Methods for detecting extracellular vesicles

Abstract

The present invention aims to provide a method for efficiently detecting extracellular vesicles in a sample. [Solution] The method for detecting extracellular vesicles in a sample according to the present invention is characterized by comprising the steps of: adsorbing the extracellular vesicles onto a positively charged carrier by contacting the sample with the carrier; binding an antibody that is specific to the extracellular vesicles and labeled with a labeling substance to the extracellular vesicles; and detecting the extracellular vesicles by a reaction involving the labeling substance.

Description

[Technical Field]

[0001] This invention relates to a method for efficiently detecting extracellular vesicles in a sample. [Background technology]

[0002] Extracellular vesicles are a general term for vesicles with a heterogeneous lipid bilayer structure secreted from almost all living cells, and are broadly classified into exosomes, microvesicles, and apoptotic bodies based on differences in their production mechanisms.

[0003] Exosomes are formed by inward budding of the late endosomal membrane, and then fuse with the cell membrane to become complete particles, which are then secreted extracellularly by exocytosis. Exosomes are extracellular vesicles with a diameter of approximately 30-150 nm released by eukaryotic cells in animals, plants, and fungi, and are known to play an important role in intercellular communication, such as encapsulating proteins, mRNA, and miRNA in body fluids. In recent years, it has been suggested that exosomes are involved in the enhancement of cancer metastasis and cancer progression, and diagnostic markers focusing on exosomes are being developed, such as biomarkers that use miRNA expressed from cancer-derived exosomes as tumor markers to predict cancer progression, metastasis, and prognosis.

[0004] Microvesicles are generated when the cell membrane buddings outward and separates. Microvesicles vary considerably in size, ranging from 100 to 1,000 nm in diameter. Apoptotic bodies are produced when cells undergo apoptosis, and their size also varies widely, from 50 to 5,000 nm in diameter. Similar applications to exosomes are expected for microvesicles and apoptotic bodies.

[0005] Therefore, various methods for detecting extracellular vesicles from a sample have been investigated, such as using ELISA or flow-through assays. However, in these methods, a primary antibody is used to capture the target extracellular vesicle, and then a secondary antibody is used to detect the captured vesicle. Depending on the type and amount of the antigen marker expressed on the extracellular vesicle, capture may not be possible. Furthermore, these methods are complicated and time-consuming, with ELISA, for example, taking about 6 hours. In addition, there is a risk of epitope competition and steric hindrance between antibodies when using two types of antibodies.

[0006] Furthermore, the method for detecting extracellular vesicles in a sample described in Patent Document 1 can be said to be ELISA itself. Patent Document 2 discloses a method for detecting exosomes that can improve the accuracy and precision of diagnosis by increasing disease specificity, and a method for isolating exosomes and obtaining detection signals using three detectable substances possessed by exosomes. Patent Document 3 discloses a method for detecting membrane proteins such as CD20, and these are also methods related to ELISA. Patent Document 4 discloses a method for detecting extracellular vesicles in a sample by labeling extracellular vesicles with a metal labeling reagent for labeling nucleic acids and a metal labeling reagent for labeling surface antigens, and identifying each labeling reagent by mass spectrometry. Patent Document 5 discloses a method for capturing extracellular vesicles in a sample on a carrier with polylysine, etc., and detecting them with a detection probe. Patent Document 6 discloses a method for capturing extracellular vesicles in a sample on a carrier with a sphingomyelin-binding protein and detecting them with a Western blot detection probe. Patent Document 7 also discloses a method for detecting membrane-bound proteins in a sample, such as CD20, using a capture antibody and a detection antibody. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Special Publication No. 2020-523555 [Patent Document 2] Japanese Patent Publication No. 2020-20810 [Patent Document 3] Special Publication No. 2022-524327 [Patent Document 4] International Publication No. 2020 / 235424 brochure [Patent Document 5] Japanese Patent Publication No. 2021-99291 [Patent Document 6] Japanese Patent Publication No. 2023-87936 [Patent Document 7] Japanese Patent Publication No. 2023-182682 [Overview of the project] [Problems that the invention aims to solve]

[0008] As mentioned above, various methods have been investigated for detecting extracellular vesicles in samples, but these methods, such as ELISA itself, modified ELISA, or the use of mass spectrometers, have not been easily implemented. Therefore, the present invention aims to provide a method for efficiently detecting extracellular vesicles in a sample. [Means for solving the problem]

[0009] The inventors of this invention conducted extensive research to solve the above problems. As a result, they discovered that by using a positively charged carrier, it is possible to selectively adsorb extracellular vesicles in a sample, and to easily detect them, thus completing the present invention. The present invention is described below.

[0010] [1] A method for detecting extracellular vesicles in a sample, A step of adsorbing the extracellular vesicles onto a carrier having a positive charge by bringing the sample into contact with the carrier, A step of binding an antibody that is specific to the extracellular vesicle and labeled with a labeling substance to the extracellular vesicle, and A method characterized by comprising the step of detecting the extracellular vesicle using the labeling substance. [2] The method according to [1] above, further comprising a step of eluting the extracellular vesicles to which the antibody is bound from the carrier. [3] The method according to [2] above, wherein the extracellular vesicles are eluted from the carrier with a buffer solution containing a metal salt of 0.12 M or more and 0.8 M or less. [4] The method according to any one of [1] to [3] above, further comprising a step of washing the carrier under conditions where the extracellular vesicles do not elute from the carrier after the extracellular vesicles are adsorbed to the carrier. [5] The method according to any one of [1] to [4] above, further comprising a step of adjusting the electrical conductivity of the sample to 4 mS / cm or more and 15 mS / cm or less before contacting the carrier. [6] The method according to any one of [1] to [5] above, wherein the carrier has a tertiary amino group and / or a quaternary ammonium group. [7] The method according to any one of [1] to [6] above, wherein the extracellular vesicles are exosomes and / or microvesicles. [Effects of the Invention]

[0011] For example, according to ELISA or flow-through assay, depending on the type and expression level of the antigen of the primary antibody, such as membrane proteins present on the surface of extracellular vesicles to be detected, it may not be possible to sufficiently capture the extracellular vesicles. In contrast, according to the method of the present invention, extracellular vesicles can be effectively captured regardless of the type and amount of membrane proteins present on the surface of the extracellular vesicles to be detected, for example. Further, only one type of antibody may be used in the method of the present invention, so there are no problems of epitope competition or steric hindrance between antibodies. Furthermore, the operation of the method of the present invention is simple and does not require a large-scale device, and the measurement does not take a relatively long time. Therefore, the present invention is very excellent industrially as a method capable of efficiently detecting extracellular vesicles, which have been found to be useful in diagnostic methods and the like in recent years. [Embodiments for Carrying Out the Invention]

[0012] The method for detecting extracellular vesicles in a sample according to the present invention will be described step by step below, but the present invention is not limited to the following specific examples. In the present invention, "detection" includes qualitatively measuring the presence or absence of extracellular vesicles in a sample and quantitatively measuring the concentration and amount of extracellular vesicles in a sample.

[0013] 1. Sample preparation process In this step, a sample that may contain extracellular vesicles to be detected is prepared as needed. Performing this step is optional, and if a sample for which extracellular vesicles should be detected already exists, this step does not need to be performed. In this disclosure, the numerical range "x~y" includes both x and y.

[0014] The sample is not particularly limited as long as it may contain the extracellular vesicles to be detected, but it is preferably in liquid form. Specifically, examples include cell culture medium, blood, interstitial fluid, urine, cerebrospinal fluid, lymph, ascites, and milk. The sample may also be crudely purified. For example, the sample may be the culture supernatant obtained by lysing or disrupting cells in a cell culture medium and removing at least some of the solid components from the cell culture by filtration or centrifugation, or serum or plasma separated from blood. Furthermore, if the sample is not liquid, such as organ tissue, it may be micronized and then dissolved or dispersed in a liquid such as a buffer. At least some of the solid components may be removed from such dispersion by filtration or centrifugation.

[0015] A cell culture includes the target cell culture and the materials that make up the culture medium for the cell culture. Examples of cell cultures include commonly available, typically commercially available cell lines, such as cultured tumor cell lines derived from humans or non-human mammals. For example, cultured tumor cells from uterine cancer, ovarian cancer, and breast cancer can be used, and HEK293 cells derived from human fetal kidneys may also be used.

[0016] Extracellular vesicles are a general term for vesicles with a heterogeneous lipid bilayer structure secreted from almost all living cells. Extracellular vesicles are broadly classified into three types based on differences in their intracellular production mechanisms: exosomes, microvesicles, and apoptotic bodies. Exosomes are 30-150 nm in diameter and are formed by inward budding of the late endosomal membrane. They then fuse with the cell membrane to form complete particles and are secreted extracellularly by exocytosis. Microvesicles, on the other hand, are produced by outward budding and separation of the cell membrane. They are 100-1,000 nm in diameter. Apoptotic bodies are produced when cells undergo systematic cell death (apoptosis). They are 50-5,000 nm in diameter.

[0017] In this invention, "extracellular vesicles" may be extracellular vesicles contained in a sample or extracellular vesicles produced by cultured cells, and there are no particular restrictions on the size or morphology of the extracellular vesicles themselves. Furthermore, they may be natural or non-natural (artificial) extracellular vesicles.

[0018] In this process, unwanted contaminants such as cell debris may be removed. For example, the cell culture may be subjected to centrifugation, microfiltration, ultrafiltration, or dialysis. Centrifugation is performed, for example, at 4°C, 10,000 × g, for 15 to 30 minutes. The supernatant from which cell debris and other contaminants have been removed by centrifugation is collected. Additional centrifugation may be performed to further remove contaminants. If additional centrifugation is performed, the centrifugal acceleration (g) may be increased to remove cell debris of medium size or smaller. If the target of detection is exosomes or microvesicles, apoptotic vesicles may be removed.

[0019] Microfiltration is a process that uses a membrane filter with a pore size of 0.2 to 0.5 μm, and by recovering the filtrate, it is possible to remove medium-sized cell debris and other impurities, similar to centrifugation.

[0020] The supernatant obtained by centrifugation and / or the filtrate obtained by microfiltration may be treated by ultrafiltration and / or dialysis using a membrane filter with a pore size of 0.1 μm or less, more preferably 0.05 μm or less. Ultrafiltration and / or dialysis can remove impurities smaller than extracellular vesicles and concentrate extracellular vesicles.

[0021] This process may include ultracentrifugation. In the ultracentrifugation process, extracellular vesicles can be precipitated by centrifugal force of 100,000 × g or more, thereby removing contaminants smaller than extracellular vesicles and concentrating extracellular vesicles.

[0022] 2. Contact process with carrier In this process, the sample is brought into contact with a positively charged carrier, thereby adsorbing the extracellular vesicles onto the carrier.

[0023] In this process, it is preferable to adjust the electrical conductivity of the sample to 4 mS / cm or higher and 15 mS / cm or lower. Generally, the sample has an osmotic pressure close to that of physiological saline, in which cells are likely to survive. The concentration and osmotic pressure of physiological saline are 0.154 mmol / L and 308 mOsm / L, respectively. The osmotic pressure of the sample is mostly derived from the ionic components in the solution, and therefore a positive correlation is observed between the electrical conductivity of the sample and the salt concentration, or between electrical conductivity and osmotic pressure. By adjusting the electrical conductivity to 15 mS / cm or lower, the adsorption efficiency of extracellular vesicles to positively charged carriers can be further increased because extracellular vesicles have a relatively weak negative charge. On the other hand, by adjusting the electrical conductivity to 4 mS / cm or higher, the denaturation of extracellular vesicles can be more reliably suppressed.

[0024] If the electrical conductivity is 15 mS / cm or less, the concentration of negatively charged ions such as chloride ions in the salt-containing buffer component of the sample is reduced. This reduces the proportion of ion exchange groups on the carrier that are ionically bound to negatively charged ions such as chloride ions, and increases the number of ion exchange groups that are effective in adsorbing negatively charged extracellular vesicles. The increased density of effective ion exchange groups on the carrier surface generates a zeta potential on the carrier surface that can attract extracellular vesicles, which have a lower diffusion coefficient compared to low-molecular-weight ions, to the carrier surface. As a result, particles with a particularly small degree of negative charge among extracellular vesicles are more easily adsorbed to the carrier. On the other hand, if the electrical conductivity is 4 mS / cm or higher, the osmotic pressure of the aqueous solution component in the sample does not decrease excessively, and damage to extracellular vesicles due to the osmotic pressure difference between the inside and outside of the lipid bilayer membrane of the extracellular vesicles can be suppressed. Therefore, it is important to adjust the electrical conductivity to the appropriate range described above. A more preferable electrical conductivity is 9 mS / cm or higher and 14 mS / cm or lower.

[0025] The electrical conductivity is preferably adjusted by adjusting the salt concentration. When the electrical conductivity is between 9 mS / cm and 14 mS / cm, the NaCl concentration in 10 mM Tris-HCl is between 79 mmol / L and 130 mmol / L.

[0026] To reduce the electrical conductivity and salt concentration of a sample, one method, though not particularly limited, is to dilute the sample with a buffer solution that has lower electrical conductivity and salt concentration than the sample. In this case, the buffer solution used is not particularly limited, but examples include 10 mM Tris-HCl buffer, phosphate-buffered saline (PBS), and Tris-buffered saline (TBS). In another embodiment, the electrical conductivity and salt concentration of the sample can be adjusted by replacing the buffer solution by ultrafiltration or by desalting by dialysis.

[0027] The basic skeletal material of the carrier is not particularly limited, but examples include cellulosic resins such as cellulose acetate and cellulose triacetate; and polysulfone resins. The carrier may also be a sheet-like separation membrane such as a flat membrane or a hollow fiber separation membrane, or it may be a particulate porous body or a non-porous body.

[0028] In the present invention, the method for imparting a positive charge to the support is not particularly limited, but one method is to impart a cationic compound after the support has been molded. More specifically, one method is to impart a positive charge by contacting a support having a hydroxyl group with a tertiary amine and / or quaternary ammonium having a glycidyl group as a reactive substituent. Alternatively, a column packed with commercially available diethylaminoethyl (DEAE) cellulose resin may be used. DEAE cellulose is an anion exchanger in which diethylaminoethyl groups are introduced into cellulose, and can be obtained from companies such as Sigma-Aldrich.

[0029] In the present invention, it is preferable to use a cellulose-based ion exchange membrane as a positively charged carrier, in which at least some of the hydroxyl groups or acetyl groups at the 2nd, 3rd, and 6th positions of a porous substrate membrane containing a cellulose-based polymer are replaced with a positively charged compound.

[0030] Porous substrate membranes containing cellulose polymers can be manufactured by a wet process. The wet process involves mixing the cellulose polymer with a solvent and a non-solvent to prepare a film-forming solution, then extruding it in a hollow form and guiding it into a solidification bath for phase separation. Alternatively, dry processes, heat-induced phase separation methods, and stretching methods may also be used. On the other hand, flat membranes such as porous substrate membranes can be manufactured by dissolving the cellulose polymer in a solvent, uniformly applying the solution to a substrate such as glass, immersing it in a solidification solution to solidify it, washing it, and drying it if necessary.

[0031] Polar solvents such as γ-butyrolactone, N-methylpyrrolidone, and dimethylacetamide are preferred as solvents for preparing the film-forming solution. These solvents may be used individually or in combination. If necessary, non-solvents such as water, glycerin, ethylene glycol, triethylene glycol, polyethylene glycol 200, and polyethylene glycol 400 may also be added. The mixing ratio of solvent to non-solvent in the film-forming solution (solvent / non-solvent ratio) is preferably 90 / 10 to 10 / 90.

[0032] It is preferable to use a mixture of a solvent and a non-solvent such as water as the coagulation solution. Furthermore, it is preferable to match the solvent / non-solvent ratio in the coagulation solution to the solvent / non-solvent ratio in the film-forming solution. By matching the solvent / non-solvent ratio of the film-forming solution and the coagulation solution, compositional fluctuations of the coagulation solution can be suppressed even during continuous film formation. In the case of flat membranes, it is preferable that the weight ratio of N-methylpyrrolidone to water in the mixed solution of N-methylpyrrolidone and water is in the range of 30:70 to 50:50. By having the coagulation bath have such a composition, the pore size of the membrane surface on the side that first comes into contact with the coagulation bath becomes larger than the pore size of the membrane surface on the side that comes into contact with the substrate, and an asymmetric structure is formed with respect to the thickness direction. When the concentration of N-methylpyrrolidone in the coagulation bath is 25% by mass or more and less than 30% by mass, the pore size of the membrane surface on the side that first comes into contact with the coagulation bath becomes equal to the pore size of the membrane surface on the side that comes into contact with the substrate, and the asymmetric structure of the membrane tends to be mitigated. Furthermore, when the concentration of N-methylpyrrolidone in the coagulation bath is less than 25% by mass, a dense layer without distinct pores is more likely to form on the membrane surface that first comes into contact with the coagulation bath. The temperature of the coagulation solution is preferably 5 to 60°C.

[0033] The resulting porous substrate film is washed with warm water to remove excess solvent and other contaminants. After washing, the porous substrate film may be stored in water, or it may be dried after filling the pores with a pore-retaining agent such as an aqueous glycerin solution.

[0034] Positively charged ion exchange groups are introduced into the porous substrate membrane. Specifically, after deacetylation treatment, a charge transfer treatment is performed on the hydroxyl groups. As an example, a method for producing a cationic cellulose membrane, which is an ion exchange membrane, by substituting positively charged groups for the hydroxyl groups of a cellulose-based porous substrate membrane is to immerse the cellulose-based porous substrate membrane in an insoluble solvent in the presence of alkali, add a solution of a positively charged compound dropwise, react under heating conditions, and quench with alcohol.

[0035] The aforementioned insoluble solvent is not particularly limited as long as it is a solvent that does not dissolve the cellulose-based porous substrate film and is capable of dissolving positively charged compounds. For example, water, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc., are good examples and may be used individually or in combination depending on the solubility of the raw materials.

[0036] As alkalis used for deacetylation, hydroxides such as lithium hydroxide, potassium hydroxide, sodium hydroxide, and cesium hydroxide, as well as carbonates and organic amines, can be used. Among these, sodium hydroxide, which is industrially inexpensive, is preferred. The alkali concentration used is preferably in the range of 0.05% by mass or more and 5.0% by mass or less. If the alkali concentration is 0.05% by mass or more, the amount of charged groups introduced can be increased, and the amount of extracellular vesicles adsorbed per unit volume can be increased more reliably. On the other hand, if the alkali concentration is 5.0% by mass or less, the degree of membrane swelling can be suppressed, so that the structure such as the pore size of the base membrane and the physical properties such as membrane strength can be more reliably maintained in the ion exchange membrane after the introduction of charged groups. Therefore, the alkali concentration is more preferably 0.1% by mass or more and 2.5% by mass or less, and even more preferably 0.2% by mass or more and 2.0% by mass or less.

[0037] The amount of charged groups imparted to the cellulose membrane can be controlled by adjusting the amount of positively charged compounds added. For example, when using a tertiary amine compound containing epoxy groups, the concentration of the tertiary amine compound in the insoluble solvent is preferably in the range of 0.01 ml / L to 3.0 ml / L. In this case, the ion exchange capacity corresponds to 0.05 meq / g to 0.9 meq / g per cellulose membrane weight. When using a quaternary ammonium compound containing epoxy groups, the concentration of the quaternary amine compound in the insoluble solvent is preferably in the range of 0.01 ml / L to 1.0 ml / L. In this case, the ion exchange capacity corresponds to 0.05 meq / g to 0.4 meq / g per cellulose membrane weight. If too many positively charged groups are imparted, the content of positively charged groups per polymer molecule increases, which can lead to increased water solubility and weakened membrane strength in water. Conversely, if too few positively charged groups are imparted, the capture efficiency of extracellular vesicles may decrease.

[0038] In the present invention, the positively charged carrier, such as an ion exchange membrane, is preferably in the form of a device housed in a container equipped with an inlet for introducing the liquid to be treated and an outlet for discharging the sample, which is the liquid to be treated after ion exchange. This device has a first chamber and a second chamber separated by an ion exchange membrane, and the sample, which is the liquid to be treated, introduced into the first chamber moves to the second chamber by permeating through the ion exchange membrane. At this time, extracellular vesicles in the sample, which is the liquid to be treated, are adsorbed onto the surface and pore surface of the positively charged carrier.

[0039] In the present invention, the ion exchange membrane, which is a positively charged carrier, may be a cellulose-based ion exchange membrane in which at least a portion of the hydroxyl groups or acetyl groups at the 2nd, 3rd, and 6th positions of the cellulose-based polymer are substituted with a compound having a positive charge.

[0040] In the present invention, examples of cellulosic polymers include cellulose acetate, cellulose propionate, cellulose butyrate, cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate laurate, cellulose acetate oleate, and cellulose acetate stearate. However, cellulose acetate is preferred due to its ease of introducing positively charged compounds. Cellulose acetates with different degrees of acetate and molecular weights are commercially available, and it is more preferable to use cellulose acetate and / or cellulose triacetate with a degree of acetate of about 52 to 62.

[0041] In the present invention, it is preferable to use a tertiary amine represented by the following formula (I) or a quaternary ammonium compound represented by the following formula (II) as the positively charged compound. In the formula, R 1 ~R 3 These are, independently, hydrogen atoms, or C 1-10 The formula represents an alkyl group, where n is an integer between 1 and 5. In the formula, X represents one or more selected from the group consisting of a halogen group of fluoro, chloro, bromo, or iodine; a leaving group such as a sulfonic acid ester such as tosylate, triflate, or mesylate; a silyl group such as alkoxysilyl or silanol; an epoxide group; an isocyanate group; or a carboxylic acid group. Any substituent X may be used for a positively charged compound, but from the standpoint of ease of obtaining raw materials, compounds in which X is a chloro, silyl, or epoxide group are preferred.

[0042] [ka]

[0043] [ka]

[0044] "C 1-10An "alkyl group" refers to a linear or branched monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, n-hexyl, n-octyl, n-hexyl, n-decyl, etc. Preferably C 1-8 Alkyl or C 1-6 It is an alkyl group, more preferably C 1-4 It is an alkyl group, and more preferably C 1-2 It is an alkyl group.

[0045] In the present invention, the cellulose-based ion exchange membrane may be a so-called homogeneous membrane in which the pore diameter is substantially constant from one surface to the other, or it may be a so-called asymmetric membrane in which the pore diameter changes continuously or discontinuously from one surface to the other. Furthermore, the minimum pore diameter of the ion exchange membrane is preferably 50 nm or more and 1000 nm or less. In an asymmetric membrane, the minimum pore diameter layer is preferably located near one of the surfaces. If the minimum pore diameter is large, the probability of contact between the membrane surface and / or the pore surface of extracellular vesicles such as exosomes decreases, which may result in a low adsorption rate of extracellular vesicles in the sample. Also, it may not be possible to remove cell debris, etc. On the other hand, if the minimum pore diameter is small, the recovery rate of extracellular vesicles that have entered the inside of the membrane may decrease, or it may take a long time to recover them. The minimum pore diameter of the ion exchange membrane is defined as the polystyrene particle diameter at which the rejection rate is 80% or more when measured using a polystyrene particle dispersion.

[0046] The average pore size of the surface of the ion exchange membrane, for example, the surface without the minimum pore size layer, is preferably between 100 nm and 5000 nm. If the average pore size of the surface is 100 nm or more, the sample can be processed at a sufficiently fast rate. If the average pore size of the surface is 5000 nm or less, the adsorption of extracellular vesicles to the carrier can be sufficiently promoted, and the recovery rate can be maintained. Furthermore, if the average pore size of the surface falls within the above range, the effect of depth filtration can be more reliably achieved. The average pore size of the surface can be calculated using the image processing software Image J based on images taken of both sides of the membrane at a magnification of 1000 to 20,000 times using a scanning electron microscope (SEM).

[0047] In this process, the sample to be treated may be processed by cross-flow or by dead-end processing. Furthermore, when using an asymmetric membrane, the sample to be treated may be introduced to either the large-pore side or the small-pore side. For example, in samples with a low solid component concentration and a high extracellular vesicle concentration, such as culture supernatant, by using the side with the larger pore diameter as the primary side and the side with the smaller pore diameter as the secondary side, it is possible to prevent the membrane pores from becoming clogged with adsorbed extracellular vesicles when loading the sample onto the membrane and allowing the extracellular vesicles to be charged and adsorbed onto the membrane. On the other hand, in samples with a relatively high solid component concentration relative to the extracellular vesicle concentration, such as serum, plasma, urine, and milk, by using the side with the smaller pore diameter as the primary side and the side with the larger pore diameter as the secondary side, impurities can be removed by surface filtration, and membrane clogging due to pore blockage can be suppressed.

[0048] In the present invention, the ion exchange capacity of the positively charged carrier is preferably 0.05 meq / g or more and 1.5 meq / g or less. If the ion exchange capacity is 0.05 meq / g or more, the zeta potential of the membrane is stable, and even substances to be purified with a small diffusion coefficient, such as extracellular vesicles, can be sufficiently attracted to the membrane, and the adsorption amount is sufficiently large, so a large-capacity device is not necessary. On the other hand, if the ion exchange capacity is 1.5 meq / g or less, the strength of the membrane is sufficient, and the adsorption amount per unit volume of the membrane becomes appropriate, suppressing membrane clogging. When using a tertiary amine compound, the ion exchange capacity is preferably 0.03 meq / g or more and 0.9 meq / g or less per carrier mass. When using a quaternary ammonium compound, the ion exchange capacity is preferably 0.03 meq / g or more and 0.4 meq / g or less per carrier mass.

[0049] In the present invention, the form of the carrier may be a flat membrane or a hollow fiber membrane, but the membrane thickness is preferably 10 μm or more and 1000 μm or less. If the membrane thickness is 10 μm or more, the membrane strength is sufficiently high, and handling convenience in film formation and device fabrication is high. On the other hand, if the membrane thickness is 1000 μm or less, the recovery time of extracellular vesicles can be suppressed and the recovery rate can be maintained. In the case of a hollow fiber membrane, the inner diameter is preferably 50 μm or more and 1000 μm or less. If the inner diameter is 50 μm or more, the shear stress of the liquid flowing through the hollow portion is sufficiently small, and damage to extracellular vesicles can be sufficiently suppressed. On the other hand, if the inner diameter is 1000 μm or less, the membrane area per device can be sufficiently large.

[0050] In this invention, the zeta potential of the carrier is preferably in the range of 10 mV or more and 70 mV or less under pH conditions of 6.5 to 7.5. If the zeta potential is 10 mV or more, the adsorption force to extracellular vesicles with low diffusion coefficients is sufficiently high, ensuring adsorption. If the zeta potential is 70 mV or less, the membrane strength is sufficiently high, and when particles such as extracellular vesicles are to be purified, the amount of adsorption per unit volume of membrane becomes appropriate, suppressing membrane clogging. The zeta potential of a sheet-like carrier can be measured using a plate zeta potential measurement cell.

[0051] In the present invention, the total specific surface area of pores with a pore size of 30 nm or more in the carrier is preferably 3 m 2 / g or more, and more preferably 8 m 2 / g or more. If such a specific surface area is 3 m 2 / g or more, a sufficient adsorption amount of extracellular vesicles can be ensured. The specific surface area is generally measured by a gas adsorption method or a mercury intrusion method. In the gas adsorption method, the specific surface area of micropores of approximately 2 nm or less and mesopores of 2 nm to 50 nm can be detected, whereas in the mercury intrusion method, pores of 50 nm or more can be detected, which is suitable for measuring pores suitable for the adsorption of extracellular vesicles. The specific surface area is preferably larger in order to ensure the adsorption amount of extracellular vesicles. However, since extracellular vesicles cannot enter the pores with a pore size of less than 30 nm, in order to increase the adsorption amount of extracellular vesicles per unit volume of the membrane, the specific surface area composed of pores with a pore size of 30 nm or more needs to be sufficiently large. Although the specific surface area of the pores is not a problem even if it is too large, it is preferably 150 m 2 / g or less, and more preferably 120 m 2 / g or less.

[0052] The porosity of the carrier is preferably 40% or more and 90% or less. If the porosity is 40% or more, a sufficient region for extracellular vesicles to adsorb can be ensured. If the porosity is 90% or less, the strength of the carrier can be sufficiently ensured. The porosity is more preferably 45% or more.

[0053] When adsorbing extracellular vesicles onto the carrier of the present invention, it is preferable to adjust the zeta potential, specific surface area, and porosity to a specific range. Among extracellular vesicles, exosomes are extracellular vesicles with a size of 30 nm to 150 nm and a negative charge on their surface. For example, when efficiently adsorbing and desorbing exosomes in a sample such as cell culture supernatant, if there are too many pores smaller than 30 nm, the extracellular vesicles cannot enter the pores, and even if the specific surface area of ​​the carrier is increased, the adsorption area and adsorption capacity cannot be increased. Also, if there are too many macropores, the distance between the membrane surface and the extracellular vesicles may be too great, making it difficult for electrical attraction to work. On the other hand, ion exchange membranes sometimes benefit from having a certain amount of micropores. Samples contain many small molecular weight substances, many of which are charged. By adsorbing such small molecular weight substances into micropores, it becomes possible to effectively utilize the adsorption sites of mesopores and macropores for the adsorption of extracellular vesicles. The specific surface area of ​​micropores and mesopores with a pore diameter of less than 30 nm is 30 m². 2 / g or more, 80m 2 Preferably less than / g

[0054] The ion exchange capacity of the positively charged support is preferably 0.08 meq / g or more and 0.3 meq / g or less. If the ion exchange capacity is 0.3 meq / g or less, the content of positively charged groups per polymer molecule constituting the support is not too high, and the water solubility of the support is not excessively high, so sufficient support strength in water can be ensured. Furthermore, if the ion exchange capacity is 0.08 meq / g or more, the capture efficiency of extracellular vesicles can be more reliably maintained at a high level. The positively charged group is preferably a tertiary amine group or a quaternary ammonium group. Diethylamine can be used as a tertiary amine compound for introducing a tertiary amine group, and trimethylammonium can be used as a quaternary ammonium compound for introducing a quaternary ammonium group.

[0055] In this invention, "contact" refers to immersing the carrier in the sample. Furthermore, if the carrier has a porous membrane structure, it also includes creating a pressure difference between the primary and secondary sides of the membrane so that the sample enters the porous membrane, thereby causing the sample to pass through the membrane from the primary to the secondary side. Additionally, if the carrier is a particulate resin, it also includes passing the sample through a resin-filled column from the inlet to the outlet using a pressure difference.

[0056] The amount of extracellular vesicles that the carrier can process depends on the amount of impurities in the sample, but when the carrier's anion exchange capacity is 0.08-0.3 meq / g, it is approximately 10 9 ~10 12 The sample density is 1 / mL, and the amount of sample processed must not exceed this value relative to the amount of carrier material.

[0057] 3. Washing process In this step, the sample is brought into contact with a positively charged carrier to adsorb extracellular vesicles onto the carrier. The carrier is then washed to the extent that the extracellular vesicles do not elute from the carrier, thereby removing impurities other than extracellular vesicles. Performing this step is optional; for example, if the sample contains very few impurities, this step may not be necessary. However, from the viewpoint of removing impurities that may interfere with subsequent steps, it is preferable to perform this step.

[0058] The washing conditions should be adjusted as appropriate to remove impurities while maintaining the adsorption of extracellular vesicles to the carrier, but it is preferable to separate the carrier after contacting it with a buffer solution. The buffer solution is not particularly limited, but for example, Tris-HCl, TBS, or PBS can be used. The salt concentration of the buffer solution is preferably adjusted to be less than or equal to the salt concentration of the sample in order to prevent the target extracellular vesicles from detaching from the carrier and eluting together with other contaminants. Specifically, it is preferable to have a salt concentration of 50 mmol / L or more and 150 mmol / L or less (0.05 M or more and less than 0.12 M), and 6.08 mS / cm or more and 16.01 mS / cm or less. The salt concentration is more preferably 0.11 M or less, and even more preferably 0.1 M or less. The pH of the buffer solution is preferably adjusted to 6.5 or more and 8 or less in order to suppress damage to the extracellular vesicles. The amount of buffer solution used for washing is preferably in the range of 5 to 100 times the volume of the carrier or the column packed with the carrier.

[0059] 4.Labeling process In this process, an antibody that is specific to extracellular vesicles adsorbed onto a carrier and labeled with a labeling substance is selectively bound to the extracellular vesicles, thereby forming a complex of extracellular vesicles and antibodies. By labeling the extracellular vesicles while they are adsorbed onto the carrier, the separation of the labeled extracellular vesicles from the antibodies that did not bind to the vesicles becomes easier.

[0060] The antibodies used in this process are antibodies that selectively bind to substances present on the surface of target extracellular vesicles. The antibodies can be selected according to the extracellular vesicles being detected. Examples of exosome marker molecules include CD81, CD63, and CD9, which are membrane protein families with structures that span the cell membrane four times; and fluorosilin, an endosome-related protein. Examples of microvesicle marker molecules include integrins, selectins, and CD40. Examples of apoptotic vesicle marker molecules include annexin V and phosphatidylserine. In this process, antibodies against these marker molecules can be used depending on the type of extracellular vesicle being detected, and such antibodies are commercially available. In addition, disease-specific marker molecules present on the surface of extracellular vesicles can also be used. For example, markers present on the surface of extracellular vesicles derived from cancer cells can be used.

[0061] Substances used to label antibodies include, for example, enzymes, radioisotopes, chemiluminescent substances, fluorescent substances, and particles. Examples of labeling enzymes include alkaline phosphatase (ALP) and peroxidase (HRP). Methods for binding labeling enzymes to antibodies include the periodic acid method, glutaraldehyde method, maleimide method, and N-hydroxysuccinimide (NHS) method. The periodic acid method involves oxidizing the sugar chain portion of the enzyme to a formyl group with periodic acid and binding it to the amino group in the enzyme via a reductive amination reaction. The glutaraldehyde method involves introducing a formyl group into the enzyme using excess glutaraldehyde and binding it to the amino group in the enzyme via a reductive amination reaction. The maleimide method involves introducing a maleimide group into the labeling enzyme and reacting it with the -SH group of the enzyme. The NHS method involves selectively activating the carboxyl group of the enzyme with NHS and reacting it with the amino group of the antibody. Kits for labeling proteins such as antibodies with ALP or HRP using these methods are commercially available, and anti-extracellular vesicle antibodies labeled with ALP or HRP are also commercially available in some cases. The method of detecting a target using an enzyme-labeled antibody is called enzyme immunoassay (EIA).

[0062] Radioisotopes (RIs) used to label antibodies include: 111 In 90 Y is one example. RIs can be introduced into antibodies using methods such as CCAP (Chemical conjecture by affinity peptide), which uses peptides that bind to specific sites on the antibody, or amine coupling. The method of detecting a target using an antibody labeled with an RI is called the RIA method.

[0063] Chemiluminescent and fluorescent substances can be bound to reactive groups such as amino groups in antibodies via linker groups. The method of detecting a target using antibodies labeled with chemiluminescent substances is called chemiluminescent immunoassay (CIA), and the method of detecting a target using antibodies labeled with fluorescent substances is called fluorescence immunoassay (FIA).

[0064] Examples of particles used to label antibodies include latex particles, gold colloids, gold nanoplates, and platinum colloids. Both chemical bonding and physical adsorption can be used to bind the particles to the antibody.

[0065] Conventional methods can be used to selectively bind antibodies to extracellular vesicles. For example, the membrane containing the captured extracellular vesicles can be immersed in the antibody solution, or the antibody solution can be passed through the membrane containing the captured extracellular vesicles.

[0066] As the solvent for the antibody solution, the buffer used for washing in washing step 3 can be used so as to prevent the adsorbed extracellular vesicles from eluting from the carrier. The concentration of the antibody in the solution depends on the amount of extracellular vesicles contained in the sample and the type of labeling substance, but can be, for example, 0.05 μg / mL or more and 5 μg / mL or less. If the concentration is 0.05 μg / mL or more, the labeled antibody can be reliably bound to the extracellular vesicles, and if it is 5 μg / mL or less, the residue of labeled antibody that is not bound to the extracellular vesicles can be more reliably suppressed. The concentration is preferably 0.1 μg / mL or more, preferably 2 μg / mL or less, more preferably 1 μg / mL or less, and even more preferably 0.5 μg / mL or less.

[0067] The amount of labeled antibody used should be adjusted as appropriate to ensure that the extracellular vesicles adsorbed to the carrier are sufficiently labeled. For example, depending on the dilution ratio of the sample, 1 mL of blood may contain trillions of exosomes, so 1 × 10⁶ antibodies per 1 mL of sample are used. 3 More than molecules, 2×10 13 Labeled antibodies of a molecular weight or less can be used.

[0068] 5. Washing process In this step, excess labeled antibody that is not bound to extracellular vesicles is removed by washing the carrier to which the labeled antibody is adsorbed. Performing this step is optional, and it is not necessary if the excess labeled antibody has been removed from the carrier in the labeling step 4. However, it is preferable to perform this step for more accurate detection.

[0069] This step is preferably carried out under the same conditions as the washing step 3 in order to suppress the elution of extracellular vesicles from the carrier. However, as long as the elution of extracellular vesicles from the carrier is suppressed, the conditions of this step do not need to be exactly the same as the actual conditions of the washing step 3.

[0070] 6.Elution process In this step, extracellular vesicles bound to the labeled antibody are eluted from the carrier. This step is optional, and the labeled extracellular vesicles may be detected while still adsorbed to the carrier. However, in that case, quantitative measurement of the extracellular vesicles may be difficult. Therefore, it is preferable to perform this step and quantitatively detect the extracellular vesicles eluted from the carrier in liquid.

[0071] To elute extracellular vesicles from a carrier while the labeled antibody remains bound, the carrier can be brought into contact with a buffer containing a metal salt of an appropriate concentration as the eluent. Tris-HCl, TBS, or PBS can be used as the buffer, and the pH of the buffer is preferably adjusted to 6.5 or higher and 8 or lower from the viewpoint of suppressing damage to the extracellular vesicles. However, when using substances that become more active under specific conditions, such as alkaline phosphatase, for the detection of extracellular vesicles, it is preferable to further adjust the type of solute and pH of the buffer as appropriate to optimize the detection conditions.

[0072] Examples of metal salts that can be included in the eluate include NaCl, Na2SO4, MgCl2, and MgSO4, with NaCl being preferred. The concentration of the metal salt in the eluate can be adjusted as appropriate, but for example, it can be 0.12M or higher and 0.8M or lower. If the concentration is 0.12M or higher, extracellular vesicles can be more reliably eluted from the carrier, and if it is 0.8M or lower, the dissociation of extracellular vesicles from the labeled antibody and damage to the extracellular vesicles can be more reliably suppressed. The metal salt concentration is preferably 0.15M or higher, more preferably 0.2M or higher, preferably 0.6M or lower, and more preferably 0.5M or lower.

[0073] Depending on the purpose, such as separating and recovering extracellular vesicles by type based on differences in their charge, it may be preferable to increase the salt concentration of the eluate while bringing it into contact with the carrier using methods such as stepwise elution or linear gradient elution.

[0074] To enhance the storage stability of extracellular vesicles eluted from the carrier, the salt concentration and electrical conductivity of the eluate containing the extracellular vesicles may be adjusted. The salt concentration and electrical conductivity should be within a range close to the electrolyte concentrations of living organisms, for example, they can be adjusted to 140 mmol / L or higher, 160 mmol / L or lower, and greater than 15 mS / cm and 17.01 mS / cm or lower. A preferred electrical conductivity is 15.01 mS / cm or higher. One method for measuring the salt concentration is to measure the electrical conductivity of the recovered fraction and calculate the NaCl concentration. Based on the obtained salt concentration, dilution can be performed by adding 10 mmol / L Tris-HCl (pH 7.5). Other methods include buffer replacement by ultrafiltration or desalting by dialysis.

[0075] 7. Detection process In this process, extracellular vesicles are detected according to the labeling substance present on the antibody bound to them. For example, if the labeling substance is an enzyme, the extracellular vesicles bound to the labeled antibody can be measured qualitatively or quantitatively by utilizing the reaction catalyzed by the enzyme. For example, if the labeling substance is alkaline phosphatase (ALP), since ALP catalyzes the hydrolysis of phosphate esters in an alkaline environment, p-nitrophenyl phosphate is added and the absorbance at 405 nm due to the resulting yellow p-nitrophenol is measured. If the labeling substance is peroxidase (HRP), 3,3',5,5'-tetramethylbenzidine (TMB) is added, and after the oxidation reaction by peroxidase, a stop solution is added and the absorbance at 450 nm is measured. Detection reagents for these enzymes are commercially available. If the labeling substance is a radioisotope (RI), the intensity of radioactivity emitted from the RI should be measured. If the labeling substance is a chemiluminescent or fluorescent substance, the intensity of luminescence or fluorescence corresponding to those substances should be measured. If the labeled substance is a particle, the change in absorbance corresponding to the aggregation of the particles can be measured, or if it is gold bound to extracellular vesicles via an antibody, it can be measured by surface plasmon resonance.

[0076] Depending on the labeling substance, the presence or absence of extracellular vesicles in a sample can be measured qualitatively, but it can also be measured quantitatively. For example, by creating a calibration curve in advance, it is possible to determine the concentration and absolute amount of extracellular vesicles in the sample and the sample being measured. [Examples]

[0077] The present invention will be described in more detail below with reference to examples, but the present invention is not limited by the following examples, and it is certainly possible to implement it with appropriate modifications within the scope that is consistent with the spirit of the preceding and following descriptions, and all such modifications are included within the technical scope of the present invention.

[0078] Example 1 (1) Preparation of anion exchange membrane A cellulose acetate membrane filter (manufactured by ADVANTEC) with a nominal pore size of 0.2 μm was deacetylated by immersion in a 0.1 M sodium hydroxide aqueous solution at room temperature for 4 hours. The resulting deacetylated membrane was then immersed in a 0.4 M glycidyl diethylamine aqueous solution at 65°C for 4 hours to obtain an anion exchange membrane as a positively charged support. The performance of the obtained anion exchange membrane is shown in Table 1.

[0079] [Table 1]

[0080] (2) Preparation of detection antibody Using a commercially available antibody / protein labeling kit ("LK13 Alkaline Phosphatase Labeling Kit-SH," manufactured by Dojin Chemical Laboratories), an anti-CD9 antibody ("Anti CD9, Human (Mouse) Unlabeled, 12A12," manufactured by Cosmo Bio) (50 μg) was labeled with alkaline phosphatase.

[0081] (3) Recovery of extracellular vesicles The anion exchange membrane prepared in (1) above (specific surface area: 9.6 m² 2The sample ( / g) was fixed to a holder, and 6 mL of 10 mM Tris-HCl buffer containing 0.1 M sodium chloride was passed through as a washing solution. Separately, pooled plasma was diluted 20-fold with the buffer to prepare a sample (4 mL). The obtained sample was permeated through an anion exchange membrane at a rate of 2 mL / min. Subsequently, 10 mL of the washing solution was passed through to remove impurities from inside the membrane. The detection antibody prepared in (2) above was diluted to 0.3 μg / mL with the buffer solution, and the resulting detection antibody solution (2 mL) was permeated through the anion exchange membrane at a rate of 0.2 mL / min. Subsequently, a washing solution (10 mL) was passed through to remove unbound antibodies from inside the membrane. The permeated washing solution was collected in 0.5 mL portions and designated as washing fractions 1 to 20. Subsequently, 2 mL of 10 mM Tris-HCl buffer containing 0.3 M sodium chloride was permeated as the eluate to elute the extracellular vesicle-detection antibody complex. The permeated eluate was collected in 0.5 mL portions and designated as elution fractions 1-4.

[0082] (4) Detection of extracellular vesicles 100 μL each of eluent, washing fractions 1 and 20, and elution fraction 1 were added to an ELISA plate. Then, 100 μL of p-nitrophenyl disodium phosphate solution (Sigma-Aldrich) was added, and the mixture was reacted at 37°C for 20 minutes. Next, the absorbance at 405 nm was measured using a microplate reader. The results are shown in Table 2.

[0083] [Table 2]

[0084] As is clear from the results in Table 2, unbound antibodies were sufficiently removed even at the stage of the first washing fraction 1, and the absorbance of the 20th washing fraction 20 was almost the same as the absorbance of the eluate itself, indicating that almost all unbound antibodies were removed by passing 10 mL of washing solution through the sample. On the other hand, subsequent elution after washing revealed that the absorbance of the first elution fraction was sufficiently high, indicating that the extracellular vesicle-detection antibody complex had been sufficiently eluted. Furthermore, while the general sandwich ELISA method requires approximately 6 hours for measurement, the method of the present invention required just over 1 hour from sample preparation to absorbance measurement.

Claims

1. A method for detecting extracellular vesicles in a sample, A step of adsorbing the extracellular vesicles onto a carrier having a positive charge by bringing the sample into contact with the carrier, A step of binding an antibody that is specific to the extracellular vesicle and labeled with a labeling substance to the extracellular vesicle, and A method characterized by comprising the step of detecting the extracellular vesicle using the labeling substance.

2. The method according to claim 1, further comprising the step of eluting the extracellular vesicle to which the antibody is bound from the carrier.

3. The method according to claim 2, wherein the extracellular vesicles are eluted from the carrier with a buffer containing a metal salt of 0.12 M or more and 0.8 M or less.

4. The method according to claim 1, further comprising the step of washing the carrier under conditions in which the extracellular vesicles do not elute from the carrier after adsorbing the extracellular vesicles onto the carrier.

5. The method according to claim 1, further comprising the step of adjusting the electrical conductivity of the sample to 4 mS / cm or more and 15 mS / cm or less before bringing it into contact with the carrier.

6. The method according to claim 1, wherein the carrier has a tertiary amino group and / or a quaternary ammonium group.

7. The method according to claim 1, wherein the extracellular vesicle is an exosome and / or a microvesicle.

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