JP7406789B2 - Methods for isolating bacterial membrane vesicles, isolation kits, and methods for their removal - Google Patents

Description

The present invention relates to a method for isolating bacterial membrane vesicles, an isolation kit, and a method for removing the same.

All bacteria, regardless of the type of bacteria, such as Gram-positive bacteria, Gram-negative bacteria, pathogenic bacteria, and resident bacteria, secrete membrane vesicles with a diameter of about 20 to 400 nm. Hereinafter, membrane vesicles secreted by bacteria will be referred to as membrane vesicles. Membrane vesicles have a structure similar to the outer membrane of bacteria because the outer membrane of bacteria protrudes outward and becomes a vesicle that is released outside the cell. Membrane vesicles mainly play a role in mediating communication between cells, and contain nucleic acids, proteins, etc. derived from the bacteria that produced the membrane vesicles. Membrane vesicles are known to contribute to the pathogenicity of bacteria and interactions between organisms, and because they have a structure similar to the cell surface, they are involved in immune regulation, and are expected to be applied to drug delivery systems and vaccines. ing.

In addition, membrane vesicles have been found to promote the production of IgA antibodies in mucosal immune systems such as the eyes, nose, mouth, respiratory tract, and digestive tract, just like live bacteria. Vaccines using membrane vesicles have a safety advantage over vaccines using detoxified or attenuated bacteria, and are therefore expected to be applied particularly to mucosal immunization vaccines. Therefore, techniques for isolating large quantities of membrane vesicles are becoming increasingly important.

It is also possible to encapsulate physiologically active substances produced by bacteria in membrane vesicles and use them for drug delivery. Therefore, the importance of techniques for isolating membrane vesicles with less damage (intact state) is increasing.

As a method for isolating membrane vesicles, ultracentrifugation using an ultracentrifuge is generally used. In the ultracentrifugation method, membrane vesicles contained in the sample are precipitated and isolated by ultracentrifuging a sample containing membrane vesicles (for example, centrifuging at 110,000 x g for 120 minutes 2 to 3 times). It is a technology that However, a problem with this method is that large-scale purification is difficult.

Note that Patent Document 1 discloses a method for isolating exosomes, which are a type of extracellular vesicles, in an intact state with extremely little damage.

International Publication WO2019/039179 Publication

However, ultracentrifugation, which is a conventionally common isolation or recovery technique for membrane vesicles, has the following problems. In the ultracentrifugation method, the centrifugation operation takes time and is difficult to scale up (large-scale purification). Furthermore, the ultracentrifugation method has a problem in that the recovery rate varies. Furthermore, problems such as membrane vesicles being damaged by strong centrifugal force have also been pointed out. Therefore, for future research on membrane vesicles and the spread of treatments using membrane vesicles, there is a need for the development of isolation methods that have higher reproducibility and can handle large quantities and multiple samples.

On the other hand, according to the method described in Patent Document 1, by using a carrier, it is possible to isolate exosomes, which are a type of extracellular vesicles, in an intact state with extremely little damage. However, it is unclear whether this method can also isolate membrane vesicles with different lipid bilayer membrane compositions in the same way as exosomes.

An object of the present invention is to provide a membrane vesicle isolation method and a membrane vesicle isolation kit that can easily isolate membrane vesicles in an intact state (or in a near-intact state). Another object of the present invention is to provide a method for removing membrane vesicles that can easily remove membrane vesicles from a sample containing membrane vesicles.

As a result of intensive studies to solve the above problems, the present inventors found that a peptide or polymyxin B containing consecutive lysines can bind to membrane vesicles. Furthermore, the present inventors have found that when using a carrier carrying a peptide containing consecutive lysines or polymyxin B, membrane vesicles bound to the carrier can be dissociated from the carrier under mild conditions.

That is, one embodiment of the present invention has the following configuration.
[1] A method for isolating membrane vesicles, comprising isolating the membrane vesicles from a sample containing membrane vesicles derived from bacteria, the method comprising: the sample and a peptide or polymyxin B containing 20 to 40 consecutive lysines; a complex forming step in which the membrane vesicle and the carrier are bonded to form a complex by contacting the membrane vesicle with a carrier carrying one or more of the following; A method for isolating membrane vesicles, the method comprising: contacting the complex with a dissociation buffer that dissociates the membrane vesicle from the complex.
[2] The method for isolating membrane vesicles according to [1], wherein the dissociation buffer contains a metal cation.
[3] A membrane vesicle isolation kit for carrying out the membrane vesicle purification method described in [1] or [2], the carrier carrying at least one of the peptide or polymyxin B. A membrane vesicle isolation kit comprising: and the dissociation buffer.
[4] A method for removing membrane vesicles from a sample containing membrane vesicles derived from bacteria, the method comprising removing the membrane vesicles from a sample and any one of a peptide or polymyxin B containing 20 to 40 consecutive lysines. a complex forming step in which the membrane vesicle and the carrier are bonded together to form a complex by contacting the membrane vesicle and the carrier carrying one or more of the carriers; A method for removing membrane vesicles, comprising a separation step of separating the complex from the sample to obtain a separated liquid from which at least a portion of the membrane vesicles have been removed from the sample.

According to one embodiment of the present invention, it is possible to easily isolate membrane vesicles in an intact state (or in a near-intact state).

FIG. 1 is a diagram showing an outline of a membrane vesicle. FIG. 2 is a diagram showing an outline of a carrier supporting a peptide in each example. FIG. 3 is a diagram showing an overview of experimental operating procedures for each example. FIG. 2 is a diagram showing an outline of an experimental procedure for an ultracentrifugation method as Comparative Example 1. FIG. 2 is a diagram illustrating an outline of the experimental procedure of an isolation method using magnetic beads carrying lysine, which is Comparative Example 2. 3 is an electrophoretic image showing the test results of Comparative Example 2. 1 is an electrophoretic image showing the test results of Example 1. 2 is an electrophoretic image showing the test results of Example 2. 3 is an electrophoretic image showing the test results of Example 3 and Example 4. 3 is an electrophoretic image showing the test results of Example 5 and Example 6. FIG. 3 is a diagram showing the results of electron microscopic observation of membrane vesicles isolated in Example 5. FIG. 3 is a diagram showing the particle size distribution of membrane vesicles contained in the eluate of Example 5.

An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various changes can be made within the scope of the claims. The present invention also includes embodiments or examples obtained by appropriately combining technical means disclosed in different embodiments or examples.

Note that all academic literature and patent literature described in this specification are incorporated as references in this specification. In addition, unless otherwise specified in this specification, the numerical range "A to B" is intended to be "above A (including A and greater than A) and less than or equal to B (including B and less than B)."

As used herein, the term "peptide" is used interchangeably with "polypeptide" and refers to a compound in which two or more amino acids are linked by a peptide bond. Furthermore, the term "peptide bond" includes not only the bond between the amino group at the α-position and the carboxyl group in an amino acid, but also the bond between the amino group included in the side chain of the amino acid and the carboxyl group at the α-position. For example, the peptide bond may be a bond between an amino group at the ε position and a carboxyl group at the α position in lysine. In this specification, the one-letter or three-letter notation defined by IUPAC and IUB is used as appropriate for the notation of amino acids.

[1. Isolation method of membrane vesicles]
A method for isolating membrane vesicles according to an embodiment of the present invention (hereinafter referred to as "isolation method of the present invention" as appropriate) is a method for isolating membrane vesicles in which membrane vesicles are isolated from a sample containing membrane vesicles. And,
By bringing the sample into contact with a carrier carrying one or more of a peptide containing 20 to 40 consecutive lysines or polymyxin B, the membrane vesicle and the carrier are bound to each other. a complex forming step of forming a complex,
The method includes a dissociation step of bringing the complex obtained by the complex formation step into contact with a dissociation buffer that dissociates the membrane vesicle from the complex.

Since the method for isolating membrane vesicles according to an embodiment of the present invention has the above-mentioned configuration, there is no need to use a conventional ultracentrifuge or the like, so that membrane vesicles can be isolated in a short time and easily. can be isolated.

Furthermore, in the isolation method of the present invention, when a dissociation buffer containing metal cations is used, the membrane structure of membrane vesicles and the structure of proteins present in the membrane are not affected (or are less affected) under mild conditions. , the membrane vesicles can be dissociated from the complex. Therefore, membrane vesicles can be isolated from the complex without damaging the membrane vesicles (in other words, in an intact state or a near-intact state).

By isolating intact (or nearly intact) membrane vesicles, it is possible to analyze the substances contained in the membrane vesicles. Membrane vesicles contain proteins, nucleic acids, signal substances, and the like. As shown in Figure 1, membrane vesicles released from bacteria are thought to be involved in the transport of various substances as described above. The substances contained in membrane vesicles vary depending on the state of the bacteria producing the membrane vesicles. Therefore, it is thought that by analyzing the substances contained in membrane vesicles, information such as the state of the bacteria in which the membrane vesicles were produced can be obtained. Using this information, it is expected that it will be possible to clarify, for example, by what mechanism substances contained in bacteria are incorporated into membrane vesicles, and how the substances are released from membrane vesicles. This is expected to advance the development of drug delivery systems, vaccines, etc. that utilize the properties of membrane vesicles.

Among these, membrane vesicles are thought to have the property of inducing antibody production or cell-mediated immunity, and are expected to be used as vaccines. In fact, vaccine development using membrane vesicles is underway; for example, in Europe and the United States, membrane vesicles created using Escherichia coli with target proteins as antigens have been certified as meningococcal vaccines.

It is also known that membrane vesicles, which have an immunoregulatory function for the nasal cavity, promote induction of bacteria-specific secretory IgA derived from membrane vesicles on mucous surfaces throughout the body. Therefore, membrane vesicles are expected to be applied particularly as mucosal immune vaccines.

Conventional injectable vaccines are effective against pathogens that have entered the body, but they are effective against the mucous membranes of the eyes, nose, mouth, respiratory tract, and digestive tract, which are the entry points for most infectious disease-causing microorganisms. It does not induce an immune response and cannot be said to be effective against infectious disease-causing microorganisms. On the other hand, in the case of mucosal immunization vaccines, immunity is induced also on the mucosal surface, so it is possible to prevent the invasion of pathogenic microorganisms themselves. Additionally, vaccines using membrane vesicles have an advantage in safety compared to vaccines using detoxified or attenuated bacteria. Furthermore, since no injection needle is required, there are other advantages such as (1) reducing the burden on infants when administering vaccines, and (2) reducing medical accidents such as accidental stabbing.

Below, the materials used in the method for isolating membrane vesicles according to one embodiment of the present invention will be explained first, and then each step of the method for isolating membrane vesicles will be explained.

[1-1. material〕
(sample)
In the isolation method of the present invention, a "sample containing membrane vesicles" (herein also simply referred to as "sample") is a mixture containing membrane vesicles derived from bacteria, and other components are not particularly limited. Not limited. For example, Gram-negative bacteria such as Acinetobacter baumannii, Bacteroides succinogences, Escherichia coli, Helicobacter pylori, and Pseudomonas aeruginosa are known to produce membrane vesicles, and Gram-positive bacteria include Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, and Streptomyces coelicolor etc. are known. Therefore, a sample containing membrane vesicles may be, for example, a culture supernatant of these bacteria.

(peptide)
The peptides used in the isolation method of the present invention (hereinafter referred to as "peptides of the present invention") are peptides containing 20 to 40 consecutive lysines (also referred to as "lysines") (hereinafter referred to as appropriate). (referred to as "polylysine"), which is a peptide that can bind to membrane vesicles in a sample.

Although the present invention is not limited to a particular theory, the peptide of the present invention, polylysine, has a positive charge, and the membrane of a membrane vesicle is negatively charged like a cell membrane. Therefore, the inventors believe that it can bind to membrane vesicles. Therefore, it is believed that the peptides of the present invention can bind membrane vesicles derived from any bacteria, including Gram-positive and Gram-negative bacteria.

If the peptide of the present invention contains 20 to 40 consecutive lysines, it can bind to membrane vesicles with a binding strength sufficient to isolate membrane vesicles. The peptide of the present invention preferably contains 20 or more consecutive lysines, more preferably 25 or more consecutive lysines, more preferably 30 or more consecutive lysines, and 35 or more consecutive lysines. It is more preferable that the lysine contains 1 or more lysines. Further, the number of consecutive lysines contained in the peptide of the present invention is preferably 40 or less. Since the peptide of the present invention has the above structure, the peptide of the present invention can easily bind to membrane vesicles.

The peptide of the present invention is not particularly limited in other configurations as long as it contains polylysine. The peptide of the present invention may, for example, consist only of polylysine, or may have amino acids other than lysine added thereto. Furthermore, the peptide of the present invention may be a complex peptide that further includes a structure other than the peptide, such as a sugar chain or an isoprenoid group. Amino acids contained in the peptides of the present invention may be modified. Furthermore, the amino acids contained in the peptide of the present invention may be of L type or D type.

The peptides of the invention may be readily made according to any technique known in the art, and may be expressed, for example, as natural peptides produced by microorganisms. For example, microorganisms that produce continuous lysine are described in the reference (Shima, S. and Sakai H. (1977). Polylysine produced by Streptomyces. Agricultural and Biological Chemistry 41: 1807-1809). Furthermore, the peptide of the present invention may be expressed by a transformant into which a peptide expression vector has been introduced, or may be chemically synthesized. That is, polynucleotides encoding the peptides of the invention are also within the scope of the invention. As a chemical synthesis method, a liquid phase method can be mentioned. Commercially available polylysine can also be used in the isolation method of the present invention.

Since polylysine, which is a peptide of the present invention, can consist of at least 20 lysines, the lower limit of the molecular weight of polylysine is 2581.42 [=(146.19×20)−(18.02×19)]. I can say that. Although the upper limit of the molecular weight of the peptide of the present invention is not particularly limited, it can be said that about 300,000 is the upper limit from the viewpoint of operability such as solubility and viscosity.

The peptide of the present invention may be composed of a peptide having a single molecular weight, or may be composed of a mixture of peptides having different molecular weights.

(Polymyxin B)
In the isolation method of the present invention, polymyxin B can also bind to membrane vesicles in a sample, similar to the peptide of the present invention.

Although the present invention is not limited to any particular theory, it is believed that polymyxin B combines the hydrophobic portion of lipopolysaccharide (LPS) in the outer membrane of membrane vesicles secreted by Gram-negative bacteria and the hydrophobic chain of polymyxin B. The inventors believe that polymyxin B can bind to membrane vesicles due to the hydrophobic binding of .

Pomixin B can be readily produced according to any technique known in the art, and may be expressed as a naturally occurring peptide produced by a microorganism, for example. For example, microorganisms that produce polymyxin B are known to be produced from Bacillus polymyxa, but are not limited thereto. Further, polymyxin B may be expressed by a transformant into which a polymyxin B expression vector has been introduced, or may be chemically synthesized. Thus, polynucleotides encoding polymyxin B are also within the scope of the invention. As a chemical synthesis method, a liquid phase method can be mentioned. Commercially available polymyxin B can also be used in the isolation method of the present invention.

(carrier)
The carrier used in the isolation method of the present invention (hereinafter appropriately referred to as "the carrier of the present invention") means a carrier capable of supporting the peptide of the present invention and polymyxin B. The carrier of the present invention can bind to the peptide of the present invention or polymyxin B bound to a membrane vesicle to form a complex in which the membrane vesicle - the peptide of the present invention or polymyxin B - the carrier of the present invention are bound in this order.

In the isolation method of the present invention, membrane vesicles can be isolated efficiently and easily by using a carrier.

The structure of the carrier is not particularly limited as long as it is a structure that can directly or indirectly support (retain) the peptide or polymyxin B of the present invention. The carrier of the present invention is preferably a support that does not inhibit the functions of the peptide of the present invention and polymyxin B bound to the carrier, such as cellulose particles, agarose particles, glass, nylon membranes, semiconductor wafers, latex particles, Examples include porous carriers such as microbeads, silica beads, magnetic beads, and monoliths. The carrier of the present invention is preferably cellulose particles or agarose particles, since membrane vesicles can be easily collected and isolated. Cellulose particles or agarose particles are widely used in the separation and purification of proteins, DNA, cells, etc., and are carriers that can be fully understood by those skilled in the art.

The mode of binding the peptide or polymyxin B of the present invention to the carrier is not particularly limited, and may be any known mode. Furthermore, the peptide or polymyxin B of the present invention may be bound to the carrier either directly or indirectly. Furthermore, the peptide or polymyxin B of the present invention may be bound to the carrier via a linker consisting of a polypeptide.

The type of peptide that binds to the carrier may be one type or a combination of multiple types. For example, only the peptide of the invention may be coupled to the carrier, or only polymyxin B may be coupled to the carrier. Additionally, both the peptide of the invention and polymyxin B may be coupled to a carrier. Furthermore, a peptide other than the peptide of the present invention and polymyxin B may be bound to a carrier. Further, the carrier of the present invention carrying the peptide of the present invention and polymyxin B may include both a carrier to which only the peptide of the present invention is bound and a carrier to which only polymyxin B is bound.

Note that the peptide of the present invention and polymyxin B may be biotinylated and bound to streptavidin held on the carrier of the present invention.

(dissociation buffer)
The dissociation buffer used in the isolation method of the present invention (hereinafter referred to as the "dissociation buffer of the present invention") refers to a dissociation buffer capable of dissociating membrane vesicles from complexes and isolating membrane vesicles. do.

Other configurations are not particularly limited as long as the buffer can dissociate membrane vesicles from the complex by bringing the complex and the dissociation buffer into contact in the dissociation step described below. As the dissociation buffer of the present invention, it is particularly preferable to use a dissociation buffer containing metal cations. By containing metal cations in the dissociation buffer of the present invention, membrane vesicles can be dissociated from complexes under mild conditions without containing protein denaturants, etc., so that membrane vesicles can be dissociated from complexes in an intact state. It can be isolated in a near-intact state. The present inventors are the first to discover that the binding between the peptide or polymyxin B of the present invention and membrane vesicles can be dissociated using such a mild dissociation buffer.

The metal cations contained in the dissociation buffer of the present invention are not particularly limited, and include, for example, monovalent metal cations such as sodium ions, potassium ions, lithium ions, silver ions, and copper (I) ions, and magnesium ions. , divalent metal cations such as calcium ions, zinc ions, nickel ions, barium ions, copper (II) ions, iron (II) ions, tin (II) ions, cobalt (II) ions and lead (II) ions. , and trivalent metal cations such as aluminum ions and iron (III) ions. Among these metal cations, sodium ions, potassium ions, magnesium ions, zinc ions, and nickel ions are preferred as the metal cations contained in the dissociation buffer of the present invention, since they have high water solubility. Furthermore, since the membrane vesicles can be sufficiently dissociated from the complex, it is preferable that the dissociation buffer of the present invention contains sodium ions, potassium ions, or magnesium ions among the above-mentioned metal cations, either alone or in combination. .

The purpose of the isolation method of the present invention is to easily isolate membrane vesicles in an intact state (or in a near-intact state). Therefore, in the isolation method of the present invention, "isolating membrane vesicles in an intact state (or in a near-intact state)" means to isolate membrane vesicles in a sample without (almost without) damaging the membrane vesicles. It means to separate and isolate from at least a part of substances other than membrane vesicles (for example, proteins, etc.) in a sample. In other words, in the isolation method of the present invention, "isolating membrane vesicles in an intact state (or in a near-intact state)" means that the membrane structure (specifically, the lipid bilayer membrane structure) of the membrane vesicle is isolated. Membrane vesicles in the sample are separated from substances other than membrane vesicles in the sample while the structure of the proteins present in the membrane is maintained (almost preserved). , means to isolate.

The dissociation buffer containing metal cations may contain substances other than metal cations as long as they do not affect the membrane structure of the membrane vesicle and the structure of proteins present in the membrane.

The concentration of the metal cation in the dissociation buffer is not particularly limited as long as it can dissociate the membrane vesicle from the complex and does not affect the membrane structure of the membrane vesicle and the structure of the protein present in the membrane. In other words, the optimal concentration of metal cations may vary depending on the binding strength between the peptide of the present invention and membrane vesicles used, the type (valence) of metal cations, etc., so after considering the optimal concentration as appropriate, All you have to do is decide. Note that the concentration of metal cations in the dissociation buffer of the present invention is not particularly limited, but for example, it is preferably 0.01 to 5M, more preferably 0.05 to 2M, and It is more preferably from .1 to 1M, even more preferably from 0.2 to 0.7M, particularly preferably from 0.2 to 0.4M.

Further, the pH of the dissociation buffer containing metal cations can be set as appropriate. The pH of the dissociation buffer containing metal cations is preferably 5 to 10, more preferably 6 to 9, even more preferably 7 to 8, and even more preferably 7.3 to 7.5. is particularly preferred. It is preferable that the pH of the dissociation buffer containing metal cations is within the above range because it does not affect the membrane structure of the membrane vesicle and the structure of proteins present in the membrane.

Furthermore, the dissociation buffer of the present invention may contain a surfactant such as sodium deoxycholate.

[1-2. Process]
(Complex formation process)
The complex formation step included in the isolation method of the present invention (hereinafter referred to as the "complex formation step of the present invention") involves a sample containing membrane vesicles and a peptide of the present invention supported on a carrier of the present invention. Alternatively, by contacting with polymyxin B, a complex formed by binding the membrane vesicle and the peptide or polymyxin B supported on the carrier (membrane vesicle - peptide of the present invention or polymyxin B - carrier of the present invention) is formed. This is a complex forming step in which a complex is formed by bonding in this order (hereinafter appropriately referred to as "complex of the present invention").

A method for contacting a sample with the peptide or polymyxin B of the present invention supported on the carrier of the present invention is such that membrane vesicles in the sample and the peptide or polymyxin B of the present invention supported on the carrier of the present invention are brought into contact with each other. Other methods are not limited as long as they are carried out under conditions that allow binding and formation of the complex of the present invention.

For example, in contacting a sample containing membrane vesicles with the peptide or polymyxin B of the present invention supported on the carrier of the present invention, adding the carrier of the present invention carrying the peptide or polymyxin B of the present invention to the sample, An example is a method of mixing. By contacting a sample with the peptide of the present invention or polymyxin B supported on the carrier of the present invention, a complex of the present invention comprising a membrane vesicle and a carrier on which the peptide of the present invention or polymyxin B is supported can be formed. .

The contact time between the sample and the carrier carrying the peptide of the present invention or polymyxin B is not limited as long as it is sufficient to form the complex of the present invention, and the optimal conditions may be examined as appropriate. can be determined above.

Further, in contacting a sample containing membrane vesicles with the peptide or polymyxin B of the present invention supported on the carrier of the present invention, the peptide or polymyxin B of the present invention is supported on, for example, a columnar or cylindrical container (column). A method using a column prepared by packing the carrier of the present invention (herein referred to as a "peptide column") can be mentioned. By passing a solution containing a sample through the peptide column, it is possible to contact the sample with the peptide or polymyxin B of the present invention supported on the carrier of the present invention, thereby obtaining the complex of the present invention.

(Washing process)
Preferably, the isolation method of the present invention includes a washing step.

The washing step is a step of washing the complex of the present invention an appropriate number of times using an appropriate washing solution. As long as the washing step is carried out under conditions that do not dissociate the complex, the type of washing liquid used, the number of washings, etc. are not particularly limited.

Examples of the washing liquid used in the washing step include physiological saline and phosphate buffered saline (PBS).

The washing step includes, for example, a method in which the above-mentioned washing liquid is added to a container containing the complex of the present invention and mixed, and the complex is separated from the washing liquid by centrifugation or the like. Furthermore, when a peptide column is used in the complex formation step of the present invention, an appropriate amount of the washing solution may be passed through the column after the complex of the present invention has been formed.

(Dissociation step)
The dissociation step included in the isolation method of the present invention (hereinafter referred to as the "dissociation step of the present invention") is a step in which the complex of the present invention obtained by the complex formation step and the dissociation buffer of the present invention are separated. This is a step of dissociating membrane vesicles from the complex of the present invention through contact.

The method for bringing the complex of the present invention into contact with the dissociation buffer of the present invention is not limited to any other method as long as it is carried out under conditions that allow membrane vesicles to be dissociated from the complex of the present invention. For example, a method may be mentioned in which the dissociation buffer of the present invention is added to a container containing the complex of the present invention and mixed. The contact time between the complex of the present invention and the dissociation buffer of the present invention is not particularly limited as long as it is sufficient time for the membrane vesicles to dissociate from the complex. By contacting the complex of the invention with the dissociation buffer of the invention, membrane vesicles are dissociated from the complex of the invention and transferred to the dissociation buffer. Then, by separating the dissociation buffer and the carrier of the present invention and isolating the dissociation buffer, membrane vesicles can be isolated.

In addition, when a column is used in the complex formation step, the complex of the present invention and the dissociation of the present invention can be separated by passing the dissociation buffer of the present invention through the column on which the complex of the present invention has been formed. It is possible to contact it with a buffer. When the column and the dissociation buffer of the present invention are brought into contact, membrane vesicles are dissociated from the complex and migrate to the dissociation buffer. Therefore, membrane vesicles can be isolated by isolating the dissociation buffer after passing through the column.

In the dissociation step of the present invention, the peptide of the present invention and polymyxin B may remain bound to the carrier of the present invention, or may be dissociated from the carrier of the present invention. Furthermore, after the separation, the peptide of the present invention and polymyxin B may be contained in the isolated dissociation buffer. From the viewpoint of isolating membrane vesicles with higher purity, it is preferable that the peptide of the present invention and polymyxin B remain bound to the carrier of the present invention in the dissociation step of the present invention, and after the separation, the isolated dissociated Preferably, the peptide of the invention and polymyxin B are not contained in the buffer.

[2. How to remove membrane vesicles]
A method for removing membrane vesicles according to an embodiment of the present invention (hereinafter referred to as "removal method of the present invention" as appropriate) is a method for removing membrane vesicles, which removes membrane vesicles from a sample containing membrane vesicles, comprising:
By bringing the sample into contact with a carrier carrying one or more of polymyxin B and a peptide containing 20 to 40 consecutive lysines, the membrane vesicle and the carrier are bonded. a complex forming step of forming a complex, and separating the complex formed by the complex forming step from the sample to obtain a separated liquid from which at least a portion of the membrane vesicles have been removed from the sample. It includes a separation step to obtain

Since the method for removing membrane vesicles according to one embodiment of the present invention has the above-mentioned configuration, it is not necessary to use an ultracentrifuge or the like, so that membrane vesicles can be removed easily in a short time. Therefore, a separated liquid with extremely few remaining membrane vesicles can be easily obtained by a simple method.

Such a method for removing membrane vesicles is useful, for example, for producing a medium from which membrane vesicles have been removed. A culture medium prepared using a specific bacterium or an enzyme derived from the specific bacterium may be contaminated with membrane vesicles derived from the specific bacterium. When preparing a medium for culturing a target bacterium different from the specific bacteria for the purpose of isolating membrane vesicles, it is preferable to remove membrane vesicles derived from the specific bacteria. Otherwise, there is a problem that membrane vesicles derived from the specific bacteria, which are not derived from the target bacteria, will eventually become contaminated. According to the removal method of the present invention, a medium containing membrane vesicles can be easily prepared by contacting the peptide or polymyxin B of the present invention with a medium containing membrane vesicles and removing the formed complex.

[2-1. material〕
The removal method according to one embodiment of the present invention can be carried out using the carrier of the present invention on which the peptide of the present invention and polymyxin B described above are supported. Further, the "sample containing membrane vesicles" in the removal method of the present invention is the same as the sample used in the isolation method of the present invention. Therefore, regarding materials [1-1. Materials] can be used.

[2-2. Process]
(Complex formation process)
The removal method of the present invention includes a complex formation step and a separation step. The complex forming step is the same as the complex forming step included in the isolation method of the present invention described above, so it is similar to [1-2. The explanation of (complex formation step) in step] can be referred to.

(separation process)
The separation step included in the removal method of the present invention (hereinafter appropriately referred to as "separation step of the present invention") is as follows:
The carrier of the present invention carrying the complex of the present invention formed in the complex formation step and the sample after contact with the carrier of the present invention carrying the peptide or polymyxin B of the present invention are separated, and This is a separation step to obtain a separated liquid from which at least a portion of membrane vesicles have been removed from the sample.

As a method for separating the complex of the present invention and the sample, the complex of the present invention can be separated from the sample by, for example, centrifugation. For example, the complex of the present invention can be easily separated from a sample by centrifugation under conditions of 500 to 4000 xg.

Furthermore, when a peptide column is used in the complex formation step of the present invention, the separated liquid can be collected as a flow-through, and the complex of the present invention remains within the column. Therefore, the complex of the present invention and the sample can be separated.

Furthermore, when the carrier contained in the complex of the present invention is a magnetic bead, the complex can be easily separated from the sample by applying magnetic force from the outside using a magnetic material such as a conventionally known magnetic stand. can.

The removal method of the present invention may include steps other than the above-described complex forming step and separation step. Other steps include, for example, a dissociation step.

(Dissociation step)
The method of the dissociation step is the same as the dissociation step included in the isolation method of the present invention described above, and therefore [1-2. The explanation of (dissociation step) in [Process] can be referred to. The dissociation step allows the carrier of the present invention to be regenerated to the state before contact with the sample containing membrane vesicles. In addition, if the peptide of the present invention and polymyxin B remain bound to the carrier of the present invention after the dissociation step, the peptide of the present invention and polymyxin B can also be reused while bound to the carrier of the present invention. can.

In the dissociation step, by using the dissociation buffer of the present invention, membrane vesicles can be dissociated from the complex of the present invention under mild conditions. Less susceptible to damage. Therefore, the carrier of the present invention, the peptide of the present invention, and polymyxin B can be used in the removal method of the present invention multiple times by undergoing a dissociation step.

[3. Membrane vesicle isolation kit]
The membrane vesicle isolation kit according to one embodiment of the present invention (hereinafter referred to as the "kit of the present invention") is a kit for carrying out the above-described isolation method of the present invention or removal method of the present invention. , the carrier of the present invention carrying the above-mentioned peptide of the present invention and polymyxin B, and the dissociation buffer of the present invention. Therefore, regarding the explanation of the configuration of the kit, please refer to [1. [Method for isolating membrane vesicles]] can be referred to.

In addition to the above-described configuration, the kit of the present invention may also include a container (for example, a bottle, plate, tube, dish, column, etc.) containing a specific material. Kits of the invention may include containers containing diluents, solvents, wash solutions, or other reagents. The term "comprising" used in the description of the kit of the present invention may mean that the kit is contained in any of the individual containers constituting the kit.

The kit of the present invention may also include instructions for carrying out the isolation method of the present invention or the removal method of the present invention.

EXAMPLES Hereinafter, one embodiment of the present invention will be described in more detail with reference to Examples, but the present invention is not limited only to these Examples.

<Isolation and removal of membrane vesicles>
〔material and method〕
(Preparation of sample)
Using Pseudomonas aeruginosa PAO1 strain (National Institute of Technology and Evaluation, Biotechnology Center (NBRC)) as a bacterium that produces membrane vesicles, samples were prepared by the following method. The medium used was LB medium (Sigma-Aldrich).
(1) Pseudomonas aeruginosa PAO1 strain was inoculated into 5 mL of LB medium, and cultured with shaking at 37°C overnight.
(2) 1% of the culture solution was inoculated into 200 mL of LB medium, and cultured overnight at 37°C.
(3) 50 mL of 200 mL of the culture solution was transferred into a centrifuge tube and centrifuged at 10,000×g for 10 minutes to collect the culture supernatant containing membrane vesicles.
(4) The culture supernatant was filtered with a 0.45 μm filter and a 0.22 μm filter to remove remaining bacterial cells and impurities, and a sample (hereinafter referred to as "supernatant sample") was obtained. ).

(Isolation and removal of membrane vesicles using a carrier carrying polylysine)
Membrane vesicles from the supernatant sample are isolated using a carrier bound to polylysine (peptide containing 20 to 40 consecutive lysines) (hereinafter referred to as "polylysine immobilized carrier") as shown in Figure 2. Isolation and removal were performed. The polylysine-immobilized carrier is a porous spherical carrier on which polylysine consisting of 20 to 40 lysine residues is immobilized, prepared by the method described in the reference (Kobunshi Ronshu, 64, 12, 821-829 (2007)). Cellulose particles were used.

An isolation method using a polylysine immobilized carrier will be described below with reference to FIG.
(1) 100 μL of polylysine fixed carrier was suspended in 300 μL of PBS (Nacalai Tesque), centrifuged at 700×g for 2 minutes, and washed by removing the supernatant.
(2) The washing step in (1) was performed three times, and the polylysine-fixed carrier was suspended in 100 μL of PBS.
(3) The polylysine immobilized carrier and the supernatant sample were combined by adding it to 5 mL of supernatant sample and performing rotary mixing for 2 hours.
(4) Centrifugation was performed at 700×g for 2 minutes to precipitate the polylysine-fixed carrier.
(5) The supernatant was removed, and the polylysine-fixed carrier was recovered and suspended in 300 μL of PBS.
(6) Centrifugation was performed at 700×g for 2 minutes, and the supernatant was removed for washing.
(7) The operation in (6) was performed three times, 50 μL of dissociation buffer was added, and the membrane vesicles bound to the polylysine fixed carrier were eluted at 37° C. for 30 minutes. During this time, vortexing was performed every 10 minutes.
(8) Centrifugation was performed at 700×g for 2 minutes to collect 40 μL of supernatant, which was used as an elution sample.

(Isolation and removal of membrane vesicles using a carrier carrying polymyxin B)
Membrane vesicles were isolated and removed from the supernatant sample using a polymyxin B-bound carrier (hereinafter referred to as "polymyxin B immobilized carrier") as shown in FIG. 2. As the polymyxin B-immobilized carrier, an agarose carrier on which polymyxin B was immobilized was used. For example, an agarose carrier on which polymyxin B is immobilized can be prepared by the method described in the reference (Biochemistry, 16, 1642-1648 (1977)).

An isolation method using a polymyxin B immobilized carrier will be described below with reference to FIG.
(1) 50 μL of polymyxin B immobilized carrier was suspended in 300 μL of PBS, centrifuged at 700×g for 2 minutes, and washed by removing the supernatant.
(2) The washing step in (1) was performed three times, and the polymyxin B-immobilized carrier was suspended in 100 μL of PBS.
(3) The polymyxin B immobilized carrier and the supernatant sample were combined by adding it to 5 mL of the supernatant sample and performing rotary mixing for 2 hours.
(4) Centrifugation was performed at 700×g for 2 minutes to precipitate the polymyxin B-immobilized carrier.
(5) The supernatant was removed and the polymyxin B-immobilized carrier was recovered and suspended in 300 μL of PBS.
(6) Centrifugation was performed at 700×g for 2 minutes, and the supernatant was removed for washing.
(7) The operation in (6) was performed three times, 50 μL of dissociation buffer was added, and the membrane vesicles bound to the polymyxin B immobilized carrier were eluted at 37° C. for 30 minutes. During this time, vortexing was performed every 10 minutes.
(8) Centrifugation was performed at 700×g for 2 minutes to collect 40 μL of supernatant, which was used as an elution sample.

(Isolation of membrane vesicles using ultracentrifugation)
As Comparative Example 1, membrane vesicles were isolated by ultracentrifugation. A specific method of ultracentrifugation will be described below with reference to FIG. 4.
(1) A 40 mL supernatant sample was added to Vivaspin 20 (100 kDa MWCO, GE Healthcare), centrifuged at 2580×g, 60 min, 4° C., and ultrafiltered to about 4 mL to obtain a concentrated sample.
(2) 1 mL of PBS was added to 2 mL of the concentrated sample, and ultracentrifugation was performed at 110,000×g, 120 min, and 4° C.
(3) The supernatant was removed, suspended in 3 mL of PBS, and ultracentrifuged again under the same conditions.
(4) The supernatant was removed and suspended in 200 μL of PBS to obtain an ultracentrifuged sample.

(Isolation of membrane vesicles using lysine-supported magnetic beads according to comparative example)
As Comparative Example 2, a peptide containing eight consecutive lysines was bound (supported) to magnetic beads, and membrane vesicles were isolated using the magnetic beads supporting the lysine peptides. As the carrier, magnetic beads on which streptavidin was immobilized (manufactured by VERITAS, Dynabeads M-280 streptavidin) (herein also referred to as "streptavidin magnetic beads") were used.

The peptide used was a peptide (Biotin-K8) consisting of the amino acid sequence GGGSGGGSGGGSKKKKKKKK (SEQ ID NO: 1), consisting of a linker peptide and eight consecutive lysines, and whose N-terminus was modified with biotin. All amino acids used in Comparative Example 2 are L-type amino acids. Note that Biotin-K8 used in Comparative Example 2 was produced by contract synthesis by Eurofin Genomics.

Magnetic beads carrying Biotin-K8 were prepared by utilizing the biotin-streptavidin interaction, and membrane vesicles were isolated. This will be explained below with reference to FIG.
(1) Add 100 μL (1.0 mg) of streptavidin magnetic beads to a 1.5 mL microtube (also simply referred to as a “tube”), set the tube on a magnetic stand, let it stand for 1 minute, and then Removed the clear water.
(2) The tube was removed from the magnetic stand, and 0.6 mL of PBS was added and mixed. The tube was set on a magnetic stand, left to stand for 1 minute, and the supernatant was removed. The operation (2) (washing) was performed three times in total.
(3) 400 μL of PBS was added to the tube, and the streptavidin magnetic beads were resuspended in PBS to obtain washed streptavidin magnetic beads (400 μL).
(4) Add 30 μL of Biotin-K8 (100 μM) to the tube.
(5) Biotin-K8 was bound (ie, supported) to the streptavidin magnetic beads by mixing the tube at room temperature for 30 minutes and bringing Biotin-K8 into contact with the streptavidin magnetic beads.
(6) The tube was set on a magnetic stand, left to stand for 1 minute, and the supernatant was removed.
(7) The same operation (washing) as in (2) above was performed three times in total.
(8) 100 μL of PBS was added to the tube, and the streptavidin magnetic beads were resuspended in PBS to obtain streptavidin magnetic beads (1.0 mg/100 μL) carrying Biotin-K8. The streptavidin magnetic beads carrying Biotin-K8 were referred to as "K8-immobilized magnetic beads."
(9) 100 μL of K8-immobilized magnetic beads were added to 40 mL of supernatant sample, and the K8-immobilized magnetic beads were combined with the supernatant sample by rotary mixing for 2 hours.
(10) The tube was set on a magnetic stand, left to stand for 1 minute, and the supernatant was removed.
(11) Perform the following operations to wash: (11-1) Remove the tube from the magnetic stand, add 1 mL of PBS to the tube, and mix; (11-2) Set the tube on the magnetic stand. The mixture was allowed to stand for 1 minute, and the supernatant was removed.
(12) The operation in (11) was performed two more times (3 times in total), 50 μL of 1% SDS was added, and the membrane vesicles bound to the K8-immobilized magnetic beads were eluted at 37° C. for 30 minutes. During this time, vortexing was performed every 10 minutes.
(13) The tube was set on a magnetic stand and left to stand for 1 minute, and 45 μL of supernatant was collected to be used as a K8 sample.

(Comparison of isolation methods for membrane vesicles)
The elution samples, ultracentrifugation samples, and K8 samples obtained by each of the above-mentioned isolation methods were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolation results were compared. The samples were subjected to SDS-PAGE using a 12.5% polyacrylamide gel. Thereafter, silver staining was performed using EzStain Silver (ATTO) to detect proteins present in the eluted sample. For ultracentrifuged samples, mix 20 μL of 2× SDS sample buffer (4% SDS, 10% sucrose, 10% 2-mercaptoethanol) with 20 μL of ultracentrifuged samples, of which 20 μL (equivalent to 1 mL of culture supernatant) was used. Since the K8 sample contains SDS in the dissociation buffer, 20 μL (equivalent to 16 mL of culture supernatant) of the K8 sample was used as it was.

When membrane vesicles are included in the sample, a band corresponding to OprF, which is reported to be abundant in membrane vesicles of Pseudomonas aeruginosa, is observed around a molecular weight of 38 kDa in the gel. Therefore, the isolation results of each isolation method were compared based on the presence or absence of OprF bands.

[Result: Isolation of membrane vesicles]
(Comparative example 1)
As Comparative Example 1, when an ultracentrifuged sample obtained by isolation of membrane vesicles using ultracentrifugation was subjected to SDS-PAGE, a band corresponding to OprF was observed at around 38 kDa. When this band was cut out and subjected to LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry) analysis, it was identified as OprF.
(Comparative example 2)
As Comparative Example 2, SDS-PAGE was performed on a K8 sample obtained by a membrane vesicle isolation method using K8-immobilized magnetic beads. FIG. 6 shows the SDS-PAGE results of Comparative Example 2. As shown in FIG. 6, no band was observed in Comparative Example 2, indicating that membrane vesicles did not bind to the K8-immobilized magnetic beads.

In Patent Document 1 mentioned above, it has been shown that exosomes bind to K8-immobilized magnetic beads, but it has been shown that K8-immobilized magnetic beads do not bind to membrane vesicles that have the same lipid bilayer membrane as exosomes. Ta. The inventors believe that this is because the production methods for exosomes and membrane vesicles are completely different, and the compositions of the lipid bilayer membranes are significantly different between animal-derived exosomes and bacterial-derived membrane vesicles.

(Example 1)
In the method for isolating membrane vesicles using the polylysine-immobilized carrier of the present invention, the membrane vesicles bound to the polylysine-immobilized carrier are prepared using 1× SDS sample buffer (2% SDS, 5% sucrose, 5% 2- The binding of membrane vesicles was compared by dissolving and dissociating them with mercaptoethanol (Example 1). In Example 1, membrane vesicles were dissolved and dissociated from the polylysine-fixed carrier using SDS, so SDS-PAGE was directly performed using 10 μL (equivalent to 1 mL of culture supernatant) of the elution sample.

FIG. 7 shows the SDS-PAGE results of Example 1 and Comparative Example 1. As shown in FIG. 7, in Example 1, a band corresponding to OprF was observed around 38 kDa, indicating that Example 1 contained membrane vesicles. Therefore, it was shown that the K8-immobilized magnetic beads did not bind to membrane vesicles, whereas the polylysine-immobilized carrier bound to membrane vesicles.

(Example 2)
Example 2 is an example in which a 1% sodium deoxycholate solution was used as a dissociation buffer in a method for isolating membrane vesicles using a polymyxin B immobilized carrier. In Example 2, 40 μL of the eluted sample was mixed with 40 μL of 2×SDS sample buffer, of which 20 μL (equivalent to 1 mL of culture supernatant) was used for SDS-PAGE.

FIG. 8 shows the SDS-PAGE results of Example 2 and Comparative Example 1. As shown in FIG. 6, a limited number of bands were observed in Example 2. The limited number of bands also included a band around 38 kDa that corresponds to OprF. Therefore, in Example 2, in which 1% sodium deoxycholate solution was used as the dissociation buffer, membrane vesicles could be isolated, and there were fewer contaminants than in Comparative Example 1, which was the ultracentrifugation method. It was shown that membrane vesicles can be isolated in a highly pure state.

(Example 3 and Example 4)
In the method for isolating membrane vesicles using a polylysine immobilized carrier, Example 3 is an example in which 1M NaCl is used as a dissociation buffer, and Example 4 is an example in which 1M MgCl ₂ is used as a dissociation buffer. In Examples 3 and 4, membrane vesicles were isolated using a dissociation buffer containing metal cations under mild conditions that do not contain protein denaturants that may destroy the membrane structure. I tried to separate. In Examples 3 and 4, 40 μL of 2×SDS sample buffer was mixed with 40 μL of the elution sample, and 20 μL of the mixture (equivalent to 1 mL of culture supernatant) was used for SDS-PAGE.

FIG. 9 shows the SDS-PAGE results of Example 1, Example 3, Example 4, and Comparative Example 1. A limited number of bands were observed in Examples 3 and 4. The limited number of bands also included a band around 38 kDa that corresponds to OprF. Therefore, in Examples 3 and 4, which used dissociation buffers containing metal cations, membrane vesicles could be isolated, and there were fewer contaminants than in the ultracentrifugation method of Comparative Example 1. It was shown that membrane vesicles can be isolated in a highly pure state. Furthermore, compared with Example 1, Examples 3 and 4, which used a mild dissociation buffer that does not contain a protein denaturant that could destroy the membrane structure of membrane vesicles, contained fewer impurities. It was shown that membrane vesicles can be isolated with high purity.

(Example 5 and Example 6)
In the method for isolating membrane vesicles using a polymyxin B immobilized carrier, Example 5 is an example in which 0.5M NaCl is used as a dissociation buffer, and Example 6 is an example in which 0.5M MgCl ₂ is used as a dissociation buffer. In Examples 5 and 6, membrane vesicles were isolated using a dissociation buffer containing metal cations under mild conditions that do not contain protein denaturants that may destroy the membrane structure. I tried to separate. In Examples 5 and 6, 40 μL of 2×SDS sample buffer was mixed with 40 μL of the elution sample, of which 20 μL (equivalent to 1 mL of culture supernatant) was used for SDS-PAGE.

FIG. 10 shows the SDS-PAGE results of Example 2, Example 5, Example 6, and Comparative Example 1. In Examples 5 and 6, similar to Example 2, a limited number of bands were observed. The limited number of bands also included a band around 38 kDa that corresponds to OprF. Therefore, in Examples 5 and 6, which used dissociation buffers containing metal cations, membrane vesicles could be isolated, and there were fewer contaminants than in the ultracentrifugation method of Comparative Example 1. It was shown that membrane vesicles can be isolated with high purity. Furthermore, even when compared with Example 2, which contains a surfactant that may destroy the membrane structure of membrane vesicles, Examples 5 and 6, which used a mild dissociation buffer, produced membranes with similar purity. It was shown that vesicles could be isolated.

(Electron microscopy observation of membrane vesicles isolated by the isolation method of the present invention)
The membrane vesicles of Example 5 isolated using a polymyxin B immobilized carrier and 0.5 M NaCl as a dissociation buffer were observed using an electron microscope. This confirmed whether intact (or nearly intact) membrane vesicles could be isolated by the isolation method of the present invention.

The elution sample obtained in Example 5 was coated with carbon using a carbon coater (Meiwaforsys), and membrane vesicles were observed using a field emission scanning electron microscope (FE-SEM, SIGMA VP, Carl Zeiss). I did it.

The results are shown in FIG. FIG. 11 is a diagram showing the results of electron microscopic observation of isolated membrane vesicles. As shown in FIG. 11, a structure that appeared to be a membrane vesicle with a size of about 100 nm could be observed. Since the observed membrane vesicles maintained a spherical shape, it is thought that the membrane structure (specifically, the lipid bilayer membrane) was maintained (or almost maintained). From this result, it was found that the isolation method of the present invention was able to isolate membrane vesicles in an intact state (or in a near-intact state).

In addition, the size of particles believed to be membrane vesicles was measured from the electron microscope images taken, and a particle size distribution map was created. The results are shown in FIG. As shown in FIG. 12, the isolated membrane vesicles had an average particle size of 97±17 nm (mean±SD). This indicates that less damaged membrane vesicles were mainly isolated. Therefore, it was shown that membrane vesicles with less damage can be obtained with high purity according to the isolation method of the present invention.

[Result: Removal of membrane vesicles]
As shown in FIGS. 7 to 10, the elution samples of Examples 1 to 6 showed that membrane vesicles bound to the polylysine-immobilized carrier and the polymyxin B-immobilized carrier and were eluted. Therefore, it can be said that the supernatant after binding membrane vesicles to a polylysine-fixed carrier and a polymyxin B-fixed carrier and centrifuging them is a separated liquid in which at least a portion of membrane vesicles have been removed from a sample containing membrane vesicles. This indicates that membrane vesicles can be removed from the sample with high efficiency according to the removal method of the present invention.

According to the present invention, membrane vesicles can be easily isolated in an intact state (nearly intact state). Therefore, according to the present invention, it becomes possible to easily and precisely analyze substances contained inside membrane vesicles. Therefore, the present invention is useful in various technical fields, particularly in the pharmaceutical and medical fields.

Claims (3)

A method for isolating membrane vesicles, comprising isolating membrane vesicles from a sample containing membrane vesicles derived from bacteria, the method comprising:
By bringing the sample into contact with a carrier carrying one or more of polymyxin B and a peptide containing 20 to 40 consecutive lysines, the membrane vesicle and the carrier are bonded. a complex forming step of forming a complex,
a dissociation step of bringing the complex obtained by the complex formation step into contact with a dissociation buffer that dissociates the membrane vesicle from the complex ,
A method for isolating membrane vesicles, wherein the dissociation buffer contains a metal cation . A membrane vesicle isolation kit for carrying out the membrane vesicle isolation method according to claim 1 , comprising:
A kit for isolating membrane vesicles, comprising the carrier on which at least one of the peptide or polymyxin B is supported, and the dissociation buffer. A method for removing membrane vesicles, the method comprising: removing membrane vesicles from a sample containing bacterially derived membrane vesicles, the method comprising:
By bringing the sample into contact with a carrier carrying one or more of polymyxin B and a peptide containing 20 to 40 consecutive lysines, the membrane vesicle and the carrier are bonded. a complex forming step of forming a complex,
A membrane method comprising: separating the complex formed in the complex forming step from the sample to obtain a separated liquid from which at least a portion of the membrane vesicles have been removed from the sample. How to remove vesicles.

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