, and a separation method capable of protecting the integrity of exosome is urgently needed to be developed.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides an immunomagnetic bead separation method capable of obtaining complete exosomes, which is characterized in that separation liquid is prepared by using trypsin with mild and controllable digestion effect as a main body, antibody protein which plays a role in connection between immunomagnetic beads and exosomes is digested, the immunomagnetic beads and the exosomes are separated, and meanwhile, the damage to exosome structures and the damage to exosome internal protein in the digestion process are avoided.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
an immunomagnetic bead separation method capable of obtaining complete exosomes is characterized by comprising the following steps:
s1, adding trypsin into the immunomagnetic bead-exosome compound resuspension according to the proportion that 1mg of immunomagnetic bead-exosome compound corresponds to 2-100 mu g of trypsin, and uniformly mixing to obtain a digestion composition;
s2, placing the digestion composition in a temperature range of 20-40 ℃ (constant temperature water bath can be adopted) for digestion reaction for 1-10 minutes to obtain a first separation composition;
s3, adding trypsin inhibitor with the same amount as that of the added trypsin into the first separation composition, and uniformly mixing to obtain a second separation composition;
and S4, carrying out magnetic separation on the second separation composition to obtain a supernatant part, namely the exosome solution.
Further, the immunomagnetic bead-exosome complex resuspension comprises resuspension with PBS solvent.
Further, the step S1 includes adding trypsin according to a ratio of 1mg immunomagnetic bead-exosome complex to 25 μ g trypsin, and mixing to obtain the digestion composition.
Further, the step S2 includes placing the digestion composition in a constant temperature of 37 ℃ for a digestion reaction lasting 3 minutes to obtain a first separated composition.
Further, the step S1 of adding trypsin includes adding an aqueous solution of trypsin; the aqueous trypsin solution comprises an aqueous solution free of divalent metal ions in which trypsin is dissolved.
Further, the trypsin comprises porcine trypsin, bovine trypsin, ovine trypsin or genetically engineered trypsin; the trypsin inhibitor comprises soybean trypsin inhibitor.
Further, the immunomagnetic beads comprise immunomagnetic beads with the particle size of 10nm to 10 μm; the immunomagnetic beads comprise a magnetic material with the following surface coating antibodies: CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, FLOT1, ICAM1, ALIX, GM130, ICAM-1, SNAP, MHC I/II, HLA-G, Integrins, Claudins, Tim4, and the antibody comprises a monoclonal or polyclonal antibody.
Further, the method also comprises the application of the method in separating exosome in plasma, serum, urine, milk, cerebrospinal fluid, tumor ascites, saliva and cell culture supernatant.
The invention has the beneficial effects that:
the immunomagnetic bead captured with the exosome is processed by the immunomagnetic bead separation method capable of obtaining the complete exosome, and the exosome is separated from the immunomagnetic bead by performing targeted digestion on an antibody on the surface of the immunomagnetic bead by using trypsin with a specific concentration, so that the integrity and the biological activity of the exosome are maintained, and the contained contents such as protein, nucleic acid and the like are not damaged, so that the immunomagnetic bead separation method can be further used for research on the form, the biological activity and the like of the exosome and characteristic detection methods such as TEM, NTA and the like. The separation method provided by the invention has the advantages of mild operation environment, simple operation method and high controllability, the exosome obtained by separation has high purity, and various reagents added in the separation process cannot influence subsequent detection and analysis, so that the separation method is a rapid and efficient immunomagnetic bead separation method.
Drawings
FIG. 1 is a schematic diagram of the operation process of an immunomagnetic bead capture exosome.
FIG. 2 is a schematic flow chart of an immunomagnetic bead separation method for obtaining complete exosomes according to the present invention.
FIG. 3 is a graph comparing the B2M content of exosomes obtained in the present example with the prior art.
FIG. 4 is a development pattern of HSP70 protein of exosomes obtained in example of the present invention.
FIG. 5 is a development pattern of CD9 protein of exosomes obtained in the example of the present invention.
FIG. 6 is a first TEM micrograph of exosomes obtained in the example of the present invention.
FIG. 7 is a second TEM micrograph of exosomes obtained in the example of the present invention.
FIG. 8 is a particle size-concentration diagram of exosomes obtained in example of the present invention.
Detailed Description
For a clearer understanding of the contents of the present invention, reference will be made to the accompanying drawings and examples.
At present, the method for capturing exosomes by using immunomagnetic beads is usually to coat antibodies such as anti-CD 9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, FLOT1, ICAM1, ALIX, GM130, ICAM-1, SNAP, MHC I/II, HLA-G, Integrins, Claudins, Tim4 and the like on the surfaces of the magnetic beads, and the antibodies are utilized to perform specific binding with exosome surface specific proteins, so as to capture exosomes. The general process is shown in fig. 1, and comprises adding immunomagnetic beads into a solution containing exosomes for incubation, and allowing the immunomagnetic beads to capture exosomes; performing magnetic separation on the solution, removing supernatant, and adding separation liquid (or called eluent) into the rest to separate immunomagnetic beads from exosomes; and carrying out magnetic separation again, and taking a supernatant part to obtain the required exosome. However, in this procedure, since most of the separation solution is high-concentration saline solution, very low-pH saline solution, or strong protein denaturant, the cell membrane structure of the obtained exosome is destroyed or even lysed, and the incomplete exosome is difficult to be examined by TEM, NTA, or other characteristics, and further study of the form, biological activity, or the like of the exosome cannot be performed.
The process of the immunomagnetic bead separation method capable of obtaining the complete exosome provided by the invention is shown in figure 2, and comprises the following steps: s1, adding trypsin into an immunomagnetic bead-exosome compound resuspension solution adopting a PBS solvent according to the proportion that 1mg of immunomagnetic bead-exosome compound corresponds to 2-100 mu g of trypsin, and carrying out vortex mixing to obtain a digestion composition, preferably adding a trypsin aqueous solution dissolved with trypsin and not containing divalent metal ions according to the proportion that 1mg of immunomagnetic bead-exosome compound corresponds to 25 mu g of trypsin, and mixing, wherein the mass of the immunomagnetic bead-exosome compound is determined by the mass of the added immunomagnetic bead, the mass of exosome per se is negligible, for example, 1mg of immunomagnetic bead is added into 1mL of plasma, and the mass of the immunomagnetic bead-exosome compound obtained after incubation is 1 mg; s2, placing the digestion composition in a constant-temperature water bath with the temperature range of 20-40 ℃ for digestion reaction for 1-10 minutes to obtain a first separation composition, and preferably, continuously performing digestion reaction for 3 minutes in the constant-temperature water bath with the temperature of 37 ℃; s3, adding trypsin inhibitor with the same amount as that of the trypsin into the first separation composition, and uniformly mixing to immediately inhibit the activity of the trypsin to obtain a second separation composition; and S4, carrying out magnetic separation on the second separation composition, wherein the obtained supernatant part is the separated complete exosome. Wherein, the digestion reaction is to cut off antibody protein on the immunomagnetic beads (for example, trypsin can cut off carboxyl side of lysine and arginine residues in polypeptide chains) and/or protein on the surface of an exosome membrane by using protease, and break the antibody protein connection between the exosome and the immunomagnetic beads, thereby separating the exosome from the magnetic beads. The trypsin can be porcine trypsin, bovine trypsin, ovine trypsin or trypsin expressed by genetic engineering; the trypsin inhibitor can be soybean trypsin inhibitor. The method can be used for processing immunomagnetic beads with the particle size of 10nm to 10 mu m; the immunomagnetic bead surface-coated antibody optionally comprises CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, FLOT1, ICAM1, ALIX, GM130, ICAM-1, SNAP, MHC I/II, HLA-G, Integrins, Claudins, Tim4, and the antibody comprises a monoclonal antibody or a polyclonal antibody. Since trypsin is also used for digesting cells in cell culture experiments, the digestion capacity is mild, the toxicity to cells is low, the integrity of the cells and the biological activity of the cells can be maintained, and the method can be suitable for treating plasma, serum, urine, milk, cerebrospinal fluid, tumor ascites, saliva and the requirement of separating exosome in cell culture supernatant.
The invention is further illustrated by the following specific example.
Example (b): centrifuging 12000g of blood plasma for 10min to obtain a supernatant, adding 1mg of CD63 and CD9 immunomagnetic beads into 1mL of blood plasma, shaking and incubating for 30min at 37 ℃, carrying out magnetic separation, removing the supernatant, adding 1mL of PBS (10mM) for washing once, carrying out magnetic separation again, resuspending the immunomagnetic beads with 200 mu L of PBS (10mM), adding 10 mu L of 2.5mg/mL trypsin aqueous solution, carrying out water bath at 37 ℃ for 3min at constant temperature, adding 10 mu L of 2.5mg/mL soybean trypsin inhibitor into the mixture solution, mixing uniformly, terminating digestion, carrying out magnetic separation, collecting the supernatant, and obtaining the supernatant which is the exosome solution eluted from the immunomagnetic beads. The separation of the exosome-magnetic bead can be completed in a few minutes in the whole process, and the separation condition is very mild, so that the integrity and the biological activity of the exosome cannot be damaged.
In order to demonstrate that effective exosomes were obtained using the method of the present invention, two prior art separation techniques were provided as comparative examples.
Comparative example 1: adopting the same operation steps of capturing exosomes by immunomagnetic beads as in the example, centrifuging 12000g of blood plasma for 10min to obtain supernatant, adding CD63 and CD9 immunomagnetic beads into 1mL of blood plasma, shaking and incubating for 30min at 37 ℃, performing magnetic separation, discarding the supernatant, adding 1mL of PBS (10mM) for washing once, performing magnetic separation again, adding Lysis-M (Roche cell Lysis solution, product number: 04719964001) Lysis solution to directly lyse exosomes on the magnetic beads, mixing uniformly, standing at room temperature for Lysis for 30min, and performing magnetic separation to collect the supernatant.
Comparative example 2: an exosome capturing operation procedure using immunomagnetic beads same as in example was carried out by centrifuging 12000g of plasma for 10min to obtain a supernatant, adding CD63 and CD9 immunomagnetic beads to 1mL of plasma, shaking and incubating at 37 ℃ for 30min, carrying out magnetic separation, discarding the supernatant, adding 1mL of PBS (10mM) to wash once, carrying out magnetic separation again, adding QIAzol lysine Reagent (QIAGEN lysate, cat # 56304569) to directly lyse exosomes, mixing, standing at room temperature for 30min to lyse, and carrying out magnetic separation to collect the supernatant.
The supernatants obtained in examples, comparative examples 1 and 2 were each subjected to RNA extraction using miRNeasy Serum/Plasma advanced kit (RNA extraction kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu.L of the reverse-transcribed cDNA, 1. mu. L B2M (a housekeeping gene) primer, probe mixture, 1. mu.L of enzyme-free water, and 10. mu.L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene B2M quantification in exosomes was performed, and the content of exosomes was reflected by the content of housekeeping gene B2M. The comparative results are shown in FIG. 3, and ddPCR assay was performed after separating exosomes from magnetic beads by trypsin digestionThe result of B2M content measurement is basically consistent with the results of lysine-M and QIAzol lysine treatment, which indicates that the exosome can be efficiently separated from the immunomagnetic beads by trypsin, and the subsequent PCR detection cannot be influenced by the trypsin treatment.
The complete exosome obtained by the example can be used for detecting HSP70 protein and CD90 protein contained in the exosome, and the specific method comprises the following steps: adding 40 μ L RIPA lysate into the above exosome solution, adding 10 μ L lloding Buffer after 30min of lysis, and boiling at 95 deg.C for 5 min; after magnetic separation, taking 14 mu L of supernatant for sampling; electrophoresis at 80V for 30min and at 120V for 1 h. After electrophoresis is finished, membrane is switched, and then 5% (w/v) of PBST solution of milk powder is used for sealing, and sealing is carried out for 1h at room temperature; according to the following steps: adding monoclonal antibody of mouse anti-CD 9 and HSP70 (protein enriched by tumor cell-derived exosome) at the ratio of 1000, and incubating overnight at 4 ℃; after washing 3 times with WB (Western blot) wash, incubation with HRP-labeled goat anti-mouse IgG (immunoglobulin G) at 1:10000 for 1h at room temperature; after washing with WB (Western blot) washing solution 3 times, the mixture was developed by adding a developing solution. The results of the resulting visualizations are shown in fig. 4 and 5, indicating HSP70 protein and CD90 protein, respectively.
Further, the complete exosome obtained by using the method described in the example can be used for TEM characterization to observe the morphology, and the specific operation is as follows: a small amount of exosome solution is dripped into a copper mesh, uranyl acetate is added for fixed dyeing, and the exosome shape is observed by using a field emission transmission electron microscope (Tecnai Spirit G2 BioTWIN, FEI company in America) after drying. The observed exosome morphology is shown in fig. 6 and fig. 7, and it can be clearly observed that the exosome morphology obtained after trypsinization separation is complete, the exosome particle size is normal, and the background is clean. The separation method provided by the invention is proved to be capable of completely separating the exosome from the immunomagnetic beads, thereby being beneficial to the subsequent morphological and biological activity research.
The effectiveness of the method can be proved by measuring the particle size and the quantity of the obtained exosomes aiming at the complete exosomes obtained in the embodiment. The method for detecting the exosome solution obtained in example 1 by using NTA specifically comprises the following steps: 0.1mL of the above exosome solution was diluted 10-fold with 0.9mL of PBS (10mM) solution using Nanosight NS300 (Malvern) NTA detection, the obtained results are shown in figure 8, and it is known that the exosome obtained by elution has normal particle size, the average particle size is about 70nm, and the exosome concentration is 4.56 × 109About one/mL (final concentration after multiplying by an exosome dilution factor of 10).
In conclusion, the method provided by the invention can quickly, efficiently and completely elute the exosomes from the immunomagnetic beads. The exosome obtained by elution by the method provided by the invention can be used for carrying out subsequent PCR and Western Blot detection and simultaneously carrying out characteristics detection such as TEM, NTA and the like, thereby providing a simple and effective exosome separation method for researching the form and the bioactivity of the exosome.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. An immunomagnetic bead separation method capable of obtaining complete exosomes is characterized by comprising the following steps:
s1, adding trypsin into the immunomagnetic bead-exosome compound resuspension according to the proportion that 1mg of immunomagnetic bead-exosome compound corresponds to 2-100 mu g of trypsin, and uniformly mixing to obtain a digestion composition;
s2, placing the digestion composition at a temperature range of 20-40 ℃ for digestion reaction for 1-10 minutes to obtain a first separation composition;
s3, adding trypsin inhibitor with the same amount as that of the added trypsin into the first separation composition, and uniformly mixing to obtain a second separation composition;
and S4, carrying out magnetic separation on the second separation composition to obtain a supernatant part, namely the exosome solution.
2. The method of claim 1, wherein the resuspension of immunomagnetic bead-exosome complexes comprises resuspension in a PBS solvent.
3. The method according to claim 1, wherein the step S1 comprises adding trypsin to the immunomagnetic bead-exosome complex in a ratio of 1mg to 25 μ g of trypsin, and mixing to obtain the digestion composition.
4. The method of claim 1, wherein step S2 comprises subjecting the digested composition to a digestion reaction at a constant temperature of 37 ℃ for a duration of 3 minutes to produce a first separated composition.
5. The method of claim 1 or 4, wherein the step of adding trypsin for mixing in S1 comprises adding an aqueous solution of trypsin; the aqueous trypsin solution comprises an aqueous solution free of divalent metal ions in which trypsin is dissolved.
6. The method of claim 1, wherein the trypsin comprises porcine trypsin, bovine trypsin, ovine trypsin, or genetically expressed trypsin; the trypsin inhibitor comprises soybean trypsin inhibitor.
7. The method of claim 1, wherein the immunomagnetic beads comprise immunomagnetic beads having a particle size of 10nm to 10 μ ι η; the immunomagnetic beads comprise a magnetic material with the following surface coating antibodies: CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, FLOT1, ICAM1, ALIX, GM130, ICAM-1, SNAP, MHCI/II, HLA-G, Integrins, Claudins, Tim4, and the antibody comprises a monoclonal or polyclonal antibody.
8. Use of the method according to any one of claims 1 to 8 for the isolation of exosomes in plasma, serum, urine, milk, cerebrospinal fluid, tumor ascites, saliva, cell culture supernatant.
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| CN113281137A (en) * | 2021-02-26 | 2021-08-20 | 中国计量科学研究院 | Method for removing magnetic beads of bacteria sample to be detected after enrichment of immunomagnetic beads |
| CN114276992A (en) * | 2021-12-14 | 2022-04-05 | 南通举健生物科技有限公司 | Complete exosome separation and purification kit and detection analysis method |
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| CN113960313B (en) * | 2021-12-22 | 2022-04-12 | 上海思路迪医学检验所有限公司 | Exosome ALK fusion protein magnetic immunochemiluminescence detection kit |
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Application publication date: 20200825 |