WO2021143577A1 - 一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物 - Google Patents

一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物 Download PDF

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WO2021143577A1
WO2021143577A1 PCT/CN2021/070289 CN2021070289W WO2021143577A1 WO 2021143577 A1 WO2021143577 A1 WO 2021143577A1 CN 2021070289 W CN2021070289 W CN 2021070289W WO 2021143577 A1 WO2021143577 A1 WO 2021143577A1
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lectin
exosomes
glycosylated
macromolecule carrier
centrifuge tube
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PCT/CN2021/070289
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English (en)
French (fr)
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林长青
陈天圣
郝昆
黄鹤
高琦
李艳召
许智慧
郄霜
赵广珍
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北京热景生物技术股份有限公司
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Priority to EP21740720.4A priority Critical patent/EP4092417A4/en
Priority to JP2022543094A priority patent/JP7493602B2/ja
Publication of WO2021143577A1 publication Critical patent/WO2021143577A1/zh
Priority to US17/864,572 priority patent/US20220349901A1/en

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Definitions

  • the present invention requires a Chinese patent application filed in the Chinese Patent Office with application number 202010060063.1 and the title of the invention "a lectin-macromolecule carrier conjugate complex used to separate glycosylated exosomes in clinical samples"
  • a lectin-macromolecule carrier conjugate complex used to separate glycosylated exosomes in clinical samples.
  • the invention belongs to the technical field related to biomedicine and bioseparation, and relates to a lectin-macromolecule carrier coupling complex used for separating glycosylated exosomes in clinical samples.
  • Exosomes are tiny vesicles with biological activity that are released when the multivesicular endosomes of cells fuse with the plasma membrane, with a diameter of about 30 to 150 nm, and are one of the important media for intercellular communication; under healthy physiological conditions , Exosomes can transport a variety of biologically active substances such as DNA, protein, mRNA, miRNA, etc. between cells, participate in various processes such as cell communication, cell migration, and tumor cell growth, and complete the transfer of intercellular substances and information.
  • Literature studies have found that almost all types of cells secrete exosomes, and they can be extracted from body fluids such as blood, saliva, urine, cerebrospinal fluid, effusion, and milk.
  • Exosomes are related to many diseases such as tumors, chronic infectious diseases, and autoimmune diseases.
  • the composition, content, and properties of exosomes secreted by cells may change; glycosylated exosomes may change
  • the detection and research of exosomes can effectively monitor the occurrence and development of diseases; because the content of exosomes in the sample is relatively low, the isolation of exosomes The study of enrichment of exosomes is crucial.
  • ultracentrifugation or gradient density centrifugation is based on the small difference in the density of exosomes and other biological components. This method requires special equipment, the sample demand is relatively large, it takes a long time, and because the characteristics of different clinical samples are different The separation effect of this method is poor, and further ultracentrifugation can easily cause vesicle damage and affect subsequent experiments.
  • the principle of the immunomagnetic bead method is that the specific proteins on the surface of exosomes such as CD9/CD63/CD81 and the like It is combined with the immunomagnetic beads with corresponding antibodies for magnetic separation.
  • the immunomagnetic beads are small in size, with a particle size of nanometers and less than or equal to the diameter of the exosomes, the space between the immunomagnetic beads and the exosomes is The resistance is large, the binding of exosomes is insufficient, and the separation efficiency is not high.
  • the exosomes separated by the immunomagnetic bead method are eluted with an acidic eluent during elution, resulting in incomplete exosomes morphology.
  • the present invention provides a lectin-macromolecule carrier conjugate complex for separating glycosylated exosomes in clinical samples, and the present invention also provides a lectin-containing lectin- A glycosylated exosome separation composition of a macromolecular carrier coupling complex, and a method and application for separating glycosylated exosomes using the lectin-macromolecule carrier coupling complex.
  • the lectin-macromolecule carrier conjugate complex provided by the present invention can accurately separate glycosylated exosomes in clinical samples, has high separation efficiency, and the separated exosomes have complete morphology, no rupture or fragmentation, and can be used directly In the detection of glycosylated exosomes liquid, or directly perform immunological related tests, or directly extract relevant nucleic acids from exosomes for genetic detection and analysis.
  • the abundant sugar chains on the outer surface of exosomes can be combined with lectins, and the glycosylated exosomes can be separated by effective centrifugal cleaning and effective elution methods. It is very simple, fast, and reproducible.
  • the isolated exosomes are complete in shape without rupture or fragmentation. They can be used for detection, monitoring and diagnosis during the occurrence and development of diseases.
  • the present invention provides a lectin-macromolecule carrier conjugate complex for separating glycosylated exosomes in clinical samples, a macromolecular carrier; and a lectin coupled to the outside of the macromolecular carrier; wherein:
  • the lectins include: wood pineapple agglutinin, peanut agglutinin, pea agglutinin (VVA and/or VVL), jack bean agglutinin, lentil agglutinin, wheat germin, soybean agglutinin, kidney bean agglutinin, snail agglutinin Any one or two or more of HAA and/or HPA;
  • the macromolecular carrier includes any one or two or more of dextran microspheres, agarose microspheres, resin or epoxy resin microspheres, and polystyrene microspheres.
  • the particle size distribution of the macromolecular carrier ranges from 1 ⁇ m to 200 ⁇ m, preferably from 10 ⁇ m to 200 ⁇ m, and more preferably from 30 ⁇ m to 150 ⁇ m.
  • lectin-macromolecule carrier conjugate complex 1-20 mg, preferably 5-15 mg, is coupled to each 1ml of the macromolecular carrier. , More preferably 10-15 mg of lectin.
  • the clinical sample includes any one of serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, and lymph.
  • the lectin includes: wood pineapple agglutinin, peanut agglutinin, pea agglutinin (VVA and/or VVL), canavalia agglutinin, small Any one or two or more of lentil agglutinin, wheat germin, soybean agglutinin, and kidney bean agglutinin.
  • the glycosylated exosomes include: N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes. Any one or two or more of the exosomes.
  • the lectin-macromolecule carrier conjugate complex is stored in a storage solution, and the obtained lectin-macromolecule carrier conjugate complex is stored
  • the storage concentration (volume ratio) of the lectin-macromolecule carrier coupling complex in the solution is 10%-60%, preferably the ratio is 30%-50%, and more preferably 40%-50%;
  • the volume of the storage solution of the carrier coupling complex refers to the total volume of the lectin-macromolecule coupling complex after being dispersed in the storage solution and the storage solution.
  • the storage solution of the lectin-macromolecule carrier coupling complex is dispensed in a separation device including an affinity adsorption centrifuge tube;
  • the affinity The adsorption centrifuge tube includes two parts: an upper centrifuge tube and an outer protective sleeve; the diameter of the upper centrifuge tube is smaller than the diameter of the outer protective sleeve, and the upper centrifugal tube is sleeved in the outer protective sleeve and has a convex outer side.
  • the raised annular edge or pillar is used to support the opening of the upper centrifuge tube higher than the outer protective sleeve;
  • the upper centrifuge tube includes a centrifuge tube cover, a centrifuge tube wall and a bottom of the filter membrane fixedly connected to the wall of the centrifuge tube, and the bottom of the filter membrane
  • the pore size of the filter membrane used is smaller than the particle size of the lectin-macromolecule carrier coupling complex, and at the same time larger than the particle size of the exosomes, the lectin-macromolecule carrier coupling complex preservation solution is stored in the upper centrifugation,
  • the pore size of the filter membrane is preferably 150nm-1000nm.
  • the lectin-macromolecule carrier coupling complex is divided into the lectin-macromolecule carrier coupling complex in the upper centrifuge tube.
  • the lectin-macromolecule carrier coupling complex in the preservation solution The volume accounts for 1/2-1/4 of the volume of the upper centrifuge tube.
  • glycosylated exosome separation composition includes: the above-mentioned lectin-macromolecule carrier coupling complex, and also includes The cleaning solution for removing non-specifically bound, unglycosylated exosomes and other impurities during the separation process and/or used to elute the glycosylation specifically bound to the lectin-macromolecule carrier coupling complex The eluate of exosomes.
  • the composition here does not mean that it is mixed as a whole, but that it is present in a set at the same time.
  • the cleaning solution is a metal salt ion-free cleaning buffer solution or purified water, and optionally a metal salt ion-free cleaning buffer solution for cleaning, removing, and separating processes.
  • a metal salt ion-free cleaning buffer solution for cleaning, removing, and separating processes.
  • the eluent is a borate buffer with sugar dissolved in it, optionally a borate buffer with mannose dissolved in it.
  • the selected pH value is 6.5 ⁇ 0.2, which is used to elute the glycosylated exosomes bound to the lectin-macromolecule carrier coupling complex, and the eluted exosomes are intact in appearance, without damage or lysis.
  • a method for separating glycosylated exosomes which includes the separation step of separating glycosylated exosomes using the above-mentioned lectin-macromolecule carrier coupling complex, and the separation step includes:
  • the ratio of the sample to be tested and the lectin-macromolecule carrier conjugate complex is 1:1-3;
  • the ratio of the volume of a single addition of the cleaning solution to the volume of the lectin-macromolecule carrier coupling complex that has been bound to glycosylated exosomes is 1-3:1;
  • the cleaning step of the cleaning solution is repeated 1-3 times;
  • the ratio of the volume of the added eluent to the volume of the washed lectin-macromolecule carrier coupling complex is 0.5-2:1, preferably 0.5-1:1.
  • the preservation solution part in the preservation solution of the lectin-macromolecule carrier coupling complex is completely removed, the sample is removed, the washing solution is removed, and/or, the elution is collected
  • the supernatant was centrifuged at a speed of 3000 rpm or less and at room temperature for 20 seconds.
  • incubate at room temperature for 10-30 min, preferably 10-15 min.
  • the method for separating glycosylated exosomes described above includes the following steps:
  • the cleaning solution is a metal salt-ion-free cleaning buffer or purified water, optionally a metal-salt-ion-free cleaning buffer, and further can be pH
  • the value of 7.6 ⁇ 0.2 is a washing buffer without metal salt ions.
  • the eluent is a borate buffer with sugar dissolved, optionally a borate buffer with mannitol dissolved, and further optional
  • the ground is a borate-mannose buffer with a pH of 6.5 ⁇ 0.2.
  • the incubation conditions are: room temperature incubation for 1-30 min, preferably 5-20 min, and more preferably 10-15 min.
  • the aforementioned method for separating glycosylated exosomes includes a separation step of using the aforementioned lectin-macromolecule carrier conjugate complex to separate glycosylated exosomes from a separation device, and the separation step includes:
  • the affinity adsorption centrifuge tube loaded with the lectin-macromolecule carrier coupling complex completely removes the storage solution part of the lectin-macromolecule carrier coupling complex preservation solution, and retains the lectin-macromolecule carrier coupling complex, And replace with a new external casing;
  • the present invention also provides an application of exosomes isolated using the above method, the application including: glycosylated exosomes liquid detection, exosomal immunological detection, or nucleosides after nucleic acid extraction from exosomes Acid fragment detection and analysis.
  • the isolated exosomes are all glycosylated exosomes with complete morphology and no damage, which can be directly used for the detection of glycosylated exosomes liquid.
  • Exosomes immunological detection, or nucleotide sequence detection and analysis after nucleic acid extraction, etc. can also be further equipped with corresponding equipment to achieve fully automated separation of glycosylated exosomes.
  • the particle size of the macromolecular carrier used is 10-1000 times the particle size of the macromolecular carrier generally used in the field of immunoassay, and a single macromolecular carrier has The larger surface area reduces the steric hindrance when the lectin is coupled to the macromolecular carrier, which facilitates the coupling of more lectins.
  • the general macromolecular carrier of the same total volume has a larger specific surface area. However, due to the larger steric hindrance, there are fewer lectins coupled.
  • lectin-macromolecule carrier coupling complex of the present invention 1-20mg of lectin can be coupled to each 1ml of the macromolecular carrier.
  • the steric hindrance of the binding of glycosylated exosomes to lectins facilitates the binding of lectins to glycosylated exosomes and greatly improves the separation efficiency of exosomes.
  • the lectin-macromolecule carrier conjugate complex of the present invention also has a good effect on clinical samples with a small amount of exosomes, such as exosomes in urine, tissue or cell culture supernatant; Clinical samples with a large amount of exosomes, such as exosomes in plasma, can be separated, and the concentration of glycosylated exosomes obtained can reach 10 11-14 /ml.
  • the lectin used is preferably plant lectin, including: wood pineapple lectin, peanut lectin, pea lectin (VVA and/or VVL), Concanavalin Lectin, lentil agglutinin, wheat germin, soybean agglutinin, kidney bean agglutinin, plant agglutinin is easier to obtain, and the raw materials are more abundant.
  • plant lectin including: wood pineapple lectin, peanut lectin, pea lectin (VVA and/or VVL), Concanavalin Lectin, lentil agglutinin, wheat germin, soybean agglutinin, kidney bean agglutinin, plant agglutinin is easier to obtain, and the raw materials are more abundant.
  • the lectin-macromolecule carrier coupling complex of the present invention can be dispensed into a specific structure of affinity adsorption centrifuge tube to form a separation device for glycosylated exosomes when leaving the factory, which is convenient for users and facilitates agglutination.
  • the selection of the pore size of the filter membrane is conducive to the smooth passage of various preservation solutions, cleaning solutions, and eluents, especially the addition of mannose to the eluent, which will cause the eluent
  • the viscosity increases, the pore size can ensure the smooth passage of the eluent, and can also ensure that the lectin-macromolecule carrier coupling complex does not leak.
  • the glycosylated exosomes separation composition of the present invention uses a cleaning buffer or purified water without metal salt ions.
  • the metal salt ions dissociate the affinity adsorption of lectin and glycosylated exosomes. It affects the affinity adsorption of lectins and glycosylated exosomes, and the higher the concentration of metal salt ions, the stronger the dissociation effect, which affects the separation effect of exosomes, and even the exosomes cannot be separated.
  • the glycosylated exosomes separation composition of the present invention uses borate buffer as the eluent, which has a better elution effect for exosomes and high elution efficiency than other buffers. , Use a small amount of eluent to complete the elution, the appearance of the eluted exosomes is complete, without damage or lysis, and because of the small amount of use, it is equivalent to completing the process of concentrating exosomes at the same time.
  • the method for separating glycosylated exosomes of the present invention has simple and easy sample pretreatment methods. Through simple centrifugation and/or concentration, samples suitable for the present invention can be effectively obtained, without the need for ultra-high-speed centrifugation to process the samples. Maximize the protection of glycosylated exosomes in the sample.
  • Figure 1 Schematic diagram of the affinity adsorption centrifuge tube of the lectin-macromolecule carrier coupling complex.
  • Figure 2 Electron microscope observation pictures of the separation of glycosylated exosomes from 4 different types of samples (cell culture supernatant, plasma, urine, and cerebrospinal fluid).
  • Figure 3 NTA analysis of glycosylated exosomes after plasma sample separation.
  • Figure 4 Four different samples (cell culture supernatant, plasma, urine, cerebrospinal fluid) the Western Blot detection results of antibodies against CD9, CD63, and CD81 specific membrane proteins of exosomes.
  • Figure 5 Western Blot detection results of four different samples (cell culture supernatant, plasma, urine, cerebrospinal fluid) exosomes-specific non-membrane protein ALIX and TSG101 antibody.
  • Figure 6 Western blot detection results of Calnexin antibodies in 4 different samples (cell culture supernatant, plasma, urine, cerebrospinal fluid).
  • Figure 7 After washing the lectin-macromolecule carrier conjugate complex bound with glycosylated exosomes with 5 different washing buffers, the eluates obtained by eluting them are exosomal-specific The results of Western Blot detection of membrane protein CD9/CD63/CD81 antibodies.
  • Figure 8 Electron microscopy results of glycosylated exosomes separated from three different eluents.
  • the lectin-macromolecule carrier coupling complex includes: a macromolecular carrier; and a lectin coupled to the outside of the macromolecular carrier, which is prepared by coupling the lectin and the macromolecular carrier under specific conditions, and is mainly used for Separate the glycosylated exosomes in the sample, and the glycosylated exosomes obtained after separation are intact.
  • Lectin selection mainly includes: Jacalin, peanut agglutinin (PNA), pea agglutinin (VVA and/or VVL), concanavalin agglutinin (ConA), lentil agglutinin (LCA), wheat germ Any one of WGA, soybean agglutinin (SBA), kidney bean agglutinin (PVL), snail agglutinin (HAA and/or HPA), or a combination of two or more lectins, the main reasons are : The combination of different lectins can realize the separation of various types of glycosylated exosomes, such as: LCA, AAL separation of fucosylated exosomes; ConA, PVL, SBA, WGA, AAL, etc.
  • N-glycosylated exosomes by lectins Separation of N-glycosylated exosomes by lectins; separation of O-glycosylated exosomes by HAA, HPA, VVA, PNA, Jacalin and other lectins; single lectins are mainly used to separate specific lectins corresponding to lectins Glycosylated exosomes, the combined use of two or more lectins can be used to separate a variety of glycosylated exosomes in different forms, or the combined use of two or more lectins can be used for specific glycosylation The exosomes are separated synergistically.
  • Macromolecular carriers mainly refer to macromolecular microspheres, which mainly include any one of dextran microspheres, agarose microspheres, resin or epoxy resin microspheres, and polystyrene microspheres, or two or more of them
  • the macromolecular carrier is used in combination, and the particle size distribution range of the macromolecular carrier is 1 ⁇ m-200 ⁇ m, preferably 10 ⁇ m-200 ⁇ m, and more preferably 30 ⁇ m-150 ⁇ m.
  • the particle size of the macromolecular carriers produced in the same batch is not uniform. Therefore, the description of the particle size of the macromolecular carrier can usually adopt either the average particle size or the particle size of the present invention.
  • the description of the way of adopting the particle size distribution range can be considered that its specific distribution conforms or basically conforms to the normal distribution within this distribution range.
  • the particle size of the macromolecular carrier used is 10-1000 times the particle size of the macromolecular carrier generally used in the field of immunoassay, and a single macromolecular carrier has The larger surface area reduces the steric hindrance when the lectin is coupled to the macromolecular carrier, which facilitates the coupling of more lectins.
  • the general macromolecular carrier of the same total volume has a larger specific surface area.
  • lectin-macromolecule carrier coupling complex of the present invention 1-20mg of lectin can be coupled to each 1ml of the macromolecular carrier.
  • the above-mentioned lectin is coupled to the macromolecular carrier to form a lectin-macromolecule carrier coupling complex.
  • Different macromolecular carriers can be combined with the lectin after adjusting the steps of soaking, coupling, cleaning, and storage.
  • the most suitable macromolecular carrier for different lectins may be different; the ratio of macromolecular carrier (ml) to lectin (mg) during the preparation of the lectin-macromolecule carrier coupling complex is 1: 1-1:20, preferably 1:5-1:15, more preferably 1:10-1:15.
  • the prepared lectin-macromolecule carrier coupling complex is stored in the preservation solution, and the storage concentration (volume) of the lectin-macromolecule carrier coupling complex in the obtained lectin-macromolecule carrier coupling complex preservation solution
  • the ratio) is 10%-60%, preferably the ratio is 30%-50%, and more preferably 40%-50%;
  • the preservation solution of the lectin-macromolecule carrier coupling complex is packed in the upper part of the affinity adsorption centrifuge tube and centrifuged
  • the affinity adsorption centrifuge tube includes two parts: an upper centrifuge tube and an outer protective sleeve; the diameter of the upper centrifuge tube is smaller than the diameter of the outer protective sleeve, and the upper centrifuge tube is sleeved in the outer protective sleeve And there is a raised annular edge or pillar on the outer side of the upper part to support the opening of the upper centrifuge tube higher than the outer protective sleeve; the upper centrifuge tube
  • agarose microspheres with a particle size distribution range of 30 ⁇ m-150 ⁇ m.
  • the above agarose microspheres are commercially available hydrogen bromide activated agarose microspheres (purchased from Pharmacia or sigma), the agarose microspheres are further preferably: agarose 4B (sephrose 4B), agarose 6B (sephrose 6B), agarose FF (sephrose FF), agarose CL-4B (sephrose CL-4B), agarose Sugar CL-6B (sephrose CL-6B), the following examples all take Sepharose 4B (sephrose 4B) microspheres as an example.
  • the preparation process of the lectin-macromolecule carrier coupling complex that is, the LCA-agarose microsphere coupling complex, is as follows:
  • step a (2) Take 15ml of 0.1-1.0M carbonate buffer solution, add lentil lectin, and then add the fully swollen agarose 4B collected in step a, where the volume (ml) of the fully swollen agarose 4B is equal to The ratio of lentil agglutinin (LCA, mg) is 1:1-1:20 (that is, 1ml of fully swollen agarose 4B is mixed with 1-20mg lentil lectin), and the reaction time is 0.5h-5h at room temperature.
  • the ratio of lentil agglutinin (LCA, mg) is 1:1-1:20 (that is, 1ml of fully swollen agarose 4B is mixed with 1-20mg lentil lectin), and the reaction time is 0.5h-5h at room temperature.
  • the concentration of the carbonate buffer solution used in this example is 0.1M, and the ratio of the volume (ml) of fully swollen Sepharose 4B to the lentil agglutinin (LCA, mg) The ratio is 1:10, that is, 15ml of carbonate buffer solution is added with 5ml of fully swollen agarose 4B and 50mg LCA, and the reaction time is 3h at room temperature.
  • microsphere storage solution is ready for use, preferably configured as 30%-50% LCA-agarose microsphere storage solution. In order to better illustrate the present invention, in this embodiment, it is configured as 50% LCA-agarose by volume. Microsphere preservation solution.
  • the affinity adsorption centrifuge tube includes two parts: an upper centrifuge tube and an outer protective sleeve; the upper centrifuge tube has a smaller diameter than the outer sleeve, and there is a convex ring on the upper part
  • the edge or pillar is used to support the opening of the upper centrifuge tube higher than the outer sleeve; the upper centrifuge tube includes a centrifuge tube cover, a centrifuge tube wall, and a bottom of the filter membrane fixedly connected to the wall of the centrifuge tube.
  • the bottom of the filter membrane is used
  • the pore size of the filter membrane is smaller than the particle size of the lectin-macromolecule carrier coupling complex and at the same time larger than the particle size of the exosomes.
  • the lectin-macromolecule carrier coupling complex preservation solution is stored in the upper centrifuge tube,
  • the pore size of the filter membrane is preferably 150 nm-1000 nm.
  • the size of the upper centrifuge tube of the affinity adsorption centrifuge tube can be designed according to the size of the specific centrifuge device.
  • the volume of the lectin-macromolecule carrier coupling complex accounts for 1/2-1/4 of the volume of the upper centrifuge tube, such as 1.5- In the upper centrifuge tube with a volume of 2ml, aliquot the lectin-macromolecule carrier coupling complex preservation solution containing 0.5ml of the lectin-macromolecule carrier coupling complex into the tube.
  • the schematic diagram of the affinity adsorption centrifuge tube is shown in Figure 1.
  • the preservation solution in the preservation solution of the lectin-macromolecule carrier coupling complex can enter the outer protective sleeve through the filter membrane and stay in the outer protective sleeve without loss.
  • the lectin-macromolecule carrier coupling complex can be combined with sufficient storage solution In contact, it will not dry out and lose its activity.
  • the present invention provides a glycosylated exosome separation composition, which specifically includes: the affinity adsorption centrifuge tube containing LCA-agarose microsphere preservation solution obtained in Example 1, namely glycosylated exosomes
  • the separation device, the cleaning solution and the eluent are separately packaged and exist in a set or kit at the same time.
  • the above-mentioned cleaning solution is a metal salt-free cleaning buffer or purified water, and a metal salt-free cleaning buffer can be selected; for example, the main component includes 10-200 mM metal salt-free TRIS-HCl buffer with a pH of 7.6 ⁇ 0.2.
  • the cleaning solution is a metal salt-free cleaning buffer, and the main component includes 100 mM metal salt-free TRIS-HCl buffer with a pH of 7.6 ⁇ 0.2, which is used to clean and remove non-specific binding during separation. Exosomes and other impurities that are not glycosylated.
  • the above-mentioned eluent is a borate buffer solution with sugar dissolved
  • the main components include 10-20 mM borate buffer solution and 100-500 mM mannose dissolved therein, and the pH value is 6.5 ⁇ 0.2.
  • the sugar-dissolved borate buffer is the main component, including 15 mM borate buffer and 300 mM mannose dissolved in it, with a pH value of 6.5 ⁇ 0.2, which is used to elute binding in agglutination.
  • the glycosylated exosomes on the protein-macromolecule carrier coupling complex, the eluted exosomes are intact in appearance and shape, without damage or lysis.
  • the present invention also provides a method for separating glycosylated exosomes, including the experimental steps of using the above-mentioned glycosylated exosome separation composition, including:
  • Self-provided equipment or equipment centrifuge, used for the centrifugation step in the process of separating glycosylated exosomes; or use the automatic agarose microsphere separation equipment of the group company and its subsidiaries, mainly used for glycosylation Exosomes are separated automatically to achieve the purpose of saving manpower.
  • the automatic separation of glycosylated exosomes from agarose microspheres is only an automatic implementation of manual methods, and can achieve the same or better function than manual separation. This example uses centrifuge manual separation for further explanation.
  • Sample pre-processing adjust the method of sample pre-processing according to the characteristics of different clinical samples.
  • step (1) Take the 200-300ul pre-processed sample in step (1), which can be mixed directly with the lectin-macromolecule carrier coupling complex without dilution of the cleaning solution, or mixed with 1-2 times the volume of the cleaning solution After evenly diluting and mixing with the lectin-macromolecule carrier coupling complex, the effect will be better after dilution; in this case, the volume of the upper centrifuge tube of the affinity adsorption centrifuge tube is 2.0ml, of which 1mL is aliquoted /Tube of lectin-macromolecule carrier coupling complex preservation solution (containing 0.5mL/tube of lectin-macromolecule carrier coupling complex), the pre-processed sample selects a volume of 200ul, and the volume of the cleaning solution is 300ul.
  • the volume after mixing and dilution is the same as the volume of the lectin-macromolecule carrier coupling complex in the affinity adsorption centrifuge tube, and the volume of the sample diluted with the cleaning solution cannot be significantly larger than the lectin-macromolecule carrier in the affinity adsorption centrifuge tube
  • the coupling effect is best when the volume of the coupling complex is basically equal.
  • step (2) Take 500ul of the diluted sample in step (2) and add it to the upper centrifuge tube, incubate at room temperature for 10-30 min, preferably 10-15 min, then centrifuge the affinity adsorption centrifuge tube at 3000 rpm for 20 seconds at room temperature to remove Sample; in this case, the incubation time is selected as 10 min.
  • the glycosylated exosomes are intact without damage or lysis.
  • the volume of the eluate is 500ul, add the upper centrifuge tube, cover the centrifuge tube cap, let stand at room temperature for 5 minutes, centrifuge at 3000 rpm for 20 seconds at room temperature; collect the elution supernatant in the outer sleeve, which is
  • the separated glycosylated exosomes solution can be used directly for detection or stored at -80 ⁇ 5°C for later use.
  • the samples to be separated that can be used in the present invention are mainly: serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, and lymph.
  • Coupling complex such as 0.5ml
  • lectin-macromolecule carrier coupling complex add the serum, plasma, saliva, cerebrospinal fluid, lymphatic fluid, tissue or cell culture supernatant, urine
  • the volume of the pre-treated sample is 50-600ul, preferably 100-300ul, and more preferably 200-300ul.
  • the sample can be added directly or diluted with the cleaning solution before adding.
  • the serum, plasma, saliva, cerebrospinal fluid, and lymph samples are only centrifuged, and the volume of the sample is basically unchanged before and after centrifugation. Therefore, the sample size is 50-600ul, preferably 100-300ul, and more preferably 200-300ul.
  • Pre-processing can obtain the required volume of pre-processed samples; and the tissue or cell culture supernatant, urine samples in the sample pre-processing process, in addition to the centrifugation step, also need to perform 10-1000 Therefore, in order to obtain sufficient pre-processed samples, 1-50ml, preferably 10-50ml, and more preferably 30-50ml of the tissue or cell culture supernatant and urine samples need to be centrifuged Only with pre-treatment of concentration can a pre-treated sample with a required volume of 50-600ul, preferably 100-300ul, and more preferably 200-300ul be obtained.
  • sample separation volumes have a slight impact on the separation effect, but can basically achieve the separation effect of glycosylated exosomes.
  • serum, For plasma, saliva, cerebrospinal fluid, and lymph samples select 200ul pre-processed samples for illustration; select the volume of tissue or cell culture supernatant, urine sample volume to be 50ml, and the volume after concentration is preferably 200ul pre-processed The sample is explained.
  • sample specificity of serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, and lymph fluid waiting to be separated four sample types including cell culture supernatant, plasma, urine, and cerebrospinal fluid are selected to further explain that after replacing other sample types, the same separation effect can be achieved.
  • Example 3 separate the glycosylated exosomes in the 200ul pre-processed cell culture supernatant, plasma, urine, and cerebrospinal fluid samples in this example, and obtain the glycosylated exosomes from 4 different sample sources. Chemical exosomes separation solution for later use.
  • This case study mainly performs transmission electron microscopy on the glycosylated exosomes isolated in case 4 to observe the morphology and particle size of glycosylated exosomes.
  • the main steps are:
  • This implementation case mainly uses the NanoSight NS300 system to perform nanoparticle tracking analysis (NTA) on the glycosylated exosomes isolated in implementation case 4.
  • NTA nanoparticle tracking analysis
  • the NTA analysis is mainly in the solution state (in-situ test)
  • the following test its high-precision particle size distribution test, can distinguish particles with a relative particle size difference of about 1:1.5 times, allowing exosomal particles to be measured closer to their original state, ensuring the measurement data Authenticity and validity, providing reliable exosomes concentration data, effectively making up for the current electron microscope direct observation and measurement of exosomes, due to the limited scope of observation at one time, the obtained particle size distribution data are often not representative Defects, and can effectively reduce the damage of exosomes caused by different methods of drying, fixing and freezing in electron microscopy.
  • glycosylated exosomes separated from plasma samples are selected for NTA detection in this example.
  • the detailed steps are: dilute about 10ul of the sample to 1mL, and use The NanoSight syringe pump is loaded, and the NanoSight NS300 system automatically analyzes the glycosylated exosomes.
  • the average particle size of glycosylated exosomes is between 30-150nm, which is in line with the average particle size range of exosomes, and the particle size is 10 11 -10 14 Pcs/ml; see Figure 3: NTA analysis chart of glycosylated exosomes after plasma sample separation;
  • Sample preparation Take 30ul glycosylated exosomes eluate, add an equal volume of 5 ⁇ SDS-PAGE loading buffer, and treat at 100°C for 10 minutes, and then set aside;
  • Transfer membrane Place PVDF membrane of appropriate size in transfer buffer, soak for 10 minutes, close the gel with PVDF membrane and sponge pad to prevent bubbles from appearing, transfer the protein to PVDF membrane with a semi-dry membrane transfer instrument Up (constant voltage 40V, constant current 200mA, transfer film for 20 minutes)
  • This implementation case is mainly for different cleaning solutions, especially common cleaning solutions containing metal salt ions, common cleaning solutions not containing metal salt ions, and the cleaning effect and separation effect of purified water in the separation process of glycosylated exosomes Do further comparison and verification to determine the suitability of the cleaning solution selected in the present invention.
  • the metal salt-free buffer cleaning solution (Tris-HCL) in the glycosylated exosome separation composition in Examples 2 and 3 was replaced with purified water and a common cleaning solution containing metal salt ions.
  • Common cleaning solutions containing metal salt ions include: phosphate buffer (PBS buffer), Tris-Triton-NaCl buffer, TBST buffer, because most common cleaning solutions containing metal salt ions contain surfactants, such as Tween -20, Triton X-100, etc., surfactants can damage the outer membrane of exosomal vesicles. Therefore, when common cleaning solutions containing metal salt ions are configured, they do not contain surfactant ingredients.
  • the common cleaning solutions containing metal salt ions are phosphate buffered saline (PBS buffer), Tris-Triton-NaCl buffer without Triton X-100, and TBST buffer without Tween-20.
  • the composition of the above PBS buffer is: potassium dihydrogen phosphate (KH 2 PO4): 0.24 g/L, disodium hydrogen phosphate (Na 2 HPO4): 1.44 g/L, sodium chloride (NaCl): 8 g/L, Potassium chloride (KCl): 0.2g/L, pH value is 7.4 ⁇ 0.2.
  • composition of the above Triton X-100-free Tris-Triton-NaCl buffer solution is: 50mM Tris-HCL, 0.6MNaCL, and the pH value is 7.6 ⁇ 0.2.
  • composition of the above-mentioned Tween-20-free TBST buffer solution is: 10mM Tris-HCL, 0.15MNaCL, and a pH of 7.6 ⁇ 0.2.
  • the five washing buffers in this example are purified water, Tris-HCL buffer, PBS buffer, Tris-Triton-NaCl buffer without Triton X-100, and TBST buffer without Tween-20. According to the above The sequence is marked as No. 1 cleaning solution, No. 2 cleaning solution, No. 3 cleaning solution, No. 4 cleaning solution, and No. 5 cleaning solution. According to the method in implementation case 3, the five cleaning solutions in this implementation case were used to separate glycosylated exosomes in plasma samples, and comparative verification and analysis were performed. Except for the different cleaning solutions, other components and steps are the same, and other detailed procedures Refer to Example 1-4 and Example 7 for the Western Blot detection and identification steps of the exosome-specific membrane protein CD9/CD63/CD81 antibody.
  • This example is mainly aimed at different common protein eluates and the borate-mannose buffer of the present invention for further comparison and verification of the separation effect of glycosylated exosomes, so as to determine the eluate selected in the present invention. Suitability.
  • Example 2 and 3 Replace the borate-mannose buffer in the glycosylated exosome separation composition in Example 2 and 3 with the commonly used protein eluate and glycoprotein eluate, specifically the protein antibody elution buffer Stripping buffer and Glycoprotein Eluting Solution (Vectorlabs, USA).
  • the above-mentioned stripping buffer composition is: 62.5 mol/L Tris ⁇ HCl, 2% SDS, 100 mmol/L ⁇ -2-mercaptoethanol, pH 6.8 ⁇ 0.2.
  • the three eluates in this implementation case were used to separate glycosylated exosomes in plasma samples, and the comparative verification analysis was performed. Except for the different eluates, other components and steps are the same.
  • implementation case 1-5 Observe the morphology and integrity of the glycosylated exosomes in the plasma samples separated by the three eluates under the electron microscope.
  • results of this example show that the eluate of the present invention can effectively separate glycosylated exosomes in the sample, and the exosomes are intact and undamaged, and the particle size is between 30-150nm; stripping buffer is used as the elution Exosomes are not observed in the liquid, which may be stripping buffer as the eluent.
  • the outer membrane of the exosomes was damaged during the elution process, and complete exosomes could not be obtained; while the glycoprotein eluting solution Glycoprotein Eluting Solution Observation Less than typical exosomal vesicles, only suspected exosomal vesicles can be observed, but the shape of the exosomes is incomplete, indicating that the exosomes caused certain damage during the isolation process, which is not conducive to subsequent detection and analysis.
  • applying the content of the present invention to the separation of glycosylated exosomes from different sample sources can achieve the same effect, and the isolated exosomes are all glycosylated exosomes, after separation and purification Exosomes are intact and undamaged, and can be directly used for glycosylated exosomes liquid detection, exosomal immunological detection, or genetic analysis after nucleic acid extraction, etc.; it can also be further purified by subsequent purification steps to obtain higher purity Exosomes.
  • the present invention relates to a lectin-macromolecule carrier coupling complex for separating glycosylated exosomes in clinical samples.
  • the lectin-macromolecule carrier coupling complex includes: a macromolecular carrier; and Lectin attached to the outside of the macromolecular carrier.
  • the lectin-macromolecule carrier conjugate complex provided by the present invention can easily, quickly and accurately separate glycosylated exosomes in clinical samples, with high separation efficiency, good reproducibility, and complete exosomes after separation. Rupture or fragmentation can be directly used for glycosylated exosomal fluid detection, or directly for immunological related detection, or directly extract related nucleic acids from exosomes for genetic detection and analysis.

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Abstract

一种凝集素-大分子载体偶联复合物，用于分离临床样本中的糖基化外泌体，复合物包括大分子载体和偶联在大分子载体外侧的凝集素。复合物可以简便、快速、精准分离临床样本中的糖基化外泌体，分离效率高，重复性好，分离后的外泌体形态完整，无破裂或碎裂，可直接用于糖基化外泌体液体检测，或直接进行免疫学相关检测，或利用直接提取的外泌体内相关核酸进行基因检测分析。

Description

一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物
交叉引用
本发明要求在中国专利局提交的、申请号为202010060063.1、发明名称为“一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
技术领域
本发明属于生物医药及生物分离相关的技术领域，涉及一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物。
背景技术
外泌体是细胞多泡内体与质膜融合时释放产生的具有生物活性的微小囊泡，其直径约为30～150nm，是细胞间通讯的重要介质之一；在人健康的生理情况下，外泌体可以在细胞之间运送DNA、蛋白质、mRNA、miRNA等多种生物活性物质，参与细胞通讯、细胞迁移、肿瘤细胞增长等多种过程，完成细胞间物质及信息的传递。文献研究发现，几乎所有类型的细胞均会分泌外泌体，并且在血液、唾液、尿液、脑脊液、积液和乳汁等体液中都能够提取到外泌体。
外泌体与肿瘤、慢性感染性疾病、自身免疫病等很多疾病相关，当机体发生病变时，细胞分泌的外泌体的成分、含量、性质等都可能发生改变；糖基化外泌体在包括肿瘤在内的多种疾病的发生、发展过程中均有着重要作用，外泌体的检测研究能有效监测疾病的发生、发展；由于外泌体在样本中含量比较低，外泌体的分离富集对外泌体的研究至关重要。
目前，外泌体的分离方法很多，主要有：超速离心法或梯度密度离心法，免疫磁珠法等。其中：超速离心法或梯度密度离心法是基于外泌体与其他生物组份密度的微小差异进行分离，该方法需要特殊设备，样品需求量比较大，耗时长，且因为不同临床样本的特点不同，采用该方法的分离效果稳定性差，进一步地超速离心还容易造成囊泡损伤从而影响后续实验；免疫磁珠法的原理是外泌体表面的特异性蛋白如：CD9/CD63/CD81等与包被有对应抗体的免疫磁珠相结合，进行磁分离，但由于免疫磁珠体积较小，粒径为纳米级别且小于等于外泌体的直径，因此免疫磁珠与外泌体结合的空间位阻较大，外泌体结合不充分，分离效率不高。此外，采用免疫磁珠法分离获得的外泌体因为洗脱时使用酸性洗脱液进行洗脱，导致外泌体形态不完整。
因此，一种能用于精准、高效分离富集样本中的糖基化外泌体的组合物、分离装置及分离方法的研究建立是必须的。
发明内容
针对现有技术的不足，本发明提供了一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物，本发明还提供了一种含有该凝集素-大分子载体偶联复合物的糖基化外泌体分离组合物，以及使用该凝集素-大分子载体偶联复合物进行糖基化外泌体分离的方法及应用。本发明提供的凝集素-大分子载体偶联复合物可以精准分离临床样本中的糖基化外泌体，分离效率高，分离后的外泌体形态完整，无破裂或碎裂，可直接用于糖基化外泌体液体检测，或直接进行免疫学相关检测，或直接提取外泌体内相关核酸进行基因检测分析。本发明提供的糖基化外泌体分离的方法中，利用外泌体外表面丰富的糖链可以和凝集素结合的原理，采用有效地离心清洗及有效地洗脱方法可以分离糖基化外泌体，并且非常简便、快速，重复性好，分离后的外泌体形态完整，无破裂或碎裂，将其应用于疾病的发生、发展等过程中检测、监控及诊断。
本发明提供了一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物，大分子载体；和偶联在大分子载体外侧的凝集素；其中：
所述凝集素包括：木菠萝凝集素、花生凝集素、豌豆凝集素(VVA和/或VVL)、刀豆凝集素、小扁豆凝集素、麦胚素、大豆凝集素、芸豆凝集素、蜗牛凝集素(HAA和/或HPA)中的任意一种或者两种及以上；
所述大分子载体包括：葡聚糖微球、琼脂糖微球、树脂或环氧树脂微球、聚苯乙烯微球中的任意一种或者两种及以上。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述大分子载体粒径分布范围为1μm-200μm，优选为10μm-200μm，更进一步优选为30μm-150μm。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述凝集素-大分子载体偶联复合物中，每1ml大分子载体上偶联有1-20mg，优选为5-15mg，进一步优选为10-15mg凝集素。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述临床样本包括:血清、血浆、唾液、组织或细胞培养上清液、尿液、脑脊液、淋巴液的任意一种。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述凝集素包括：木菠萝凝集素、花生凝集素、豌豆凝集素(VVA和/或VVL)、刀豆凝集素、小扁豆凝集素、麦胚素、大豆凝集素、芸豆凝集素中的任意一种或者两种及以上。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述糖基化外泌体包括：N-糖基化外泌体、O-糖基化外泌体、岩藻糖基化外泌体中的任意一种或两种及以上。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述凝集素-大分子载体偶联复合物保存在保存液中，所获得的凝集素-大分子载体偶联复合物保存液中的凝集素-大分子载体偶联复合物的保存浓度(体积比)为10％-60％，优选比例为30％-50％，进一步优选为40％-50％；凝集素-大分子载体偶联复合物保存液的体积是指凝集素-大分子偶联复合物分散在保存液中后，其与保存液的总体积。
上述凝集素-大分子载体偶联复合物在某些实施方式中，所述凝集素-大分子载体偶联复合物保存液分装于包括亲和吸附离心管的分离装置中；所述亲和吸附离心管包括：上部离心管和外部保护套管两部分；所述上部离心管的直径小于外部保护套管的直径，所述上部离心管套设于外部保护套管 内且在其上部外侧存在凸起的环形边缘或支柱用于支撑上部离心管的开口处高于外部保护套管；所述上部离心管包括离心管盖、离心管壁和与离心管壁固定连接的过滤膜底部，过滤膜底部所使用的过滤膜的孔径小于凝集素-大分子载体偶联复合物的粒径，同时大于外泌体的粒径，所述凝集素-大分子载体偶联复合物保存液保存于上部离心，组成糖基化外泌体的分离装置，过滤膜孔径优选为150nm-1000nm。
上述凝集素-大分子载体偶联复合物在某些实施方式中，分装于上部离心管中的凝集素-大分子载体偶联复合物保存液中的凝集素-大分子载体偶联复合物体积占上部离心管体积的1/2-1/4。
本发明另一方面还提供了一种糖基化外泌体分离组合物，所述糖基化外泌体分离组合物包括：上述凝集素-大分子载体偶联复合物，还包括用于清洗去除分离过程中非特异性结合的、未发生糖基化的外泌体及其他杂质的清洗液和/或用于洗脱特异性结合在凝集素-大分子载体偶联复合物上的糖基化外泌体的洗脱液。这里的组合物并非是指其混合为一体，而是指其同时存在于一个套装中。
上述糖基化外泌体分离组合物在某些实施方式中，所述清洗液为无金属盐离子清洗缓冲液或纯化水，可选为无金属盐离子清洗缓冲液，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质。
上述糖基化外泌体分离组合物在某些实施方式中，所述洗脱液为溶解有糖的硼酸盐缓冲液，可选地为溶有甘露糖的硼酸盐缓冲液，进一步可选地pH值为6.5±0.2，用于洗脱结合在凝集素-大分子载体偶联复合物上的糖基化外泌体，洗脱后的外泌体外观形态完整，无破损或裂解。
本发明另一方面还提供了一种糖基化外泌体分离方法，包括应用上述的凝集素-大分子载体偶联复合物分离糖基化外泌体的分离步骤，所述分离步骤包括：
临床样本前处理：
取前处理后的样本，可选地与清洗液混合均匀进行稀释，作为待检测样本；
完全去除凝集素-大分子载体偶联复合物保存液中的保存液部分，保留凝集素-大分子载体偶联复合物；
取待检测样本加入到除去保存液后的凝集素-大分子载体偶联复合物中，室温静置孵育，去除样本；
取清洗液清洗已结合有糖基化外泌体的凝集素-大分子载体偶联复合物，去除清洗液，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质；
取洗脱液，洗脱清洗后的凝集素-大分子载体偶联复合物，室温静置，收集洗脱上清液，即为分离后的糖基化外泌体溶液。
上述糖基化外泌体分离方法在某些实施方式中，待检测样本与凝集素-大分子载体偶联复合物的比例为1：1-3；
和/或，单次加入清洗液的体积与已结合有糖基化外泌体的凝集素-大分子载体偶联复合物体积的比例为1-3：1；
和/或，清洗液清洗步骤重复进行1-3次；
和/或，加入洗脱液的体积与清洗后的凝集素-大分子载体偶联复合物体积的比例为0.5-2：1，优选为0.5-1：1。
上述糖基化外泌体分离方法在某些实施方式中，完全去除凝集素-大分子载体偶联复合物保存液中的保存液部分、去除样本、去除清洗液、和/或，收集洗脱上清液的方式为3000rpm以下的速度、室温下离心20秒。
上述糖基化外泌体分离方法在某些实施方式中，室温静置孵育10-30min，优选为10-15min。
上述糖基化外泌体分离方法在某些实施方式中，样本前处理的方法包括以下步骤：
对血清、血浆、唾液、脑脊液或淋巴液样本，将样本经3000g以下速度离心10-15min，去除样本中的细胞碎片、沉淀物杂质，取离心后上清液备用；
对组织或细胞培养上清液、尿液的样本，将样本经3000g以下速度离心10-15min，去除样本中的细胞碎片、沉淀物杂质，然后将上清液通过超滤管进行10-1000倍浓缩后备用。
上述糖基化外泌体分离方法在某些实施方式中，所述清洗液为无金属盐离子的清洗缓冲液或纯化水，可选为无金属盐离子的清洗缓冲液，进一步可选为pH值7.6±0.2的无金属盐离子的清洗缓冲液。
上述糖基化外泌体分离方法在某些实施方式中，所述洗脱液为溶有糖的硼酸盐缓冲液，可选地为溶有甘露醇的硼酸盐缓冲液，进一步可选地为pH值6.5±0.2的硼酸盐-甘露糖缓冲液。
上述糖基化外泌体分离方法在某些实施方式中，孵育的条件为：室温孵育1-30min，优选为5-20min，进一步优选为10-15min。
上述糖基化外泌体分离方法在某些实施方式中，包括应用上述凝集素-大分子载体偶联复合物分离糖基化外泌体和分离装置的分离步骤，所述分离步骤包括：
临床样本前处理：
取前处理后的样本，可选地与清洗液混合均匀进行稀释，作为待检测样本；
装载有凝集素-大分子载体偶联复合物的亲和吸附离心管完全去除凝集素-大分子载体偶联复合物保存液中的保存液部分，保留凝集素-大分子载体偶联复合物，并更换新的外部套管；
取待检测样本加入到除去保存液后的上部离心管中，室温静置孵育，去除样本；
取清洗液加入上部离心管中，将亲和吸附离心管离心后将上部离心管取出，装入新的外部套管中，将原外部套管及其中液体全部弃去，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质；本步骤进行1-3次；
取洗脱液，加入上部离心管，洗脱清洗后的凝集素-大分子载体偶联复合物，室温静置，收集外部套管中的洗脱上清液，即为分离后的糖基化外泌体溶液，洗脱分离后糖基化外泌体形态完整，无破损或裂解。
本发明还提供了一种使用上述方法分离的外泌体的应用，所述应用包括：糖基化外泌体液体检测、外泌体免疫学检测，或外泌体中核酸提取后的核苷酸片段检测分析。
有益效果：
1、本发明凝集素-大分子载体偶联复合物，分离得到的外泌体均为糖基化外泌体，其形态完整，无破损，可直接用于糖基化外泌体液体检测，外泌体免疫学检测，或核酸提取后的核苷酸序列检测分析等，还可以进一步通过配合相应的设备实现糖基化外泌体的全自动分离。
2、本发明凝集素-大分子载体偶联复合物中，所使用的大分子载体的粒径为一般用于免疫检测领域的大分子载体粒径的10-1000倍，单个大分子载体上具有更大表面积，减小了凝集素与大分子载体偶联时的空间位阻，利于偶联更多的凝集素，相比之下，同样总体积的一般大分子载体虽然具有更大的比表面积，但由于空间位阻较大，偶联上的凝集素反而更少，本发明的凝集素-大分子载体偶联复合物中，每1ml大分子载体上可以偶联1-20mg凝集素，而研究发现使用体积较小的大分子载体时，每1ml大分子载体偶联凝集素的数量为ng级别，偶联效率低；同时该粒径也远远大于外泌体本身的直径，可以减少凝集素和糖基化外泌体结合的空间位阻，有利于凝集素与糖基化外泌体结合，大幅提高外泌体的分离效率。
3、本发明凝集素-大分子载体偶联复合物，对于外泌体含量较少的临床样本，如尿液、组织或细胞培养上清液中的外泌体也有很好的效果；对于外泌体含量较多的临床样本，如血浆中的外泌体进行分离，分离所得糖基化外泌体的浓度可达10 11-14个/ml。
4、本发明凝集素-大分子载体偶联复合物中，所采用的凝集素优选植物凝集素，包括：木菠萝凝集素、花生凝集素、豌豆凝集素(VVA和/或VVL)、刀豆凝集素、小扁豆凝集素、麦胚素、大豆凝集素、芸豆凝集素，植物凝集素的大量获取更容易，原料更加丰富。
5、本发明凝集素-大分子载体偶联复合物，出厂时即可分装于特定结构的亲和吸附离心管中组成糖基化外泌体的分离装置，既方便用户使用，又利于凝集素-大分子载体偶联复合物的保存，过滤膜孔径的选择利于各种保存液、清洗液、洗脱液的顺利通过，尤其是洗脱液中添加有甘露糖，会导致洗脱液的粘度增大，该孔径下能保证洗脱液的顺利通过，也能保证凝集素-大分子载体偶联复合物无漏出。
6、本发明糖基化外泌体分离组合物，所使用的是无金属盐离子的清洗缓冲液或纯化水，金属盐离子对凝集素与糖基化外泌体的亲和吸附具有解离作用，影响凝集素与糖基化外泌体的亲和吸附，且金属盐离子浓度越高，解离作用越强，从而影响外泌体的分离效果，甚至无法分离外泌体。
7、本发明糖基化外泌体分离组合物，所使用的洗脱液是硼酸盐缓冲液，相比其他缓冲液，具有针对外泌体的更好的洗脱效果，洗脱效率高，使用少量的洗脱液即可完成洗脱，洗脱后的外泌体外观形态完整，无破损或裂解，同时由于使用量较少，相当于同时完成了浓缩外泌体的过程。
8、本发明糖基化外泌体分离方法，样品前处理的方法简单易行，通过简单的离心和/或浓缩，即可有效获得适用于本发明的样本，无需超高速离心处理样本，可以最大程度地保护样本中的糖基化外泌体。
附图说明
图1：凝集素-大分子载体偶联复合物的亲和吸附离心管示意图。
图2：4种不同样本(细胞培养上清、血浆、尿液、脑脊液)类型分离糖基化外泌体电镜观察图 片。
图3：血浆样本分离后糖基化外泌体的NTA分析图。
图4：4种不同样本(细胞培养上清、血浆、尿液、脑脊液)外泌体特异性膜蛋白CD9、CD63、CD81抗体Western Blot检测结果图。
图5：4种不同样本(细胞培养上清、血浆、尿液、脑脊液)外泌体特异性非膜蛋白ALIX和TSG101抗体的Western Blot检测结果图。
图6：4种不同样本(细胞培养上清、血浆、尿液、脑脊液)外泌体Calnexin抗体的Western Blot检测结果图。
图7：5种不同清洗缓冲液对结合有糖基化外泌体的凝集素-大分子载体偶联复合物进行清洗后，再对其进行洗脱所得洗脱液的进行外泌体特异性膜蛋白CD9/CD63/CD81抗体的Western Blot检测结果图。
图8：3种不同洗脱液分离得到的糖基化外泌体溶液的电镜检测结果图。
具体实施方式
为更好的说明本发明，现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。本申请说明书和实施案例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。除非特殊说明，以下所用到的各种试剂均为商品化试剂，其中所用的化学试剂不低于分析纯。
实施案例1凝集素-大分子载体偶联复合物制备
凝集素-大分子载体偶联复合物包括：大分子载体；和偶联在大分子载体外侧的凝集素，其是通过凝集素和大分子载体在特定条件下偶联制备而成，主要用于分离样本中的糖基化外泌体，分离后得到的糖基化外泌体形态完整。其中：
凝集素选用主要包括：木菠萝凝集素(Jacalin)、花生凝集素(PNA)、豌豆凝集素(VVA和/或VVL)、刀豆凝集素(ConA)、小扁豆凝集素(LCA)、麦胚素(WGA)、大豆凝集素(SBA)、芸豆凝集素(PVL)、蜗牛凝集素(HAA和/或HPA)中的任意一种，或者两种及两种以上凝集素联合使用，主要原因为：不同凝集素之间的组合可以实现各种类型糖基化外泌体的分离，如：LCA、AAL对岩藻糖基化外泌体的分离；ConA、PVL、SBA、WGA、AAL等凝集素对N-糖基化外泌体的分离；HAA、HPA、VVA、PNA、Jacalin等凝集素对O-糖基化外泌体的分离；单一凝集素主要用于分离与凝集素对应的特定糖基化外泌体，两种及两种以上凝集素联合使用可以用于分离多种不同形态的糖基化外泌体，或者两种或两种以上凝集素联合使用可以针对特定糖基化外泌体进行协同增效地分离。
为更进一步说明本发明，以下实施案例主要采用小扁豆凝集素(LCA)(购自于美国sigma公司)对本发明进行更进一步说明。
大分子载体主要是指大分子微球，主要包括：葡聚糖微球、琼脂糖微球、树脂或环氧树脂微球、聚苯乙烯微球中的任意一种，或者两种及以上的大分子载体联合使用，所述大分子载体粒径分布范围为1μm-200μm，优选为10μm-200μm，更进一步优选为30μm-150μm。在大分子载体的生产中，同一批次生产的大分子载体的粒径也不是均一的，因此大分子载体的粒径描述通常既可以采用平均粒径的方式，也可以采用本发明中粒径分布范围的方式，对于采用粒径分布范围的方式的描述可以认为其具体分布符合或基本符合在此分布范围内的正态分布。本发明实施例凝集素-大分子载体偶联复合物中，所使用的大分子载体的粒径为一般用于免疫检测领域的大分子载体粒径的10-1000倍，单个大分子载体上具有更大表面积，减小了凝集素与大分子载体偶联时的空间位阻，利于偶联更多的凝集素，相比之下，同样总体积的一般大分子载体虽然具有更大的比表面积，但由于空间位阻较大，偶联上的凝集素反而更少，本发明的凝集素-大分子载体偶联复合物中，每1ml大分子载体上可以偶联1-20mg凝集素，而研究发现使用体积较小的大分子载体时，每1ml大分子载体偶联凝集素的数量为ng级别，偶联效率低；同时该粒径也远远大于外泌体本身的直径，可以减少凝集素和糖基化外泌体结合的空间位阻，有利于凝集素与糖基化外泌体结合，大幅提高外泌体的分离效率。
上述凝集素与大分子载体偶联结合，形成凝集素-大分子载体偶联复合物，不同大分子载体通过调节浸泡、偶联、清洗、保存等步骤后，均能达到与凝集素偶联结合的效果，当然针对不同凝集素的最适大分子载体可能是不同的；在凝集素-大分子载体偶联复合物制备过程中大分子载体(ml)与凝集素(mg)的比例为1:1-1:20，优选为1:5-1:15，进一步优选为1:10-1:15。
制备后的凝集素-大分子载体偶联复合物于保存液中进行保存，凝集素-大分子载体偶联复合物在所得凝集素-大分子载体偶联复合物保存液中的保存浓度(体积比)为10％-60％，优选比例为30％-50％，进一步优选为40％-50％；凝集素-大分子载体偶联复合物保存液分装于亲和吸附离心管的上部离心管中；所述亲和吸附离心管包括：上部离心管和外部保护套管两部分；所述上部离心管的直径小于外部保护套管的直径，所述上部离心管套设于外部保护套管内且在上部外侧存在凸起的环形边缘或支柱用于支撑上部离心管的开口处高于外部保护套管；所述上部离心管包括离心管盖、离心管壁和与离心管壁固定连接的过滤膜底部，过滤膜底部所使用的过滤膜的孔径小于凝集素-大分子载体偶联复合物的粒径，同时大于外泌体的粒径，过滤膜孔径优选为150nm-1000nm，用于装载凝集素-大分子载体偶联复合物，组成糖基化外泌体的分离装置。
为更进一步说明本发明，以下实施案例均采用粒径分布范围30μm-150μm的琼脂糖微球进行说明，上述琼脂糖微球为商品化的溴化氢活化琼脂糖微球(购自Pharmacia公司或sigma公司)，所述琼脂糖微球进一步优选为：琼脂糖4B(sephrose 4B)、琼脂糖6B(sephrose6B)、琼脂糖FF(sephrose FF)、琼脂糖CL-4B(sephrose CL-4B)、琼脂糖CL-6B(sephrose CL-6B)，以下实施案例均以琼脂糖4B(sephrose 4B)微球为例进行说明。
凝集素-大分子载体偶联复合物，即LCA-琼脂糖微球偶联复合物的制备过程详情如下：
(1)称取活化琼脂糖4B 1g，加入pH2-3的1mM HCl溶液不少于200ml，混匀浸泡直至完全溶胀，用pH2-3的1mM HCl溶液在玻璃砂芯漏斗中清洗不低于15min，清洗结束后，收集完全溶胀的琼脂糖4B备用，完全溶胀的琼脂糖4B约3-5ml。
(2)取0.1-1.0M的碳酸盐缓冲液溶液15ml，加入小扁豆凝集素，然后加入步骤a中收集的完全溶胀的琼脂糖4B，其中完全溶胀的琼脂糖4B体积(ml)与小扁豆凝集素(LCA，mg)的比例为1:1-1:20(即每1ml完全溶胀的琼脂糖4B与1-20mg小扁豆凝集素混合)，室温混匀反应时间0.5h-5h，去上清；为更好的说明本发明，本实施案例中碳酸盐缓冲液溶液的使用浓度为0.1M，完全溶胀的琼脂糖4B体积(ml)与小扁豆凝集素(LCA，mg)的比例为1:10，即15ml碳酸盐缓冲液溶液加入5ml完全溶胀的琼脂糖4B和50mg LCA，室温混匀反应时间3h。
(3)加入100ml 0.1M碳酸盐缓冲液，混匀去上清；然后加入200ml纯化水，混匀去上清。
(4)用不少于完全溶胀的琼脂糖4B体积10倍体积的0.1mol/L pH4.0的醋酸盐缓冲液(含0.5mol/L的NaCl)和0.1mol/L pH8.0的Tris-HCl缓冲液(含0.5mol/L的NaCl)依次反复洗涤至少3次。
(5)加入0.1mol/L pH8.0的TRIS-HCl缓冲液洗涤一次，收集洗涤后的完全溶胀的琼脂糖4B，加入TRIS-HCl缓冲液，配置成10％-60％的LCA-琼脂糖微球保存液备用，优选配置成30％-50％的LCA-琼脂糖微球保存液，为更好的说明本发明，在本实施案例中均配置成体积比为50％的LCA-琼脂糖微球保存液。
(6)将步骤(5)中的50％的LCA-琼脂糖微球保存液混匀后，均匀分装于包括亲和吸附离心管的分离装置中，组成糖基化外泌体的分离装置，静置密封，2-8℃保存备用；其中亲和吸附离心管包括：上部离心管和外部保护套管两部分；所述上部离心管直径小于外部套管，且在上部存在凸起的环形边缘或支柱用于支撑上部离心管的开口处高于外部套管；所述上部离心管包括离心管盖、离心管壁和与离心管壁固定连接的过滤膜底部，所述过滤膜底部所使用的过滤膜的孔径小于凝集素-大分子载体偶联复合物的粒径，同时大于外泌体的粒径，所述凝集素-大分子载体偶联复合物保存液保存于上部离心管中，可选地，过滤膜孔径优选为150nm-1000nm。亲和吸附离心管的上部离心管的尺寸可以根据具体离心设备的尺寸进行设计，凝集素-大分子载体偶联复合物体积占上部离心管体积的1/2-1/4，如选择1.5-2ml体积的上部离心管，在管中分装含有0.5ml凝集素-大分子载体偶联复合物的凝集素-大分子载体偶联复合物保存液，亲和吸附离心管示意图详见图1。凝集素-大分子载体偶联复合物保存液中的保存液可以通过过滤膜进入外部保护套管中并留存于外部保护套管中不会流失。又由于上部离心管和外部保护套管的套设结构以及凝集素-大分子载体偶联复合物保存液中的体积浓度选择，使得凝集素-大分子载体偶联复合物能够与足够的保存液接触，不会干燥失去活性。
实施案例2糖基化外泌体分离组合物制备
本发明提供了一种糖基化外泌体分离组合物，具体包括：实施案例1中所得的分装有LCA-琼脂糖微球保存液的亲和吸附离心管、即糖基化外泌体的分离装置，和清洗液、洗脱液，分别独立包装，同时存在于一个套装或试剂盒中。
上述清洗液为无金属盐离子清洗缓冲液或纯化水，可选为无金属盐离子清洗缓冲液；如：主要成分包括10-200mM无金属盐离子的TRIS-HCl缓冲液，其pH值为7.6±0.2。在本实施案例中，清洗液为无金属盐离子清洗缓冲液，主要成分包括100mM无金属盐离子的TRIS-HCl缓冲液，其pH值为7.6±0.2，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质。
上述洗脱液为溶解有糖的硼酸盐缓冲液，如：主要成分包括10-20mM的硼酸盐缓冲液和溶于其中的100-500mM的甘露糖、pH值6.5±0.2。在本实施案例中，溶解有糖的硼酸盐缓冲液为主要成分包括15mM的硼酸盐缓冲液和溶于其中的300mM的甘露糖，其pH值6.5±0.2，用于洗脱结合在凝集素-大分子载体偶联复合物上的糖基化外泌体，洗脱后的外泌体外观形态完整，无破损或裂解。
实施案例3糖基化外泌体分离组合物的使用方法
本发明还提供了一种糖基化外泌体分离方法，包括使用上述糖基化外泌体分离组合物的实验步骤，包括：
1、实验前准备
自备器材或设备：离心机，用于分离糖基化外泌体过程中的离心步骤；或使用本集团公司及旗下子公司的全自动琼脂糖微球分离仪器，主要用于糖基化外泌体全自动分离，达到节约人力的目的。全自动琼脂糖微球分离糖基化外泌体只是对手工方式的自动化实现，并能达到等同或优于手工分离的功能，本实施案例采用离心机手工分离进行进一步说明。
2、试验流程
(1)样本前处理：根据不同临床样本的特性调整样本前处理的方法。
对所述血清、血浆、唾液、脑脊液、淋巴液样本而言，将样本经3000g离心10-15min以去除样本中的细胞碎片、沉淀物等杂质，将离心后上清液备用；为了更清楚地说明，在本实施案例中离心10min；
对所述组织或细胞培养上清液、尿液的样本而言，将样本经3000g离心10-15min以去除样本中的细胞碎片、沉淀物等杂质，然后将上清液通过超滤管进行10-1000倍浓缩，将浓缩后的上清液备用；为了更清楚地说明，在本实施案例中离心10min。
(2)取步骤(1)中的200-300ul经过前处理的样本，既可以不用清洗液稀释直接与凝集素-大分子载体偶联复合物混合，也可以与1-2倍体积清洗液混合均匀进行稀释后再与凝集素-大分子载体偶联复合物混合，稀释后的效果更好；在本实施案例中，亲和吸附离心管的上部离心管体积为2.0ml，其中分装有1mL/管的凝集素-大分子载体偶联复合物保存液(内含0.5mL/管凝集素-大分子载体偶联复合物)，经过前处理的样本选择体积为200ul，清洗液体积为300ul，混匀稀释后体积与亲和吸附离心管中的凝集素-大分子载体偶联复合物体积相同，经过清洗液稀释的样本的体积不能显著大于亲和吸附离心管中的凝集素-大分子载体偶联复合物的体积，二者体积基本相等时的结合效果最好。
(3)取出装载有凝集素-大分子载体偶联复合物的亲和吸附离心管的上部离心管，装入新的外部套管中，3000rpm室温下离心20秒，去除凝集素-大分子载体偶联复合物保存液中的保存液部分，保留凝集素-大分子载体偶联复合物，并更换新的外部套管。
(4)取步骤(2)中已经稀释好的样本500ul加入上部离心管中，室温静置孵育10-30min，优选为10-15min，之后将亲和吸附离心管于3000rpm室温下离心20秒去除样本；在本实施案例中，孵育时间选择为10min。
(5)取500ul-1500ul、pH值7.6±0.2的无金属盐离子清洗液100mM TRIS-HCl缓冲液加入上部离心管中，将亲和吸附离心管于3000转室温下离心20秒，将上部离心管取出，装入新的外部套 管中，将原外部套管及其中液体全部弃去，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质；重复步骤(5)1-3次。在本实施案例中，无盐离子清洗液体积为1ml。
(6)取与亲和吸附离心管中的凝集素-大分子载体偶联复合物体积相同的、pH值6.5±0.2的硼酸盐-甘露糖缓冲液作为洗脱液加入上部离心管，其中洗脱液中包括15mM的硼酸盐缓冲液和溶于其中的300mM的甘露糖，洗脱清洗后的凝集素-大分子载体偶联复合物，室温静置5-10min，3000rpm室温下离心20秒；收集外部套管中的洗脱上清液，即为分离后的糖基化外泌体溶液，洗脱分离后糖基化外泌体形态完整，无破损或裂解。在本实施案例中，洗脱液体积为500ul，加入上部离心管，盖上离心管盖，室温静置5min，3000rpm室温下离心20秒；收集外部套管中的洗脱上清液，即为分离后的糖基化外泌体溶液，直接用于检测或于-80±5℃保存备用。
实施案例4
本实施案例主要对糖基化外泌体的分离样本作进一步说明，可用于本发明的待分离样本主要为：血清、血浆、唾液、组织或细胞培养上清液、尿液、脑脊液、淋巴液的任意一种；针对常规1.5-2ml上部离心管，可以分装有1mL/管的凝集素-大分子载体偶联复合物保存液(内含有0.1-0.6ml/管的凝集素-大分子载体偶联复合物，如0.5ml)，针对600ul的凝集素-大分子载体偶联复合物，则加入所述血清、血浆、唾液、脑脊液、淋巴液、组织或细胞培养上清液、尿液的经过前处理的样本体积50-600ul，优选为100-300ul，进一步优选为200-300ul，该样本可直接加入也可清洗液稀释后再加入。在样本前处理过程中，血清、血浆、唾液、脑脊液、淋巴液样本只经过离心，样本离心前后体积基本无变化，因此对50-600ul，优选为100-300ul，进一步优选为200-300ul的样本进行前处理即可获得所需体积的经过前处理的样本；而所述组织或细胞培养上清液、尿液的样本在样本前处理过程中，除了离心步骤之外，还需要进行10-1000倍浓缩，所以为了获得足够的经过前处理的样本，需要对1-50ml，优选为10-50ml，进一步优选为30-50ml的所述组织或细胞培养上清液、尿液的样本进行包括离心和浓缩的前处理才能获得所需体积50-600ul，优选为100-300ul，进一步优选为200-300ul的经过前处理的样本。
不同的样本分离体积对分离效果略有影响，但基本均能达到糖基化外泌体的分离效果，为更进一步说明本发明，在本实施案例中，依据实施案例3的分离步骤，血清、血浆、唾液、脑脊液、淋巴液样本均选择200ul经过前处理的样本进行说明；组织或细胞培养上清液、尿液的样本体积均选择体积为50ml，浓缩后体积优选为200ul的经过前处理的样本进行说明。
依据血清、血浆、唾液、组织或细胞培养上清液、尿液、脑脊液、淋巴液等待分离样本的样本特异性，分别选取细胞培养上清、血浆、尿液、脑脊液等4种样本类型做进一步说明，将其他样本类型替换后，也能达到相同的分离效果。
依据实施案例3的步骤，分别分离本实施案例中200ul的经过前处理的细胞培养上清、血浆、尿液、脑脊液样本中的糖基化外泌体，分别得到4种不同样本来源的糖基化外泌体分离溶液备用。
实施案例5
本实施案例主要对实施案例4中分离得到的糖基化外泌体进行透射电镜检测，用于观察糖基化外泌体的形态及粒径大小，主要步骤为：
吸取约5ul实施案例4中分离得到的糖基化外泌体样本，滴在铜网上，静置4-5min，用滤纸从铜网边缘吸去多余的液体，滴上负染色液(0.5％的醋酸铀水溶液，pH4.5)，在铜网上反应1min，用滤纸吸干，重复清洗2次，晾干后，进行Tecnai Spirit(120kV TEM)透射电镜观测。透射电镜观察实施案例4中的四种不同样本分离的糖基化外泌体，外泌体形态完整、无破损，粒径均在30-150nm之间，详见图2中4种不同样本类型分离糖基化外泌体电镜观察图片。
实施案例6：
本实施案例主要对实施案例4中分离得到的糖基化外泌体使用NanoSight NS300系统对外泌体粒度和粒径进行纳米颗粒跟踪分析(NTA)，NTA分析主要是在溶液状态(原位测试)下的测试，其高精度的粒度数量分布测试，可以分辨出相对粒径差异在1：1.5倍左右的颗粒，能够让外泌体颗粒在更接近其原始状态下进行测量，保证了测量数据的真实性和有效性，提供可靠的外泌体浓度数据，有效弥补目前电镜直接观察和测量外泌体时，由于一次所能观察到的范围有限，所获得的粒径分布数据往往不具代表性的缺陷，并能有效降低电镜观察的干燥、固定以及冷冻等不同方式的前处理带来的外泌体损伤。
基于实施案例5中电镜观察4种不同样本类型结果的一致性，在本实施案例中选取血浆样本分离的糖基化外泌体进行NTA检测，详细步骤为：将约10ul样本稀释到1mL，采用NanoSight注射泵上样，NanoSight NS300系统自动分析，糖基化外泌体较为均一，平均粒径均在30-150nm之间，符合外泌体的平均粒径范围，粒度均在10 11-10 14个/ml；详见图3：血浆样本分离后糖基化外泌体的NTA分析图；
实施案例7
本实施案例主要对实施案例4分离得到的糖基化外泌体进行外泌体特异性膜蛋白CD9/CD63/CD81、外泌体特异性非膜蛋白ALIX和TSG101、外泌体Calnexin抗体Western Blot检测和鉴定，主要检测步骤如下：
(1)样本准备：取30ul糖基化外泌体洗脱液，加入等体积5×SDS-PAGE上样缓冲液，100℃处理10分钟，备用；
(2)SDS电泳：配置凝胶(15％分离胶、5％浓缩胶)，加入1×电泳缓冲液，加样本10μl/孔，设置60V，120min的电泳条件；
(3)转膜：将合适大小PVDF膜，放置到转移缓冲液，浸泡10min，将凝胶与PVDF膜以及海绵垫紧密贴合，防止出现气泡，用半干转膜仪将蛋白转至PVDF膜上(恒压40V，恒流200mA，转膜20分钟)
(4)封闭：5％脱脂奶粉封闭液封闭1小时，TBST清洗3次，每次5分钟；
(5)孵育：分别加入一抗(Anti-CD9、Anti-CD81、Anti-CD63、Anti-TSG101、Anti-Alix、Anti-Calnexin单抗，浓度1mg/mL，1:400稀释)孵育1小时，TBST清洗3次，每次5分钟；加入二抗(羊抗鼠HRP 1mg/mL，1:5000稀释)孵育1小时，TBST清洗3次，每次5分钟；
(6)显示：使用BeyoECL Moon(极超敏ECL化学发光试剂盒)显色后进行检测，
实施案例4分离得到的4种糖基化外泌体的Western Blot检测结果分别为：
(1)外泌体特异性膜蛋白CD9、CD63、CD81抗体Western Blot检测均为阳性，说明具有外泌体特异性膜蛋白CD9、CD63、CD81；详见图4：外泌体特异性膜蛋白CD9、CD63、CD81抗体Western Blot检测结果图；
(2)外泌体特异性非膜蛋白ALIX和TSG101抗体的Western Blot检测均为阳性，说明具有外泌体特异性非膜蛋白ALIX和TSG101；详见图5：外泌体特异性非膜蛋白ALIX和TSG101抗体的Western Blot检测结果图；
(3)外泌体样本中Calnexin抗体的Western Blot检测均为阴性。详见图6：外泌体Calnexin抗体的Western Blot检测结果图。Calnexin存在于细胞内质网上，不存在于外泌体中，本实施例分离样本得到糖基化外泌体的Calnexin抗体的Western Blot检测均为阴性，说明分离后的糖基化外泌体样本中没有细胞碎片存在。
实施案例8
本实施案例主要是针对不同清洗液，尤其是包含金属盐离子的常见清洗液、不包含金属盐离子的常见清洗液、和纯化水对糖基化外泌体分离过程中的清洗效果及分离效果做进一步对比验证，以确定本发明中所选择清洗液的适宜性。
将实施案例2和3中的糖基化外泌体分离组合物中的无金属盐离子缓冲液清洗液(Tris-HCL)替换为纯化水、以及常见的含金属盐离子的清洗液。常见的含金属盐离子的清洗液包括：磷酸盐缓冲液(PBS缓冲液)、Tris-Triton-NaCl缓冲液、TBST缓冲液，由于常见含金属盐离子的清洗液大都含有表面活性剂，如Tween-20、Triton X-100等，表面活性剂会对外泌体囊泡的外膜造成破坏，因此，在配置常见的含金属盐离子的清洗液时均不含表面活性剂成分。
在本实施案例中常见的含金属盐离子的清洗液选择为磷酸盐缓冲液(PBS缓冲液)、无Triton X-100的Tris-Triton-NaCl缓冲液、无Tween-20的TBST缓冲液。
上述PBS缓冲液的组成成分为：磷酸二氢钾(KH 2PO4)：0.24g/L，磷酸氢二钠(Na 2HPO4)：1.44g/L，氯化钠(NaCl)：8g/L，氯化钾(KCl)：0.2g/L，pH值为7.4±0.2。
上述无Triton X-100的Tris-Triton-NaCl缓冲液组成成分为：50mM Tris-HCL、0.6MNaCL，pH值为7.6±0.2。
上述无Tween-20的TBST缓冲液组成成分为：10mM Tris-HCL、0.15MNaCL、pH值为7.6±0.2。
本实施案例中的5种清洗缓冲液分别为纯化水、Tris-HCL缓冲液、PBS缓冲液、无Triton X-100的Tris-Triton-NaCl缓冲液、无Tween-20的TBST缓冲液，按照上述顺序依次标记为1号清洗液、2号清洗液、3号清洗液、4号清洗液、5号清洗液。按照实施案例3中的方法应用本实施案例中的5种清洗液分别分离血浆样本中的糖基化外泌体，进行对比验证分析，除清洗液不同，其他组成及步骤均相同，其他详细过程参考实施案例1-4、以及实施案例7中的外泌体特异性膜蛋白CD9/CD63/CD81抗体的Western Blot检测和鉴定步骤。
本实施案例中5种不同清洗缓冲液清洗后，再洗脱分离得到的糖基化外泌体溶液的外泌体特异性膜蛋白CD9/CD63/CD81抗体的Western Blot检测结果详见图7。
本实施案例结果显示，不含金属盐离子缓冲液(1号清洗液和2号清洗液)对结合有糖基化外泌体的凝集素-大分子载体偶联复合物进行清洗后，再对其进行洗脱所得洗脱液的Western Blot检测结果显示：外泌体特异性膜蛋白CD9/CD63/CD81显示均为阳性，提示均能有效分离得到糖基化外泌体；而无表面活性剂的常见含有金属盐离子的常见清洗缓冲液(3号、4号、5号清洗液)对结合有糖基化外泌体的凝集素-大分子载体偶联复合物进行清洗后，再对其进行洗脱所得洗脱液的Western Blot检测结果显示：外泌体特异性膜蛋白CD9/CD63/CD81显示均为阴性，说明溶液中金属盐离子的存在影响糖基化外泌体的分离效果，其对本发明中的糖基化外泌体具有明显的解离作用。
实施案例9
本实施案例主要针对不同的常见蛋白质洗脱液和本发明的硼酸盐-甘露糖缓冲液对糖基化外泌体的分离效果做进一步对比验证，以确定本发明中所选择洗脱液的适宜性。
将实施案例2和3中的糖基化外泌体分离组合物中的硼酸盐-甘露糖缓冲液替换为常用的蛋白质洗脱液和糖蛋白洗脱液，具体为蛋白质抗体洗脱缓冲液stripping buffer和糖蛋白洗脱液Glycoprotein Eluting Solution(美国Vectorlabs公司)。
上述stripping buffer组成成分为：62.5m mol/L Tris·HCl、2％SDS、100mmol/L β-2-巯基乙醇，pH6.8±0.2。
应用本实施案例中的3种洗脱液分别分离血浆样本中的糖基化外泌体，进行对比验证分析，除洗脱液不同，其他组成及步骤均相同，详细过程参考实施案例1-5，观察3种洗脱液分别分离血浆样本中的糖基化外泌体在电镜下的外泌体形态及完整性。
本实施案例中3种不同洗脱液分离得到的糖基化外泌体溶液的外泌体电镜检测结果详见图8。
本实施案例结果显示，本发明的洗脱液能有效分离样本中的糖基化外泌体，且外泌体形态完整、无破损，粒径均在30-150nm之间；stripping buffer作为洗脱液未观察到外泌体，可能为stripping buffer做为洗脱液在洗脱的过程中对外泌体的外膜造成破坏，无法得到完整的外泌体；而糖蛋白洗脱液Glycoprotein Eluting Solution观察不到典型的外泌体囊泡，仅能观察到疑似外泌体囊泡，但外泌体形态不完整，说明在分离过程中对外泌体造成一定的破坏，不利于后续的检测分析。
综上所述，应用本发明的内容对不同样本来源的糖基化外泌体的分离，均能达到同样的效果，且分离得到的外泌体均为糖基化外泌体，分离纯化后外泌体形态完整，无破损，可直接用于糖基化外泌体液体检测，外泌体免疫学检测，或核酸提取后基因检测分析等；还可以进一步通过后续纯化步骤获得纯度更高的外泌体。
上述说明示出并描述了本发明的优选实施案例，如前所述，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施案例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。
工业实用性
本发明涉及一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物，所述凝集素-大分子载体偶联复合物包括：大分子载体；和偶联在大分子载体外侧的凝集素。本发明提供的 凝集素-大分子载体偶联复合物可以简便、快速、精准分离临床样本中的糖基化外泌体，分离效率高，重复性好，分离后的外泌体形态完整，无破裂或碎裂，可直接用于糖基化外泌体液体检测，或直接进行免疫学相关检测，或直接提取外泌体内相关核酸进行基因检测分析。

Claims (15)

  1. 一种用于分离临床样本中的糖基化外泌体的凝集素-大分子载体偶联复合物，其特征在于，包括：大分子载体；和偶联在大分子载体外侧的凝集素；其中：
    所述凝集素包括：木菠萝凝集素、花生凝集素、豌豆凝集素(VVA和/或VVL)、刀豆凝集素、小扁豆凝集素、麦胚素、大豆凝集素、芸豆凝集素、蜗牛凝集素(HAA和/或HPA)中的任意一种或者两种及以上；
    所述大分子载体包括葡聚糖微球、琼脂糖微球、树脂或环氧树脂微球、聚苯乙烯微球中的任意一种或者两种及以上。
  2. 根据权利要求1所述的凝集素-大分子载体偶联复合物，其特征在于，所述大分子载体粒径分布范围为1μm-200μm，优选为10μm-200μm，更进一步优选为30μm-150μm。
  3. 根据权利要求1所述凝集素-大分子载体偶联复合物，其特征在于，所述凝集素-大分子载体偶联复合物中，每1ml大分子载体上偶联有1-20mg，优选为5-15mg，进一步优选为10-15mg凝集素。
  4. 根据权利要求1所述的凝集素-大分子载体偶联复合物，其特征在于，所述临床样本包括：血清、血浆、唾液、组织或细胞培养上清液、尿液、脑脊液、淋巴液中的任意一种。
  5. 根据权利要求1所述凝集素-大分子载体偶联复合物，其特征在于，所述凝集素包括：木菠萝凝集素、花生凝集素、豌豆凝集素(VVA和/或VVL)、刀豆凝集素、小扁豆凝集素、麦胚素、大豆凝集素、芸豆凝集素中的任意一种或者两种及以上。
  6. 根据权利要求1所述的凝集素-大分子载体偶联复合物，其特征在于，所述糖基化外泌体包括：N-糖基化外泌体、O-糖基化外泌体、岩藻糖基化外泌体的任意一种或两种及以上。
  7. 根据权利要求1所述的凝集素-大分子载体偶联复合物，其特征在于，所述凝集素-大分子载体偶联复合物保存液分装于包括亲和吸附离心管的分离装置中；所述亲和吸附离心管包括：上部离心管和外部保护套管两部分；所述上部离心管的直径小于外部保护套管的直径，所述上部离心管套设于外部保护套管内且在其上部外侧存在凸起的环形边缘或支柱用于支撑上部离心管的开口处高于外部保护套管；所述上部离心管包括离心管盖、离心管壁和与离心管壁固定连接的过滤膜底部，过滤膜底部所使用的过滤膜的孔径小于凝集素-大分子载体偶联复合物的粒径，同时大于外泌体的粒径，所述凝集素-大分子载体偶联复合物保存液保存于上部离心管中；可选地过滤膜孔径为150nm-1000nm。
  8. 一种糖基化外泌体分离组合物，其特征在于，所述糖基化外泌体分离组合物包括：权利要求1-7之一所述的凝集素-大分子载体偶联复合物，还包括：用于清洗去除分离过程中非特异性结合的、未发生糖基化的外泌体及其他杂质的清洗液和/或用于洗脱特异性结合在凝集素-大分子载体偶联复合物上的糖基化外泌体的洗脱液。
  9. 根据权利要求8所述的糖基化外泌体分离组合物，其特征在于，所述清洗液为无金属盐离子的清洗缓冲液或纯化水；可选地为无金属盐粒子的清洗缓冲液；
    和/或，所述洗脱液为溶有糖的硼酸盐缓冲液，进一步可选地为溶有甘露糖的硼酸盐缓冲液。
  10. 一种糖基化外泌体分离方法，其特征在于：包括应用权利要求1-7之一所述的凝集素-大分子载体偶联复合物分离糖基化外泌体的分离步骤，所述分离步骤包括：
    临床样本前处理：
    取前处理后的样本，可选地与清洗液混合均匀进行稀释，作为待检测样本；
    完全去除凝集素-大分子载体偶联复合物保存液中的保存液部分，保留凝集素-大分子载体偶联复合物；
    取待检测样本加入到除去保存液后的凝集素-大分子载体偶联复合物中，室温静置孵育，去除样本；
    取清洗液清洗已结合有糖基化外泌体的凝集素-大分子载体偶联复合物，去除清洗液，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质；
    取洗脱液，洗脱清洗后的凝集素-大分子载体偶联复合物，室温静置，收集洗脱上清液，即为分离后的糖基化外泌体溶液。
  11. 根据权利要求10所述的糖基化外泌体分离方法，其特征在于：待检测样本与凝集素-大分子载体偶联复合物的比例为1：1-3；
    和/或，单次加入清洗液的体积与已结合有糖基化外泌体的凝集素-大分子载体偶联复合物体积的比例为1-3：1；
    和/或，清洗液清洗步骤重复进行1-3次；
    和/或，加入洗脱液的体积与清洗后的凝集素-大分子载体偶联复合物体积的比例为0.5-2：1，优选为0.5-1：1。
  12. 根据权利要求10所述的糖基化外泌体分离方法，其特征在于：完全去除凝集素-大分子载体偶联复合物保存液中的保存液部分、去除样本、去除清洗液、和/或收集洗脱上清液的方式为采用3000rpm以下的速度、室温下离心20秒；
    和/或，室温静置孵育10-30min，优选为10-15min；
    和/或，所述清洗液为无金属盐离子的清洗缓冲液或纯化水；可选地为无金属盐粒子的清洗缓冲液；
    和/或，所述洗脱液为溶有糖的硼酸盐缓冲液，可选地为含有甘露糖的硼酸盐缓冲液。
  13. 根据权利要求10所述的糖基化外泌体分离方法，其特征在于：样本前处理的方法包括以下步骤：
    对血清、血浆、唾液、脑脊液或淋巴液样本，将样本经3000g以下速度离心10-15min，去除样本中的细胞碎片、沉淀物杂质，取离心后上清液备用；
    对组织或细胞培养上清液、尿液的样本，将样本经3000g以下速度离心10-15min，去除样本中的细胞碎片、沉淀物杂质，然后将上清液通过超滤管进行10-1000倍浓缩后备用。
  14. 根据权利要求10所述的糖基化外泌体分离方法，其特征在于：应用权利要求7所述的凝集素-大分子载体偶联复合物分离糖基化外泌体的分离步骤包括：
    临床样本前处理：
    取前处理后的样本，可选地与清洗液混合均匀进行稀释，作为待检测样本；
    装载有凝集素-大分子载体偶联复合物的亲和吸附离心管完全去除凝集素-大分子载体偶联复合物保存液中的保存液部分，保留凝集素-大分子载体偶联复合物，并更换新的外部套管；
    取待检测样本加入到除去保存液后的上部离心管中，室温静置孵育，去除样本；
    取清洗液加入上部离心管中，将亲和吸附离心管离心后将上部离心管取出，装入新的外部套管中，将原外部套管及其中液体全部弃去，用于清洗去除分离过程中非特异性结合的未发生糖基化的外泌体及其他杂质；本步骤进行1-3次；
    取洗脱液，加入上部离心管，洗脱清洗后的凝集素-大分子载体偶联复合物，室温静置，收集外部套管中的洗脱上清液，即为分离后的糖基化外泌体溶液。
  15. 一种使用权利要求10-14之一所述的糖基化外泌体分离方法分离的外泌体的应用，其特征在于，所述应用包括：糖基化外泌体液体检测、外泌体免疫学检测，或外泌体中核酸提取后的核苷酸片段检测分析。
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