Application of lectin-macromolecule carrier coupling complex for separation of glycosylated exosomes

Abstract

The present invention is a divisional application with application number 202010060063.1, and the present invention relates to the use of a lectin-macromolecular carrier coupling complex in isolating glycosylated exosomes from clinical samples, wherein the lectin-macromolecular carrier coupling complex comprises: a macromolecular carrier; and a lectin coupled to the outside of the macromolecular carrier. The lectin-macromolecular carrier coupling complex provided by the present invention can easily, quickly and accurately separate glycosylated exosomes from clinical samples, with high separation efficiency and good repeatability. The separated exosomes are in complete morphology without rupture or fragmentation, and can be directly used for glycosylated exosome liquid detection, or directly for immunological related detection, or directly extracting related nucleic acids in exosomes for genetic detection and analysis.

Description

[0001] The present invention is a divisional application of a Chinese application with an application date of January 19, 2020, application number 202010060063.1, and invention name “A lectin-macromolecular carrier coupled complex for isolating glycosylated exosomes from clinical samples”. Technical Field

[0002] The present invention belongs to the technical field related to biomedicine and biological separation, and relates to a lectin-macromolecular carrier coupling complex for separating glycosylated exosomes in clinical samples. Background Art

[0003] Exosomes are tiny biologically active vesicles released when multivesicular endosomes fuse with the plasma membrane. They are about 30 to 150 nm in diameter 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, proteins, mRNA, and miRNA between cells, participate in a variety of processes such as cell communication, cell migration, and tumor cell growth, and complete the transfer of substances and information between cells. Literature research has found that almost all types of cells secrete exosomes, and exosomes can be extracted from body fluids such as blood, saliva, urine, cerebrospinal fluid, effusion, and breast milk.

[0004] Exosomes are related to many diseases such as tumors, chronic infectious diseases, autoimmune diseases, etc. When the body is diseased, the composition, content, and properties of the exosomes secreted by cells may change; glycosylated exosomes play an important role in the occurrence and development of many diseases including tumors. The detection and research of exosomes can effectively monitor the occurrence and development of diseases; since the content of exosomes in samples is relatively low, the separation and enrichment of exosomes is crucial to the study of exosomes.

[0005] At present, there are many methods for separating exosomes, mainly including ultracentrifugation or gradient density centrifugation, immunomagnetic bead method, etc. Among them: ultracentrifugation or gradient density centrifugation is based on the slight difference in density between exosomes and other biological components for separation. This method requires special equipment, requires a large amount of samples, and is time-consuming. In addition, due to the different characteristics of different clinical samples, the separation effect of this method is poorly stable. Further ultracentrifugation is also prone to cause vesicle damage, thus affecting subsequent experiments; the principle of the immunomagnetic bead method is that specific proteins on the surface of exosomes, such as CD9 / CD63 / CD81, are combined with immunomagnetic beads coated with corresponding antibodies for magnetic separation. However, due to the small size of the immunomagnetic beads, the particle size is nanometer-level and less than or equal to the diameter of the exosomes, so the steric hindrance of the immunomagnetic beads binding to the exosomes is large, the exosomes are not fully bound, and the separation efficiency is not high. In addition, the exosomes separated by the immunomagnetic bead method are incomplete in morphology because the acidic eluent is used for elution.

[0006] Therefore, it is necessary to research and establish a composition, a separation device, and a separation method that can be used to accurately and efficiently separate glycosylated exosomes from enriched samples. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a lectin-macromolecular carrier coupling complex for separating glycosylated exosomes in clinical samples, and the present invention also provides a glycosylated exosome separation composition containing the lectin-macromolecular carrier coupling complex, as well as a method and application of using the lectin-macromolecular carrier coupling complex to separate glycosylated exosomes. The lectin-macromolecular carrier coupling complex provided by the present invention can accurately separate glycosylated exosomes in clinical samples, with high separation efficiency, and the exosomes after separation are complete in morphology, without rupture or fragmentation, and can be directly used for glycosylated exosome liquid detection, or directly perform immunological related detection, or directly extract relevant nucleic acids in exosomes for gene detection and analysis. In the method for separating glycosylated exosomes provided by the present invention, the principle that the rich sugar chains on the outer surface of exosomes can bind to lectins is utilized, and glycosylated exosomes can be separated by effective centrifugal washing and effective elution methods, and it is very simple, fast, and has good repeatability. The exosomes after separation are complete in morphology, without rupture or fragmentation, and are applied to the occurrence and development of diseases, etc., for detection, monitoring and diagnosis.

[0008] The present invention provides a lectin-macromolecular carrier coupling complex for separating glycosylated exosomes in clinical samples, a macromolecular carrier; and a lectin coupled to the outside of the macromolecular carrier; wherein:

[0009] The lectin includes: any one or two or more of japanese lectin, peanut lectin, pea lectin (VVA and / or VVL), concanavalin A lectin, lentil lectin, wheat germ agglutinin, soybean lectin, kidney bean lectin, and snail lectin (HAA and / or HPA);

[0010] The macromolecular carrier includes any one or two or more of dextran microspheres, agarose microspheres, resin or epoxy resin microspheres, and polystyrene microspheres.

[0011] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupling complex, the particle size distribution range of the macromolecular carrier is 1 μm-200 μm, preferably 10 μm-200 μm, and more preferably 30 μm-150 μm.

[0012] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupling complex, in each ml of the macromolecular carrier, 1-20 mg, preferably 5-15 mg, and more preferably 10-15 mg of lectin is coupled to the lectin-macromolecular carrier.

[0013] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupling complex, the clinical sample includes: any one of serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, and lymph fluid.

[0014] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupling complex, the lectin includes any one or two or more of jacaranda lectin, peanut lectin, pea lectin (VVA and / or VVL), concanavalin A agglutinin, lentil lectin, wheat germ agglutinin, soybean lectin, and kidney bean lectin.

[0015] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupling complex, the glycosylated exosomes include: any one or two or more of N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes.

[0016] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupled complex, the lectin-macromolecular carrier coupled complex is stored in a storage solution, and the storage concentration (volume ratio) of the lectin-macromolecular carrier coupled complex in the obtained lectin-macromolecular carrier coupled complex storage solution is 10%-60%, preferably 30%-50%, and further preferably 40%-50%; the volume of the lectin-macromolecular carrier coupled complex storage solution refers to the total volume of the lectin-macromolecular carrier coupled complex and the storage solution after the lectin-macromolecular carrier coupled complex is dispersed in the storage solution.

[0017] In certain embodiments of the above-mentioned lectin-macromolecular carrier coupling complex, the lectin-macromolecular carrier coupling complex preservation solution is packaged in a separation device including an affinity adsorption centrifuge tube; the affinity adsorption centrifuge tube includes: 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, 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 for supporting the opening of the upper centrifuge tube to be higher than the outer protective sleeve; the upper centrifuge tube includes a centrifuge tube cover, a centrifuge tube wall and a filter membrane bottom fixedly connected to the centrifuge tube wall, the pore size of the filter membrane used at the bottom of the filter membrane is smaller than the particle size of the lectin-macromolecular carrier coupling complex, and larger than the particle size of the exosomes, the lectin-macromolecular carrier coupling complex preservation solution is stored in the upper centrifuge to form a separation device for glycosylated exosomes, and the pore size of the filter membrane is preferably 150nm-1000nm.

[0018] In certain embodiments of the above lectin-macromolecular carrier coupled complex, the volume of the lectin-macromolecular carrier coupled complex in the lectin-macromolecular carrier coupled complex preservation solution dispensed into the upper centrifuge tube occupies 1 / 2-1 / 4 of the volume of the upper centrifuge tube.

[0019] Another aspect of the present invention provides a glycosylated exosome separation composition, which comprises: the above-mentioned lectin-macromolecular carrier coupling complex, and also comprises a cleaning solution for cleaning and removing non-specifically bound, non-glycosylated exosomes and other impurities during the separation process and / or an eluent for eluting glycosylated exosomes specifically bound to the lectin-macromolecular carrier coupling complex. The composition here does not mean that it is mixed as a whole, but that it exists in one set at the same time.

[0020] In certain embodiments of the above-mentioned glycosylated exosome separation composition, the cleaning solution is a metal salt ion-free cleaning buffer or purified water, and can be a metal salt ion-free cleaning buffer, which is used to clean and remove non-specifically bound non-glycosylated exosomes and other impurities during the separation process.

[0021] In certain embodiments of the above-mentioned glycosylated exosome separation composition, the eluent is a borate buffer dissolved with sugar, optionally a borate buffer dissolved with mannose, and further optionally has a pH value of 6.5±0.2, which is used to elute the glycosylated exosomes bound to the lectin-macromolecular carrier coupling complex, and the exosomes after elution have an intact appearance and morphology without damage or lysis.

[0022] Another aspect of the present invention further provides a method for isolating glycosylated exosomes, comprising a separation step of using the above-mentioned lectin-macromolecular carrier coupling complex to separate glycosylated exosomes, wherein the separation step comprises:

[0023] Clinical sample pretreatment:

[0024] Taking the pre-treated sample, optionally mixing it evenly with a cleaning solution to dilute it, and using it as a sample to be tested;

[0025] Completely removing the preservation solution portion of the lectin-macromolecule carrier coupling complex preservation solution, and retaining the lectin-macromolecule carrier coupling complex;

[0026] Take the sample to be tested and add it to the lectin-macromolecule carrier coupling complex after removing the preservation solution, incubate it at room temperature, and remove the sample;

[0027] The cleaning solution is used to clean the lectin-macromolecule carrier coupling complex bound to the glycosylated exosomes, and the cleaning solution is removed to clean and remove the non-specifically bound non-glycosylated exosomes and other impurities during the separation process;

[0028] The eluted solution is taken to elute the washed lectin-macromolecule carrier coupling complex, and the eluted supernatant is collected, which is the separated glycosylated exosome solution.

[0029] In certain embodiments of the above-mentioned glycosylated exosome isolation method, the ratio of the sample to be detected to the lectin-macromolecule carrier coupling complex is 1:1-3;

[0030] and / or, the ratio of the volume of the washing solution added once to the volume of the lectin-macromolecular carrier coupling complex bound to glycosylated exosomes is 1-3:1;

[0031] and / or, the cleaning step of the cleaning solution is repeated 1-3 times;

[0032] And / or, the ratio of the volume of the added eluent to the volume of the washed lectin-macromolecular carrier coupling complex is 0.5-2:1, preferably 0.5-1:1.

[0033] In certain embodiments of the above-mentioned method for isolating glycosylated exosomes, the preservation solution portion in the preservation solution of the lectin-macromolecular carrier coupling complex is completely removed, the sample is removed, the washing solution is removed, and / or the elution supernatant is collected by centrifugation at a speed of less than 3000 rpm and room temperature for 20 seconds.

[0034] In certain embodiments of the above-mentioned method for isolating glycosylated exosomes, the incubation is performed at room temperature for 10-30 min, preferably 10-15 min.

[0035] In certain embodiments of the above-mentioned method for isolating glycosylated exosomes, the sample pretreatment method comprises the following steps:

[0036] For serum, plasma, saliva, cerebrospinal fluid or lymph samples, centrifuge the samples at a speed below 3000g for 10-15 minutes to remove cell debris and precipitate impurities in the samples, and take the supernatant after centrifugation for later use;

[0037] For tissue or cell culture supernatant and urine samples, centrifuge the samples at a speed below 3000g for 10-15 minutes to remove cell debris and precipitate impurities in the samples, and then concentrate the supernatant 10-1000 times through an ultrafiltration tube for later use.

[0038] In certain embodiments of the above-mentioned glycosylated exosome isolation method, the cleaning solution is a cleaning buffer solution without metal salt ions or purified water, and can be selected as a cleaning buffer solution without metal salt ions, and can further be selected as a cleaning buffer solution without metal salt ions having a pH value of 7.6±0.2.

[0039] In certain embodiments of the above-mentioned method for isolating glycosylated exosomes, the eluent is a borate buffer containing sugar, optionally a borate buffer containing mannitol, and further optionally a borate-mannose buffer with a pH value of 6.5±0.2.

[0040] In certain embodiments of the above-mentioned method for isolating glycosylated exosomes, the incubation conditions are: incubation at room temperature for 1-30 min, preferably 5-20 min, and more preferably 10-15 min.

[0041] In certain embodiments, the above-mentioned method for separating glycosylated exosomes comprises a separation step of using the above-mentioned lectin-macromolecular carrier coupling complex to separate glycosylated exosomes and a separation device, wherein the separation step comprises:

[0042] Clinical sample pretreatment:

[0043] Taking the pre-treated sample, optionally mixing it evenly with a cleaning solution to dilute it, and using it as a sample to be tested;

[0044] The affinity adsorption centrifuge tube loaded with the lectin-macromolecular carrier coupling complex completely removes the preservation solution portion of the lectin-macromolecular carrier coupling complex preservation solution, retains the lectin-macromolecular carrier coupling complex, and replaces a new outer sleeve;

[0045] Take the sample to be tested and add it to the upper centrifuge tube after removing the preservation solution, incubate at room temperature, and remove the sample;

[0046] Take the washing liquid and add it to the upper centrifuge tube. After centrifuging the affinity adsorption centrifuge tube, take out the upper centrifuge tube and put it into a new outer sleeve. Discard the original outer sleeve and the liquid therein to wash and remove the non-specifically bound non-glycosylated exosomes and other impurities during the separation process. This step is performed 1-3 times;

[0047] Take the eluate and add it to the upper centrifuge tube to elute the washed lectin-macromolecular carrier coupling complex, let it stand at room temperature, and collect the elution supernatant in the outer sleeve, which is the separated glycosylated exosome solution. After elution and separation, the glycosylated exosomes are intact in morphology without damage or lysis.

[0048] The present invention also provides an application of exosomes separated by the above method, the application including: glycosylated exosome liquid detection, exosome immunological detection, or nucleotide fragment detection and analysis after nucleic acid extraction in exosomes.

[0049] Beneficial effects:

[0050] 1. The exosomes separated from the lectin-macromolecular carrier coupling complex of the present invention are all glycosylated exosomes, which are intact in morphology and undamaged, and can be directly used for glycosylated exosome liquid detection, exosome immunological detection, or nucleotide sequence detection and analysis after nucleic acid extraction, etc., and can further realize the fully automatic separation of glycosylated exosomes by cooperating with corresponding equipment.

[0051] 2. In the lectin-macromolecular carrier coupling complex of the present invention, 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. A single macromolecular carrier has a larger surface area, which reduces the steric hindrance when the lectin is coupled with the macromolecular carrier, which is conducive to coupling more lectins. In contrast, although the general macromolecular carrier of the same total volume has a larger specific surface area, due to the larger steric hindrance, the lectin coupled is less. In the lectin-macromolecular carrier coupling complex of the present invention, 1-20 mg of lectin can be coupled to each 1 ml of macromolecular carrier. However, studies have found that when a smaller macromolecular carrier is used, the number of lectins coupled to each 1 ml of macromolecular carrier is at the ng level, and the coupling efficiency is low; at the same time, the particle size is also much larger than the diameter of the exosomes themselves, which can reduce the steric hindrance of the binding of lectins and glycosylated exosomes, which is conducive to the binding of lectins and glycosylated exosomes, and greatly improves the separation efficiency of exosomes.

[0052] 3. The lectin-macromolecule carrier coupling complex of the present invention has a good effect on clinical samples with low exosome content, such as exosomes in urine, tissue or cell culture supernatant; for clinical samples with high exosome content, such as exosomes in plasma, the concentration of separated glycosylated exosomes can reach 10 11-14 Pieces / ml.

[0053] 4. In the lectin-macromolecular carrier coupling complex of the present invention, the lectin used is preferably a plant lectin, including: japanese lectin, peanut lectin, pea lectin (VVA and / or VVL), concanavalin A lectin, lentil lectin, wheat germ agglutinin, soybean lectin, kidney bean lectin. Plant lectins are easier to obtain in large quantities and the raw materials are more abundant.

[0054] 5. The lectin-macromolecular carrier coupling complex of the present invention can be packaged in affinity adsorption centrifuge tubes with specific structures to form a separation device for glycosylated exosomes when leaving the factory, which is convenient for users to use and is conducive to the preservation of the lectin-macromolecular carrier coupling complex. The selection of the pore size of the filter membrane is conducive to the smooth passage of various preservation solutions, cleaning solutions, and eluents. In particular, the addition of mannose to the eluent will increase the viscosity of the eluent. This pore size can ensure the smooth passage of the eluent and ensure that the lectin-macromolecular carrier coupling complex does not leak out.

[0055] 6. The glycosylated exosome separation composition of the present invention uses a cleaning buffer or purified water without metal salt ions. The metal salt ions have a dissociation effect on the affinity adsorption of lectins and glycosylated exosomes, affecting the affinity adsorption of lectins and glycosylated exosomes. The higher the concentration of metal salt ions, the stronger the dissociation effect, thereby affecting the separation effect of exosomes, and even making it impossible to separate the exosomes.

[0056] 7. The glycosylated exosome separation composition of the present invention uses a borate buffer as the eluent, which has a better elution effect on exosomes than other buffers, has a high elution efficiency, and can be eluted using a small amount of eluent. The exosomes after elution have a complete appearance without damage or lysis. At the same time, due to the small amount used, it is equivalent to completing the process of concentrating exosomes at the same time.

[0057] 8. The method for isolating glycosylated exosomes of the present invention is simple and easy to perform sample pretreatment. Samples suitable for the present invention can be effectively obtained by simple centrifugation and / or concentration. There is no need to process the samples by ultra-high-speed centrifugation, and the glycosylated exosomes in the samples can be protected to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : Schematic diagram of affinity adsorption centrifuge tube of lectin-macromolecular carrier coupling complex.

[0059] Figure 2 : Electron microscopic observation images of glycosylated exosomes separated from 4 different sample types (cell culture supernatant, plasma, urine, and cerebrospinal fluid).

[0060] Figure 3 : NTA analysis of glycosylated exosomes after separation of plasma samples.

[0061] Figure 4 : Western Blot test results of exosome-specific membrane proteins CD9, CD63, and CD81 antibodies in 4 different samples (cell culture supernatant, plasma, urine, and cerebrospinal fluid).

[0062] Figure 5 : Western Blot detection results of exosome-specific non-membrane protein ALIX and TSG101 antibodies in 4 different samples (cell culture supernatant, plasma, urine, and cerebrospinal fluid).

[0063] Figure 6 : Western Blot detection results of exosomal Calnexin antibodies in 4 different samples (cell culture supernatant, plasma, urine, and cerebrospinal fluid).

[0064] Figure 7: The lectin-macromolecular carrier coupling complex bound to glycosylated exosomes was washed with 5 different washing buffers and then eluted. The eluate obtained was subjected to Western Blot detection of exosome-specific membrane protein CD9 / CD63 / CD81 antibodies.

[0065] Figure 8 : Electron microscopy results of glycosylated exosome solutions separated by three different eluents. DETAILED DESCRIPTION

[0066] To better illustrate the present invention, various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as limiting the present invention but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0067] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific implementations of the present invention without departing from the scope or spirit of the present invention. The present application description and implementation examples are merely exemplary.

[0068] The terms "include", "comprising", "having", "containing", etc. used in this article are open-ended terms, which mean including but not limited to. Unless otherwise specified, all reagents used below are commercial reagents, and the chemical reagents used are not less than analytical grade.

[0069] Example 1 Preparation of lectin-macromolecule carrier coupling complex

[0070] The lectin-macromolecule carrier coupling complex includes: a macromolecule carrier; and a lectin coupled to the outside of the macromolecule carrier, which is prepared by coupling the lectin and the macromolecule carrier under specific conditions, and is mainly used to separate glycosylated exosomes in the sample. The glycosylated exosomes obtained after separation have a complete morphology. Among them:

[0071] The selection of lectins mainly includes: any one of jacaranda lectin (Jacalin), peanut lectin (PNA), pea lectin (VVA and / or VVL), concanavalin A (ConA), lentil lectin (LCA), wheat germ agglutinin (WGA), soybean lectin (SBA), kidney bean lectin (PVL), snail lectin (HAA and / or HPA), or the combination of two or more lectins. The main reason is that the combination of different lectins can achieve the separation of various types of glycosylated exosomes, such as LCA, A AL is used to separate fucosylated exosomes; ConA, PVL, SBA, WGA, AAL and other lectins are used to separate N-glycosylated exosomes; HAA, HPA, VVA, PNA, Jacalin and other lectins are used to separate O-glycosylated exosomes; a single lectin is mainly used to separate specific glycosylated exosomes corresponding to the lectin, and the combined use of two or more lectins can be used to separate glycosylated exosomes of various different forms, or the combined use of two or more lectins can synergistically separate specific glycosylated exosomes.

[0072] To further illustrate the present invention, the following examples mainly use lentil agglutinin (LCA) (purchased from Sigma, USA) to further illustrate the present invention.

[0073] Macromolecular carriers mainly refer to macromolecular microspheres, mainly including: any one of dextran microspheres, agarose microspheres, resin or epoxy resin microspheres, polystyrene microspheres, or two or more macromolecular carriers used in combination, and the particle size distribution range of the macromolecular carrier is 1μm-200μm, preferably 10μm-200μm, and more preferably 30μm-150μm. In the production of macromolecular carriers, the particle size of macromolecular carriers produced in the same batch is not uniform, so the particle size description of the macromolecular carrier can usually be described in terms of average particle size or in terms of particle size distribution range in the present invention. For the description in terms of particle size distribution range, it can be considered that its specific distribution conforms to or substantially conforms to the normal distribution within this distribution range. In the lectin-macromolecular carrier coupling complex of the embodiment of the present invention, 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. A single macromolecular carrier has a larger surface area, which reduces the steric hindrance when the lectin is coupled to the macromolecular carrier, which is conducive to coupling more lectins. In contrast, although the general macromolecular carrier of the same total volume has a larger specific surface area, due to the larger steric hindrance, the lectin coupled is less. In the lectin-macromolecular carrier coupling complex of the present invention, 1-20 mg of lectin can be coupled to each 1 ml of macromolecular carrier. However, studies have found that when a smaller macromolecular carrier is used, the number of lectins coupled to each 1 ml of macromolecular carrier is at the ng level, and the coupling efficiency is low; at the same time, the particle size is also much larger than the diameter of the exosomes themselves, which can reduce the steric hindrance of the binding of lectins and glycosylated exosomes, which is conducive to the binding of lectins to glycosylated exosomes and greatly improves the separation efficiency of exosomes.

[0074] The above-mentioned lectin is coupled with the macromolecular carrier to form a lectin-macromolecular carrier coupled complex. Different macromolecular carriers can achieve the effect of coupling with the lectin after adjusting the steps of soaking, coupling, washing, and storage. Of course, the optimal macromolecular carrier for different lectins may be different; in the preparation process of the lectin-macromolecular carrier coupled complex, the ratio of the macromolecular carrier (ml) to the lectin (mg) is 1:1-1:20, preferably 1:5-1:15, and further preferably 1:10-1:15.

[0075] The prepared lectin-macromolecular carrier coupling complex is stored in a storage solution, and the storage concentration (volume ratio) of the lectin-macromolecular carrier coupling complex in the obtained lectin-macromolecular carrier coupling complex storage solution is 10%-60%, preferably 30%-50%, and more preferably 40%-50%; the lectin-macromolecular carrier coupling complex storage solution is dispensed into the upper centrifuge tube of the affinity adsorption centrifuge tube; the affinity adsorption centrifuge tube comprises: 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 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 to be higher than the outer protective sleeve; the upper centrifuge tube includes a centrifuge tube cover, a centrifuge tube wall and a filter membrane bottom fixedly connected to the centrifuge tube wall. The pore size of the filter membrane used at the bottom of the filter membrane is smaller than the particle size of the lectin-macromolecular carrier coupling complex and larger than the particle size of the exosomes. The pore size of the filter membrane is preferably 150nm-1000nm, which is used to load the lectin-macromolecular carrier coupling complex to form a separation device for glycosylated exosomes.

[0076] To further illustrate the present invention, the following embodiments are all described using agarose microspheres with a particle size distribution range of 30 μm-150 μm. The agarose microspheres are commercially available hydrogen bromide-activated agarose microspheres (purchased from Pharmacia or Sigma). The agarose microspheres are further preferably: sephrose 4B, sephrose 6B, sephrose FF, sephrose CL-4B, and sephrose CL-6B. The following embodiments are all described using sephrose 4B microspheres as an example.

[0077] The preparation process of the lectin-macromolecule carrier coupling complex, i.e., the LCA-agarose microsphere coupling complex, is as follows:

[0078] (1) Weigh 1 g of activated agarose 4B, add no less than 200 ml of 1 mM HCl solution at pH 2-3, mix and soak until completely swollen, and wash in a glass frit funnel with 1 mM HCl solution at pH 2-3 for no less than 15 min. After washing, collect the completely swollen agarose 4B for later use. The completely swollen agarose 4B is about 3-5 ml.

[0079] (2) Take 15 ml of 0.1-1.0 M carbonate buffer solution, add lentil agglutinin, and then add the fully swollen agarose 4B collected in step a, wherein the ratio of the volume (ml) of fully swollen agarose 4B to lentil agglutinin (LCA, mg) is 1:1-1:20 (i.e., 1-20 mg of lentil agglutinin is mixed with every 1 ml of fully swollen agarose 4B), mix and react at room temperature for 0.5 h-5 h, and remove the supernatant; to better illustrate the present invention, the concentration of the carbonate buffer solution used in this embodiment is 0.1 M, and the ratio of the volume (ml) of fully swollen agarose 4B to lentil agglutinin (LCA, mg) is 1:10, i.e., 5 ml of fully swollen agarose 4B and 50 mg of LCA are added to 15 ml of carbonate buffer solution, and the mixing and reaction time at room temperature is 3 h.

[0080] (3) Add 100 ml of 0.1 M carbonate buffer, mix well and remove the supernatant; then add 200 ml of purified water, mix well and remove the supernatant.

[0081] (4) Wash repeatedly at least three times with 0.1 mol / L pH 4.0 acetate buffer (containing 0.5 mol / L NaCl) and 0.1 mol / L pH 8.0 Tris-HCl buffer (containing 0.5 mol / L NaCl) in a volume not less than 10 times the volume of the fully swollen agarose 4B.

[0082] (5) Wash once with 0.1 mol / L TRIS-HCl buffer at pH 8.0, collect the completely swollen agarose 4B after washing, add TRIS-HCl buffer, and prepare 10%-60% LCA-agarose microsphere preservation solution for standby use, preferably 30%-50% LCA-agarose microsphere preservation solution. In order to better illustrate the present invention, in this embodiment, it is prepared into a 50% by volume LCA-agarose microsphere preservation solution.

[0083] (6) After mixing 50% of the LCA-agarose microsphere preservation solution in step (5), the solution is evenly distributed in a separation device including an affinity adsorption centrifuge tube to form a separation device for glycosylated exosomes, which is sealed and stored at 2-8°C for later use; wherein the affinity adsorption centrifuge tube comprises: an upper centrifuge tube and an outer protective sleeve; the diameter of the upper centrifuge tube is smaller than that of the outer sleeve, and there is a raised annular edge or pillar at the upper part to support the opening of the upper centrifuge tube to be higher than the outer sleeve; the upper centrifuge tube comprises a centrifuge tube cover, a centrifuge tube wall and a filter membrane bottom fixedly connected to the centrifuge tube wall, the pore size of the filter membrane used at the bottom of the filter membrane is smaller than the particle size of the lectin-macromolecular carrier coupling complex and larger than the particle size of the exosomes, the lectin-macromolecular carrier coupling complex preservation solution is stored in the upper centrifuge tube, and optionally, the pore size of the filter membrane is preferably 150nm-1000nm. The size of the upper centrifuge tube of the affinity adsorption centrifuge tube can be designed according to the size of the specific centrifuge equipment. The volume of the lectin-macromolecular carrier coupling complex accounts for 1 / 2-1 / 4 of the volume of the upper centrifuge tube. For example, if a 1.5-2 ml upper centrifuge tube is selected, 0.5 ml of the lectin-macromolecular carrier coupling complex preservation solution is dispensed into the tube. For details, see the schematic diagram of the affinity adsorption centrifuge tube. Figure 1 The preservation solution in the lectin-macromolecular carrier coupling complex preservation solution can enter the external protective sleeve through the filter membrane and remain in the external protective sleeve without being lost. Due to the sheath structure of the upper centrifuge tube and the external protective sleeve and the volume concentration selection in the lectin-macromolecular carrier coupling complex preservation solution, the lectin-macromolecular carrier coupling complex can be in contact with sufficient preservation solution and will not dry out and lose activity.

[0084] Example 2 Preparation of Glycosylated Exosome Separation Composition

[0085] The present invention provides a glycosylated exosome separation composition, which specifically comprises: an affinity adsorption centrifuge tube obtained in Example 1 and filled with LCA-agarose microsphere preservation solution, i.e., a glycosylated exosome separation device, and a washing solution and an elution solution, which are separately packaged and exist in a set or a kit at the same time.

[0086] The cleaning solution is a metal salt ion-free cleaning buffer or purified water, and can be a metal salt ion-free cleaning buffer; for example, the main component includes 10-200mM metal salt ion-free TRIS-HCl buffer, and its pH value is 7.6±0.2. In this embodiment, the cleaning solution is a metal salt ion-free cleaning buffer, the main component includes 100mM metal salt ion-free TRIS-HCl buffer, and its pH value is 7.6±0.2, which is used to clean and remove non-specifically bound non-glycosylated exosomes and other impurities during the separation process.

[0087] The above-mentioned eluent is a borate buffer solution containing dissolved sugar, such as: the main components include 10-20mM borate buffer solution and 100-500mM mannose dissolved therein, and the pH value is 6.5±0.2. In the present embodiment, the borate buffer solution containing dissolved sugar is a borate buffer solution containing 15mM borate buffer solution and 300mM mannose dissolved therein, and the 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 exosomes after elution have a complete appearance without damage or lysis.

[0088] Example 3: Method for using the glycosylated exosome separation composition

[0089] The present invention also provides a method for isolating glycosylated exosomes, comprising the following experimental steps using the above-mentioned glycosylated exosome isolation composition:

[0090] 1. Preparation before the experiment

[0091] Self-provided equipment or devices: centrifuge, used for the centrifugation step in the process of separating glycosylated exosomes; or use the fully automatic agarose microsphere separation instrument of the group company and its subsidiaries, which is mainly used for fully automatic separation of glycosylated exosomes to save manpower. Fully automatic agarose microsphere separation of glycosylated exosomes is only an automated realization of the manual method, and can achieve the same or better function than manual separation. This implementation case uses centrifuge manual separation for further explanation.

[0092] 2. Test process

[0093] (1) Sample pretreatment: Adjust the sample pretreatment method according to the characteristics of different clinical samples.

[0094] For the serum, plasma, saliva, cerebrospinal fluid, and lymph samples, the samples were centrifuged at 3000 g for 10-15 min to remove cell debris, precipitates, and other impurities in the samples, and the supernatant after centrifugation was reserved for later use; for a clearer explanation, the centrifugation was performed for 10 min in this embodiment;

[0095] For the tissue or cell culture supernatant and urine samples, the samples are centrifuged at 3000 g for 10-15 min to remove impurities such as cell debris and precipitates in the samples, and then the supernatant is concentrated 10-1000 times through an ultrafiltration tube, and the concentrated supernatant is set aside; for a clearer explanation, centrifugation is performed for 10 min in this embodiment.

[0096] (2) Take 200-300ul of the pre-treated sample in step (1), and mix it directly with the lectin-macromolecular carrier coupling complex without diluting it with the cleaning solution, or mix it with 1-2 times the volume of the cleaning solution and dilute it before mixing it with the lectin-macromolecular carrier coupling complex. The effect after dilution is better. In this embodiment, the volume of the upper centrifuge tube of the affinity adsorption centrifuge tube is 2.0ml, in which 1mL / tube of lectin-macromolecular carrier coupling complex preservation solution (containing 0.5mL / tube of lectin-macromolecular carrier coupling complex) is dispensed. The volume of the pre-treated sample is selected to be 200ul, and the volume of the cleaning solution is 300ul. After mixing and diluting, the volume is the same as the volume of the lectin-macromolecular carrier coupling complex in the affinity adsorption centrifuge tube. The volume of the sample diluted with the cleaning solution cannot be significantly larger than the volume of the lectin-macromolecular carrier coupling complex in the affinity adsorption centrifuge tube. The binding effect is best when the volumes of the two are basically equal.

[0097] (3) Take out the upper centrifuge tube of the affinity adsorption centrifuge tube loaded with the lectin-macromolecular carrier coupling complex, put it into a new outer sleeve, centrifuge at 3000 rpm at room temperature for 20 seconds, remove the preservation solution part of the lectin-macromolecular carrier coupling complex preservation solution, retain the lectin-macromolecular carrier coupling complex, and replace with a new outer sleeve.

[0098] (4) Take 500ul of the diluted sample in step (2) and add it to the upper centrifuge tube. Incubate it at room temperature for 10-30min, preferably 10-15min. Then, centrifuge the affinity adsorption centrifuge tube at 3000rpm at room temperature for 20 seconds to remove the sample. In this embodiment, the incubation time is selected to be 10min.

[0099] (5) Take 500ul-1500ul of 100mM TRIS-HCl buffer, a metal salt-free cleaning solution with a pH value of 7.6±0.2, and add it to the upper centrifuge tube. Centrifuge the affinity adsorption centrifuge tube at 3000 rpm for 20 seconds at room temperature. Take out the upper centrifuge tube and put it into a new outer sleeve. Discard the original outer sleeve and the liquid therein. Use it to wash and remove non-specifically bound non-glycosylated exosomes and other impurities during the separation process; repeat step (5) 1-3 times. In this embodiment, the volume of the salt-free cleaning solution is 1ml.

[0100] (6) Take the same volume of the lectin-macromolecule carrier coupling complex in the affinity adsorption centrifuge tube and a borate-mannose buffer with a pH value of 6.5±0.2 as the eluent and add it to the upper centrifuge tube, wherein the eluent includes 15mM borate buffer and 300mM mannose dissolved therein, the lectin-macromolecule carrier coupling complex after elution and washing, stand at room temperature for 5-10min, centrifuge at 3000rpm for 20 seconds; collect the elution supernatant in the outer sleeve, which is the separated glycosylated exosome solution, and the glycosylated exosomes are intact in shape after elution and separation, without damage or lysis. In this embodiment, the eluent volume is 500ul, added to the upper centrifuge tube, covered with the centrifuge tube cap, stood at room temperature for 5min, centrifuged at room temperature for 20 seconds at 3000rpm; collect the elution supernatant in the outer sleeve, which is the separated glycosylated exosome solution, which is directly used for detection or stored at -80±5℃ for future use.

[0101] Implementation Case 4

[0102] This embodiment mainly further illustrates the separation sample of glycosylated exosomes. The samples to be separated that can be used in the present invention are mainly: any one of serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, and lymph fluid; for conventional 1.5-2ml upper centrifuge tubes, 1mL / tube of lectin-macromolecular carrier coupling complex preservation solution (containing 0.1-0.6ml / tube of lectin-macromolecular carrier coupling complex, such as 0.5ml) can be dispensed, and for 600ul of lectin-macromolecular carrier coupling complex, a pre-treated sample volume of 50-600ul, preferably 100-300ul, and more preferably 200-300ul of the serum, plasma, saliva, cerebrospinal fluid, lymph fluid, tissue or cell culture supernatant, and urine is added. The sample can be added directly or diluted with a cleaning solution before addition. During the sample pretreatment process, serum, plasma, saliva, cerebrospinal fluid, and lymph fluid samples are only centrifuged, and the volume of the samples does not change substantially before and after centrifugation. Therefore, 50-600ul, preferably 100-300ul, and more preferably 200-300ul of samples are pretreated to obtain the required volume of pretreated samples. During the sample pretreatment process, the tissue or cell culture supernatant and urine samples, in addition to the centrifugation step, also need to be concentrated 10-1000 times. Therefore, in order to obtain sufficient pretreated samples, 1-50ml, preferably 10-50ml, and more preferably 30-50ml of the tissue or cell culture supernatant and urine samples need to be pretreated including centrifugation and concentration to obtain the required volume of 50-600ul, preferably 100-300ul, and more preferably 200-300ul of pretreated samples.

[0103] Different sample separation volumes have a slight influence on the separation effect, but basically all can achieve the separation effect of glycosylated exosomes. To further illustrate the present invention, in this embodiment, according to the separation steps of embodiment 3, 200ul of pre-treated samples are selected for serum, plasma, saliva, cerebrospinal fluid, and lymph samples for illustration; the sample volume of tissue or cell culture supernatant and urine is 50ml, and the volume after concentration is preferably 200ul of pre-treated samples for illustration.

[0104] According to the sample specificity of serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, lymph fluid and other samples to be separated, four sample types including cell culture supernatant, plasma, urine and cerebrospinal fluid were selected for further explanation. After replacing other sample types, the same separation effect can be achieved.

[0105] According to the steps of Example 3, glycosylated exosomes in 200ul of pre-treated cell culture supernatant, plasma, urine, and cerebrospinal fluid samples in this example were separated to obtain glycosylated exosome separation solutions from four different sample sources for use.

[0106] Implementation Case 5

[0107] This implementation case mainly performs transmission electron microscopy on the glycosylated exosomes separated in implementation case 4 to observe the morphology and particle size of the glycosylated exosomes. The main steps are:

[0108] About 5ul of the glycosylated exosome sample isolated in Example 4 was taken and dropped on the copper mesh. After standing for 4-5min, excess liquid was removed from the edge of the copper mesh with filter paper. A negative staining solution (0.5% uranyl acetate aqueous solution, pH 4.5) was dropped on the copper mesh for 1min. The sample was dried with filter paper and washed twice. After drying, the sample was observed by transmission electron microscopy using Tecnai Spirit (120kV TEM). The glycosylated exosomes isolated from the four different samples in Example 4 were observed by transmission electron microscopy. The exosomes were intact and undamaged, and the particle size was between 30-150nm. For details, see Figure 2 Electron microscopic observation images of glycosylated exosomes separated from 4 different sample types.

[0109] Implementation Case 6:

[0110] This implementation case mainly uses the NanoSight NS300 system to perform nanoparticle tracking analysis (NTA) on the exosome particle size and diameter of the glycosylated exosomes separated in Implementation Case 4. The NTA analysis is mainly tested in a solution state (in situ test). Its high-precision particle size number distribution test can distinguish particles with a relative particle size difference of about 1:1.5 times, allowing the exosome particles to be measured in a state closer to their original state, ensuring the authenticity and validity of the measurement data, providing reliable exosome concentration data, and effectively making up for the current defect that when directly observing and measuring exosomes with an electron microscope, the particle size distribution data obtained is often not representative due to the limited range that can be observed at one time, and can effectively reduce the exosome damage caused by different pre-treatment methods such as drying, fixation and freezing for electron microscopy observation.

[0111] Based on the consistency of the results of electron microscopy observation of 4 different sample types in Implementation Case 5, glycosylated exosomes separated from plasma samples were selected for NTA detection in this implementation case. The detailed steps were as follows: about 10ul of sample was diluted to 1mL, sample was loaded using the NanoSight syringe pump, and the NanoSight NS300 system automatically analyzed. The glycosylated exosomes were relatively uniform, with an average particle size between 30-150nm, which was consistent with the average particle size range of exosomes, and the particle size was within 10 11 -10 14 pcs / ml; see Figure 3 : NTA analysis of glycosylated exosomes after separation of plasma samples;

[0112] Implementation Case 7

[0113] This implementation case mainly performs Western Blot detection and identification of the glycosylated exosomes separated in implementation case 4 using exosome-specific membrane proteins CD9 / CD63 / CD81, exosome-specific non-membrane proteins ALIX and TSG101, and exosome Calnexin antibodies. The main detection steps are as follows:

[0114] (1) Sample preparation: Take 30ul of glycosylated exosome eluate, add an equal volume of 5× SDS-PAGE loading buffer, treat at 100℃ for 10 minutes, and set aside;

[0115] (2) SDS electrophoresis: prepare gel (15% separation gel, 5% stacking gel), add 1× electrophoresis buffer, add 10 μl / well of sample, set the electrophoresis conditions at 60 V and 120 min;

[0116] (3) Transfer: Place a PVDF membrane of appropriate size in the transfer buffer and soak for 10 min. Fit the gel tightly to the PVDF membrane and sponge pad to prevent bubbles. Use a semi-dry transfer instrument to transfer the protein to the PVDF membrane (constant voltage 40 V, constant current 200 mA, transfer for 20 min).

[0117] (4) Blocking: Block with 5% skim milk powder for 1 hour, and wash with TBST three times, 5 minutes each time;

[0118] (5) Incubation: Add primary antibodies (Anti-CD9, Anti-CD81, Anti-CD63, Anti-TSG101, Anti-Alix, Anti-Calnexin monoclonal antibodies, concentration 1 mg / mL, 1:400 dilution) and incubate for 1 hour, wash three times with TBST, 5 minutes each time; add secondary antibodies (goat anti-mouse HRP 1 mg / mL, 1:5000 dilution) and incubate for 1 hour, wash three times with TBST, 5 minutes each time;

[0119] (6) Display: Use BeyoECL Moon (ultra-sensitive ECL chemiluminescence kit) for color development and detection.

[0120] The Western Blot test results of the four glycosylated exosomes isolated in Example 4 are as follows:

[0121] (1) Western Blot tests of exosome-specific membrane proteins CD9, CD63, and CD81 antibodies were all positive, indicating that the exosomes contained specific membrane proteins CD9, CD63, and CD81; see Figure 4 : Western Blot test results of exosome-specific membrane proteins CD9, CD63, and CD81 antibodies;

[0122] (2) Western Blot detection of exosome-specific non-membrane proteins ALIX and TSG101 antibodies was positive, indicating that the exosome-specific non-membrane proteins ALIX and TSG101 were present; see Figure 5 : Western Blot detection results of exosome-specific non-membrane proteins ALIX and TSG101 antibodies;

[0123] (3) Western Blot detection of Calnexin antibodies in exosome samples was negative. Figure 6: Western Blot test result of Calnexin antibody against exosomes. Calnexin exists in the endoplasmic reticulum of cells but not in exosomes. The Western Blot test of Calnexin antibody against glycosylated exosomes obtained by separating samples in this example was negative, indicating that there were no cell debris in the separated glycosylated exosome samples.

[0124] Implementation Case 8

[0125] This embodiment mainly compares and verifies the cleaning effect and separation effect of different cleaning solutions, especially common cleaning solutions containing metal salt ions, common cleaning solutions not containing metal salt ions, and purified water in the separation process of glycosylated exosomes, so as to determine the suitability of the cleaning solution selected in the present invention.

[0126] The metal salt ion-free buffer washing solution (Tris-HCL) in the glycosylated exosome separation composition in Examples 2 and 3 was replaced with purified water and a common washing solution containing metal salt ions. Common washing solutions containing metal salt ions include: phosphate buffered saline (PBS buffer), Tris-Triton-NaCl buffer, TBST buffer. Since most common washing solutions containing metal salt ions contain surfactants, such as Tween-20, Triton X-100, etc., the surfactants will damage the outer membrane of the exosome vesicles. Therefore, when preparing common washing solutions containing metal salt ions, no surfactant components are contained.

[0127] In this embodiment, 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.

[0128] The composition of the above-mentioned PBS buffer is: potassium dihydrogen phosphate (KH2PO4): 0.24 g / L, disodium hydrogen phosphate (Na2HPO4): 1.44 g / L, sodium chloride (NaCl): 8 g / L, potassium chloride (KCl): 0.2 g / L, and the pH value is 7.4±0.2.

[0129] The composition of the Tris-Triton-NaCl buffer without Triton X-100 is: 50mM Tris-HCL, 0.6MNaCL, and the pH value is 7.6±0.2.

[0130] The composition of the TBST buffer without Tween-20 is: 10mM Tris-HCL, 0.15MNaCL, and pH value is 7.6±0.2.

[0131] The five washing buffers in this embodiment are purified water, Tris-HCL buffer, PBS buffer, Triton X-100-free Tris-Triton-NaCl buffer, and Tween-20-free TBST buffer, which are marked as washing solution No. 1, washing solution No. 2, washing solution No. 3, washing solution No. 4, and washing solution No. 5 in the above order. According to the method in embodiment 3, the five washing solutions in this embodiment are used to separate the glycosylated exosomes in the plasma samples, and a comparative verification analysis is performed. Except for the different washing solutions, the other compositions and steps are the same. For other detailed processes, refer to embodiments 1-4, and the Western Blot detection and identification steps of the exosome-specific membrane protein CD9 / CD63 / CD81 antibody in embodiment 7.

[0132] In this embodiment, after washing with 5 different washing buffers, the Western Blot test results of the exosome-specific membrane protein CD9 / CD63 / CD81 antibody of the glycosylated exosome solution obtained by elution and separation are detailed in Figure 7 .

[0133] The results of this implementation case show that after the lectin-macromolecular carrier coupling complex bound to glycosylated exosomes was washed with a buffer solution containing no metal salt ions (cleaning solution No. 1 and cleaning solution No. 2), the Western Blot test results of the eluate obtained by eluting it showed that the exosome-specific membrane proteins CD9 / CD63 / CD81 were all positive, indicating that glycosylated exosomes can be effectively separated; and the common cleaning buffer solution containing metal salt ions without surfactant (cleaning solution No. 3, No. 4, No. 5) was used to wash the lectin-macromolecular carrier coupling complex bound to glycosylated exosomes, and then the Western Blot test results of the eluate obtained by eluting it showed that the exosome-specific membrane proteins CD9 / CD63 / CD81 were all negative, indicating that the presence of metal salt ions in the solution affects the separation effect of glycosylated exosomes, and it has a significant dissociation effect on the glycosylated exosomes in the present invention.

[0134] Implementation Case 9

[0135] This embodiment mainly compares and verifies the separation effect of different common protein eluents and the borate-mannose buffer of the present invention on glycosylated exosomes to determine the suitability of the eluent selected in the present invention.

[0136] The borate-mannose buffer in the glycosylated exosome separation composition in Examples 2 and 3 was replaced with a commonly used protein elution buffer and a glycoprotein elution buffer, specifically a protein antibody elution buffer stripping buffer and a glycoprotein elution solution Glycoprotein Eluting Solution (Vectorlabs, USA).

[0137] The stripping buffer composition is: 62.5 mmol / L Tris·HCl, 2% SDS, 100 mmol / L β-2-mercaptoethanol, pH 6.8±0.2.

[0138] The three eluents in this implementation case were used to separate the glycosylated exosomes in the plasma samples, and a comparative verification analysis was performed. Except for the different eluents, the other compositions and steps were the same. The detailed process was referred to Implementation Cases 1-5, and the exosome morphology and integrity of the glycosylated exosomes in the plasma samples separated by the three eluents were observed under an electron microscope.

[0139] The results of exosome electron microscopy detection of glycosylated exosome solutions separated by three different eluents in this embodiment are detailed in Figure 8 .

[0140] The results of this implementation case show that the elution solution of the present invention can effectively separate the glycosylated exosomes in the sample, and the exosomes are intact and undamaged, with a particle size between 30-150nm; no exosomes were observed when stripping buffer was used as the elution solution, which may be because the stripping buffer damaged the outer membrane of the exosomes during the elution process and failed to obtain complete exosomes; and no typical exosome vesicles were observed with the glycoprotein elution solution Glycoprotein Eluting Solution, only suspected exosome vesicles were observed, but the exosome morphology was incomplete, indicating that the exosomes were damaged to a certain extent during the separation process, which is not conducive to subsequent detection and analysis.

[0141] In summary, the application of the present invention to the separation of glycosylated exosomes from different sample sources can achieve the same effect, and the separated exosomes are all glycosylated exosomes. After separation and purification, the exosomes are intact in morphology without damage, and can be directly used for glycosylated exosome liquid detection, exosome immunological detection, or gene detection and analysis after nucleic acid extraction, etc.; exosomes with higher purity can also be further obtained through subsequent purification steps.

[0142] The above description shows and describes the preferred implementation of the present invention. As mentioned above, it should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other implementations, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims

1. Use of a lectin-macromolecule carrier coupling complex in isolating glycosylated exosomes from clinical samples, characterized in that: include: Macromolecular carriers; and a lectin coupled to the outside of a macromolecular carrier; wherein: The lectin includes: any one or two or more of japanese lectin, peanut lectin, pea lectin, canava lectin, lentil lectin, wheat germ lectin, soybean lectin, kidney bean lectin, and snail lectin; The macromolecular carrier includes any one or two or more of dextran microspheres, agarose microspheres, resin or epoxy resin microspheres, and polystyrene microspheres; The glycosylated exosomes include: any one or two or more of N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes; The particle size distribution range of the macromolecular carrier is 1 μm-200 μm; In the lectin-macromolecular carrier coupling complex, 1-20 mg of lectin is coupled to each 1 ml of the macromolecular carrier.

2. The use according to claim 1, characterized in that: The particle size distribution range of the macromolecular carrier is 10 μm-200 μm.

3. The use according to claim 1, characterized in that: The particle size distribution range of the macromolecular carrier is 30 μm-150 μm.

4. The use according to claim 1, characterized in that: In the lectin-macromolecular carrier coupling complex, 5-15 mg of lectin is coupled to each 1 ml of the macromolecular carrier.

5. The use according to claim 1, characterized in that: In the lectin-macromolecular carrier coupling complex, 10-15 mg of lectin is coupled to each 1 ml of the macromolecular carrier.

6. The use according to claim 1, characterized in that: The clinical sample includes: any one of serum, plasma, saliva, tissue or cell culture supernatant, urine, cerebrospinal fluid, and lymph fluid.

7. The use according to claim 1, characterized in that: The lectin includes any one or two or more of japanese lectin, peanut lectin, pea lectin, concanavalin A lectin, lentil lectin, wheat germ agglutinin, soybean lectin, and kidney bean lectin.

8. The use according to claim 1, characterized in that: Pea agglutinins include VVA and / or VVL.

9. The use according to claim 1, characterized in that: Snail lectins include HAA and / or HPA.

10. The use according to claim 1, characterized in that: The lectin-macromolecule carrier coupling complex preservation solution is packaged in a separation device including an affinity adsorption centrifuge tube; The affinity adsorption centrifuge tube comprises: an upper centrifuge tube and an outer protective sleeve; the diameter of the upper centrifuge tube is smaller than that of the outer protective sleeve, the upper centrifuge tube is sleeved in the outer protective sleeve and has a raised annular edge or pillar on the outer side of the upper part for supporting the opening of the upper centrifuge tube to be higher than the outer protective sleeve; the upper centrifuge tube comprises a centrifuge tube cover, a centrifuge tube wall and a filter membrane bottom fixedly connected to the centrifuge tube wall, the pore size of the filter membrane used at the bottom of the filter membrane is smaller than the particle size of the lectin-macromolecular carrier coupling complex and larger than the particle size of the exosomes, and the lectin-macromolecular carrier coupling complex preservation solution is stored in the upper centrifuge tube.

11. The use according to claim 10, characterized in that: The pore size of the filter membrane is 150nm - 1000nm.

12. The use according to claim 10 or 11, characterized in that: The separation of glycosylated exosomes from clinical samples is performed by a method comprising the following steps: Clinical sample pretreatment: Take the pre-treated sample as the sample to be tested; Completely remove the preservation solution portion of the lectin-macromolecular carrier coupling complex preservation solution from the affinity adsorption centrifuge tube loaded with the lectin-macromolecular carrier coupling complex, retain the lectin-macromolecular carrier coupling complex, and replace with a new outer sleeve; Take the sample to be tested and add it to the upper centrifuge tube after removing the preservation solution, incubate at room temperature, and remove the sample; Take the washing liquid and add it to the upper centrifuge tube. After centrifuging the affinity adsorption centrifuge tube, take out the upper centrifuge tube and put it into a new outer sleeve. Discard the original outer sleeve and the liquid therein to wash and remove the non-specifically bound non-glycosylated exosomes and other impurities during the separation process. This step is performed 1-3 times; Take the eluate and add it to the upper centrifuge tube to elute the washed lectin-macromolecular carrier coupling complex, let it stand at room temperature, and collect the elution supernatant in the outer sleeve, which is the separated glycosylated exosome solution.

13. The use according to claim 12, characterized in that: The pre-treated sample is mixed evenly with the cleaning solution and diluted as the sample to be tested.

14. Use of a glycosylated exosome separation composition in separating glycosylated exosomes from clinical samples, characterized in that: The glycosylated exosome separation composition comprises: the lectin-macromolecular carrier coupling complex used in any one of claims 1 to 13, and further comprises: a cleaning solution for cleaning and removing non-specifically bound, non-glycosylated exosomes and other impurities during the separation process, and an eluent for eluting the glycosylated exosomes specifically bound to the lectin-macromolecular carrier coupling complex, wherein the eluent makes the eluted exosomes intact in appearance and morphology without damage or lysis; wherein, The cleaning solution is a cleaning buffer solution or purified water without metal salt ions; The eluent is a borate buffer solution containing sugar.

15. The use according to claim 14, characterized in that: The cleaning solution is a cleaning buffer solution without metal salt particles.

16. The use according to claim 14, characterized in that The eluent is a borate buffer solution containing mannose.

17. The use according to any one of claims 14 to 16, characterized in that: The separation of glycosylated exosomes from clinical samples is performed by a method comprising the following steps: 1) Pre-treatment of clinical samples: 2) Take the pre-treated sample as the sample to be tested; 3) completely removing the preservation solution portion of the lectin-macromolecule carrier coupling complex preservation solution, and retaining the lectin-macromolecule carrier coupling complex; 4) Take the sample to be tested and add it to the lectin-macromolecular carrier coupling complex after removing the preservation solution, incubate at room temperature, and remove the sample; 5) Use the cleaning solution to clean the lectin-macromolecule carrier coupling complex bound to the glycosylated exosomes, remove the cleaning solution, and use it to clean and remove the non-specifically bound non-glycosylated exosomes and other impurities during the separation process; 6) Take the eluate, elute and wash the lectin-macromolecule carrier coupling complex that specifically binds to the glycosylated exosomes, let it stand at room temperature, and collect the elution supernatant, which is the separated glycosylated exosome solution.

18. The use according to claim 17, characterized in that In step 2), the pre-treated sample is mixed evenly with the cleaning solution and diluted as the sample to be tested.

19. The use according to claim 17, characterized in that: In step 4), the ratio of the sample to be tested to the lectin-macromolecule carrier coupling complex is 1:1-3.

20. The use according to claim 17, characterized in that In step 5), the ratio of the volume of the washing solution added once to the volume of the lectin-macromolecule carrier coupling complex bound to glycosylated exosomes is 1-3:

1.

21. The use according to claim 17, characterized in that In step 5), the cleaning step with the cleaning solution is repeated 1-3 times.

22. The use according to claim 17, characterized in that: In step 6), 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.

23. The use according to claim 17, characterized in that In step 6), the ratio of the volume of the added eluent to the volume of the washed lectin-macromolecule carrier coupling complex is 0.5-1:

1.

24. The use according to claim 17, characterized in that: The method of completely removing the preservation solution portion of the lectin-macromolecule carrier coupling complex preservation solution, removing the sample, removing the washing solution, and / or collecting the elution supernatant is to centrifuge at a speed of less than 3000 rpm at room temperature for 20 seconds.

25. The use according to claim 17, characterized in that In step 4), incubate at room temperature for 10-30 minutes.

26. The use according to claim 17, characterized in that In step 4), incubate at room temperature for 10-15 minutes.

27. The use according to claim 17, characterized in that: The sample pretreatment method includes the following steps: For serum, plasma, saliva, cerebrospinal fluid or lymph samples, centrifuge the samples at a speed below 3000g for 10-15 minutes to remove cell debris and precipitate impurities in the samples, and take the supernatant after centrifugation for later use; For tissue or cell culture supernatant and urine samples, centrifuge the samples at a speed below 3000g for 10-15 minutes to remove cell debris and precipitate impurities in the samples, and then concentrate the supernatant 10-1000 times through an ultrafiltration tube for later use.

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