JP7376021B2 - Method for separating and analyzing microvesicles from human blood - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act April 30, 2019, https://www. biorxiv. org/content/10.1101/623553v1

The present invention relates to a method for efficiently separating and analyzing microvesicles present in human blood.

Exosomes (0.03 to 0.1 μm in diameter) and microvesicles (0.1 to 1 μm in diameter) are known as micromembrane fractions that separate from cells, and these are secreted by different mechanisms. It has been reported that it is secreted by many different cell types under both normal and pathological conditions (Non-Patent Document 1).

These micromembrane fractions are included in human body fluids (blood, urine, cerebrospinal fluid, etc.), and the contents contained in these micromembrane fractions are important in signal transduction functions in interactions between cells and tissues. It is believed that this role plays a role. On the other hand, it is expected that these micromembrane fractions can be used as liquid biopsy, which is a non-invasive diagnostic material, to determine the presence or absence of morbidity and disease (Patent Document 1, Patent Document 2, Non-Patent Document 2, Non-Patent Document 3).

Biomaterials used as liquid biopsies include free nucleic acids, micromembrane fractions, circulating tumor cells (CTC), and the like. The micromembrane fraction is divided into (I) exosomes: membranous vesicles with a diameter of 30 to 100 nm, and (II) ectosomes (also referred to as microvesicles and shedding microvesicles (SMVs)). (iii) Apoptotic blebs: large membranous vesicles with a diameter of 100 to 1,000 nm that are released directly from the plasma membrane; (III) apoptotic blebs: large membranous vesicles with a diameter of 50 nm that are released from dying cells ~5,000 nm vesicles.

In recent years, the clinical value of these micromembrane fractions has attracted attention. Clinical application of exosomes is progressing, and a method for diagnosing breast cancer using polynucleotides within vesicles has been developed (Patent Document 3). However, conventional diagnostic methods using exosomes have the problem that it is difficult to characterize each individual exosome and it is difficult to easily determine what tissue or cell it originates from. Microvesicles are found in large amounts in human body fluids compared to CTCs, play an extremely important role in tumor invasion (Non-Patent Document 4), and microvesicles present in blood have physiological effects. It has been reported that it is a major blood coagulation factor and is also involved in diseases such as sepsis (Non-Patent Document 1), so it is expected that it will be used effectively as a clinical test material, but it has not yet been realized. not present.

Although exosomes and microvesicles have different production processes and are defined as separate micromembrane fractions, conventional observation methods show that their sizes and the surface antigens present on the membranes that characterize them are very close and similar. Therefore, it was not possible to strictly distinguish and separate and observe these. For example, Patent Document 1 and Patent Document 2 describe separation methods and analysis methods that include both exosomes and microvesicles, and the distinction between the two is unclear.

In addition, the micromembrane fraction present in body fluids is generally derived from organs and tissues throughout the body, or the cells that make them up, so in order to find value in diagnostic and testing applications, it is necessary to Observation of micromembrane fractions focused on specific matched organs, tissues, and cells is required. Therefore, the clinical usefulness of micromembrane fractions would be further enhanced if an analytical method could be established to easily categorize the organ, tissue, or cell from which the observed micromembrane fractions originate.

As described above, in order to effectively utilize microvesicles as clinical test materials, it is necessary to separate them from exosomes and to use separation and analysis methods that can distinguish the organs, tissues, and cells from which they originate.

Patent No. 5838963 Japanese Patent Application Publication No. 2015-91251 Japanese Patent Application Publication No. 2014-117282

Andrea Piccin, William G, Owen P (2007) Circulating microparticles: pathophysiology and clinical implications. Blood Reviews 21, 157-171 Boukouris S, Mathivanan S (2015) Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin Appl 9(3-4): 358-367 IH Chen, L Xue, CC Hsu, (2017) Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. PNAS vol. 114, no. 12, 3175-3180 James W. Clancy, Alanna Sedgwick, Carine Rosse, (2015) Regulated delivery of molecular cargo to invasive tumor-derived microvesicles. Nature Communications 6, Article number: 6919

As mentioned above, microvesicles can be used as liquid biopsies as an effective clinical testing material, but it is extremely difficult to clearly distinguish and separate exosomes from microvesicles, and they can also be used in various cells and tissues. As long as microvesicles derived from blood are present, even if the entire blood microvesicles are used as a liquid biopsy (for example, to extract nucleic acids, etc.), it will be difficult to identify the disease site or obtain results that match the diagnostic purpose. Conceivable.

Therefore, we focused on microvesicles (0.1 to 1 μm in diameter) in the blood, and it is possible to easily characterize them and observe what kind of cells each microvesicle originates from. The purpose of the present invention is to construct a method for distinguishing and separating exosomes and microvesicles, which have been unclear until now, and also clarifying the cells, tissues, and organs from which the observed microvesicles originate.

Furthermore, in addition to the above-mentioned problems, the present inventors have discovered that when using microvesicles as clinical test materials, there are proteins that supplement immunoglobulins, such as complement, in blood, and that these proteins bind to immunocomplexes. It has been found that these molecules may be formed and have a molecular size close to microvesicles, and that they also interact with IgG, which may pose a problem when performing an immunochemical characterization method on blood-derived microvesicles.
In addition, in this specification, separation shall be used synonymously with fractionation, detection, etc.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have discovered a pretreatment step in which microvesicles are concentrated from a blood sample (whole blood, plasma, or serum) by centrifugation; A step of adding labeled IgG to the concentrated microvesicle fraction obtained in step 2. A step of collecting and observing the fraction with a diameter of approximately 0.1 to 1 μm using a flow cytometer. By selecting and carrying out an appropriate combination of the steps of removing a particle population that reacts with We have discovered that it is possible to accurately observe which organs, tissues, and their constituent cells have released vesicles and present them in the blood. Based on this knowledge, the present invention has completed a measurement method that can individually characterize microvesicles in blood.

That is, the present invention provides:
[1] A method for separating microvesicles in a blood sample, which includes the following steps;
(Step A) A step of adding (1) a staining reagent and (2) labeled IgG to a blood sample or its processed product in this order, in the reverse order, or at the same time;
(Step B) a step of subjecting the reaction product obtained in the step A to measurement with a flow cytometer;
(Step C) Analyze the data acquired in step B, and observe images of (1) aggregated microvesicles and (2) particles that react with labeled IgG in this order, in the reverse order, or simultaneously. a process to be excluded from;
(Step D) A step of sorting and characterizing the derived cells into microvesicle populations,
[2] The method of [1], wherein the observation range in step B is either 1 μm or less, or 200 nm or more and 1 μm or less,
[3] The method of [1] or [2], which includes the step of setting parameters of the flow cytometer in order to converge particles of a desired diameter in the observation area of the flow cytometer;
[4] Any method of [1] to [3], which uses polystyrene beads with a particle size of 0.22 to 1.35 μm to estimate the particle size in the observation area;
[5] The method of any one of [1] to [4], wherein the processed blood sample is a sample separated by centrifugation;
[6] Any method of [1] to [5], wherein the diameter of the microvesicles to be separated is 1 μm or less,
[7] The method of any one of [1] to [6], wherein the isolated microvesicles contain the proteins listed in Table 4;
[8] A method of searching for substances specifically contained in blood-derived microvesicles using the fraction obtained by any of the methods of [1] to [7];
[9] A method of aiding in the detection of a disease, drug administration effect, or other medical condition in a subject using the fraction obtained by any of the methods of [1] to [7].

According to the present invention, microvesicles present in blood can be easily and specifically separated and observed, and furthermore, individual microvesicles can be characterized using surface antigens present on the membrane (what kind of cells and tissues are present). , whether it originates from an organ). As a result, it is expected that the value of the test will improve as a test that can be used for diagnosis and testing that focuses on specific organs, tissues, and cells that match specific diseases. Furthermore, by analyzing the fractions obtained according to the present invention, it becomes possible to estimate diseases, drug administration effects, or other medical conditions in a subject.

These are flow cytometer images of a sample in which plasma was directly stained (upper side) and a sample in which the microvesicle fraction concentrated through pretreatment was stained (lower side). This is a flow cytometer observation image of the microvesicle fraction concentrated by pretreatment. This is a negative staining image of a microvesicle fraction concentrated by pretreatment using a transmission electron microscope. This is a particle size distribution histogram obtained by a nanoparticle analysis system of a microvesicle fraction concentrated by pretreatment. These are the results of protein enrichment analysis using metascape from proteins detected by shotgun proteomics analysis. These are the results of network analysis for each cluster of protein enrichment analysis results using metascape from proteins detected by shotgun proteomics analysis. This is an observation image of flow cytometer measurement data of the microvesicle fraction concentrated by pretreatment, developed in terms of pulse width and pulse area in side scattered light. This is an observation image in which the same measurement data as in FIG. 7 is expanded by the areas of forward scattered light and side scattered light. This is an observed image developed by side scattered light and APC mouse IgG from measurement data that excludes (gates out) groups that deviate significantly from the observed images shown in FIGS. 7 and 8. An explanatory diagram showing the process of selecting red blood cell-derived microvesicles from the measurement data after excluding (gating out) the APC mouse IgG positive population from the observed image shown in Figure 9, and a table showing the reactivity with the surface antigen used for characterization. It is. An explanatory diagram showing the process of selecting macrophage/monocyte/granulocyte-derived microvesicles from the measurement data after excluding (gating out) the APC mouse IgG positive population from the observation image shown in Figure 9, and the surface antigens used for characterization. This is a table showing the reactivity of . An explanatory diagram showing the process of selecting T cell/B cell-derived microvesicles from the measurement data after excluding (gating out) the APC mouse IgG positive population from the observed image shown in Figure 9, and the reaction with the surface antigen used for characterization. This is a table showing gender. An explanatory diagram showing the process of selecting platelet-derived microvesicles from the measurement data after excluding (gating out) the APC mouse IgG positive population from the observed image shown in Figure 9, and a table showing the reactivity with the surface antigen used for characterization. It is. Figure 9 is an explanatory diagram showing the process of selecting vascular endothelial cell-derived microvesicles from measurement data excluding (gating out) the APC mouse IgG positive population from the observed image, and the reactivity with the surface antigen used for characterization. This is a table showing It is a diagram superimposed on a developed view of side scattered light and Annexin5 after characterization of microvesicles in the derived cell population. After characterization of microvesicles in the derived cell populations, side scattering light and Annexin5 development diagrams are superimposed (upper side), and flow cytometer images of Annexin5-positive and Annexin5-negative populations in each derived cell population. This is an observed image (lower side). It is a graph showing the results of comparing the proportions of Annexin5-positive population and Annexin5-negative population of microvesicles derived from each cell from 10 healthy individuals using a flow cytometer.

Although embodiments of the present invention will be described in detail below, the usage method is not limited thereto.

The present invention is a separation method and an analysis method using a flow cytometer in which observation conditions specific to microvesicles in blood are set. Although a pretreatment step for removing impurities and concentrating microvesicles may be combined, it is also possible to omit the pretreatment conditions and directly separate and measure the microvesicles using a flow cytometer. Direct separation and measurement using a flow cytometer is preferable as a versatile observation system for testing applications and practical use. In this specification, pretreatment includes the meaning of concentration and recovery, and concentration and recovery are sometimes used as almost synonymous terms.

In this specification, blood includes, for example, whole blood, plasma, serum, and the like. The biological sample may be applied to the method of the present invention as is, or it may be dissolved or suspended in water, an acidic solution, an alkaline solution, a buffer solution, or a mixture thereof, and subjected to further processing as necessary. It may also be applied to the method of the invention. Those skilled in the art can appropriately select and use the acidic solution, alkaline solution, and buffer solution that can be used in the present invention.

An example of conditions for separating and analyzing microvesicles in blood using a flow cytometer will be described below, but the present invention is not limited thereto.

Furthermore, the flow cytometer that can be used in the present invention is not limited to a specific device, and any flow cytometer that employs any type of flow cell, such as a quartz cuvette type or a jet-in-air type, can be used. .

The observation method using a flow cytometer that can be used in the present invention can be carried out, for example, according to the following steps.

(1) Staining of microvesicles In this step, a blood sample or its processed material is mixed with a staining reagent to stain microvesicles. More specifically, microvesicles are stained by mixing a staining reagent, such as a fluorescently labeled antibody specific to a surface antigen, with whole blood, plasma, serum itself, or a fraction concentrated by a pretreatment method. do. As for the dyeing method, those skilled in the art can adopt a conventional method and, depending on the case, suitably examine the conditions. In order to target multiple antigens in microvesicles, detection may be performed by staining with multiple types of labeled antibodies as multicolor separation. For example, Annexin 5, which is a protein that binds to phosphatidylserine (PS), which is a membrane component of microvesicles, in the presence of metal ions, etc. can be used.

Other substances that can specifically bind to microvesicles include, for example, any substance that can bind to proteins, lipids, or sugars present on the surface of microvesicles. Substances present on the surface include, for example, proteins such as CD235a, CD59, CD44, CD33, CD45, CD144, CD66a, CD66b, CD66c, CD66e, CD41, CD61, EP-CAM, CD324, CD14, CD81, CD31, CD274. , CD63, Alix, CD105, CD133, CD279, CD15, TSG101, CD20, CD249, CD5, CD10, CD26, CD273, CD9, MUC-1, CD13, CD146, CD62E or combinations thereof can be used. For example, Annexin5, CD235a, and CD59 are microvesicles derived from red blood cells, Annexin5, CD41, and CD61 are microvesicles derived from platelets, and Annexin5, CD45, and CD15 are microvesicles derived from macrophages, monocytes, and granulocytes. By using a combination of Annexin5, CD45, and CD5 as microvesicles derived from T cells/B cells, and Annexin5, CD146, and CD105 as microvesicles derived from vascular endothelial cells, more accurate separation can be achieved. preferable.

Substances that can specifically bind to microvesicles other than those listed above include, for example, substances that have binding affinity for proteins, substrates for enzymes, coenzymes, regulatory factors, substances that specifically bind to receptors, Lectins, sugars, glycoproteins, antigens, antibodies or antigen-binding fragments thereof, hormones, neurotransmitters, phospholipid-binding proteins, proteins containing pleckstrin homology (PH) domains, cholesterol-binding proteins, or these It may be a combination. Antigen-binding fragments include antigen-binding sites, and may be, for example, single-domain antibodies, Fabs, Fab's, or scFvs.

(2) Addition of IgG Add IgG to the mixture obtained by mixing the blood sample or its processed product with the staining reagent in the previous step, or to the blood sample or its processed product before mixing with the staining reagent. Mix the fluorescently labeled materials and let them react. The origin of the IgG is not particularly limited, and for example, mouse IgG, human IgG, rat IgG, rabbit IgG, goat IgG, bovine IgG, etc. can be used. As with the microvesicle staining process, those skilled in the art can carry out the process according to standard methods. For example, it can be carried out by using allophycocyanin (APC)-labeled mouse IgG, Mix-n-Stain APC Antibody Labeling kit (Biotium), and allowing it to stand at room temperature for 30 minutes according to a regular protocol.

Proteins such as complement that supplement immunoglobulins exist in the blood, and immunocomplexes may be formed. This immunocomplex may be close in molecular size to microvesicles. Therefore, since the immunocomplex also interacts with IgG, it may pose a problem when performing an immunochemical characterization method on blood-derived microvesicles. Therefore, in order to detect the fraction that captures IgG in the observed image and remove it from the observed image, it is possible to achieve the objective by performing a step of mixing fluorescently labeled IgG and reacting with the IgG captured fraction. This method is preferable because it can remove substances other than microvesicles, and it is possible to perform more accurate measurements that do not include contaminants.
In addition, the above-mentioned step of staining microvesicles with a surface antigen-specific antibody and the step of adding IgG may be carried out first, or both steps may be carried out at the same time. It is possible to select an appropriate one according to the implementation environment.

(3) Flow cytometer settings Adjust the voltage and threshold of the photomultiplier tube for forward scattered light and side scattered light to converge particles with a specific diameter in the observation area. When setting conditions such as these parameters, it is preferable to measure unstained samples and stained samples, and determine voltage settings and sheath flow rates that satisfy the detection sensitivity for detecting each target.

Those skilled in the art can search for the optimal conditions and set the parameters of the flow cytometer as appropriate; for example, the flow rate may be 12 μL/min, the voltage of forward scattered light may be The voltage of the side scattered light can be set to 381 and 340, and the fluorescence intensity threshold can be set to 200 as the respective detection sensitivity. The wavelength and voltage of the excitation light (Ex.) and fluorescence detection filter (Em.) for each fluorescent substance can be appropriately selected and set depending on each fluorescent substance used.

Using the flow cytometer parameters set above, estimate the particle size in the observation area. Measurement can be performed according to a standard method using polystyrene beads of uniform size (eg, SPHEROTM Nano Polystyrene Size Standard Kit, Spherotech). In order to estimate the particle size in the observation area, the particle size of the polystyrene beads is preferably 0.22 μm, 0.45 μm, 0.88 μm, or 1.35 μm, for example.

The diameter set as the observation area is not particularly limited as long as it includes microvesicles, but for example, it is preferably about 1 μm or less, more preferably 100 nm to 1 μm, and even more preferably 200 nm to 1 μm. Particularly when observing these microparticles, side scattered light is preferable because it has higher resolution than forward scattered light and can be used for particle size verification.

Note that this step is not limited to the embodiment performed between the staining step and IgG addition step and the measurement/separation step described below; for example, after setting the parameters, the staining step, the IgG addition step, and the measurement/separation step are performed. Alternatively, once set, a series of steps can be performed repeatedly without setting parameters for each measurement.

(4) Measurement and separation by flow cytometry Obtain data with a flow cytometer. When performing multi-color measurement, correction is made for the leakage of fluorescence from each fluorescence to a detector other than the assigned one, and measurement results are obtained under that condition.

Microvesicles may aggregate, but in order to exclude aggregated microvesicles from the observed image, 1) the pulse width of the side scattered light is very high and deviates from the group, 2) the forward From the plot of scattered light and side scattered light, it is possible to mark and gate out those where both are out of the group and have high values. This is particularly effective when microvesicles aggregate due to pretreatment. Furthermore, in order to remove immune complexes with the IgG capture fraction, a positive population that reacts with fluorescently labeled IgG can be marked and gated out.
In addition, the above-mentioned step of excluding aggregated microvesicles and the step of removing immune complexes captured by IgG may be carried out first, or both may be carried out at the same time. It is possible to select an appropriate one according to the environment in which the vendor operates.

By performing a gating operation on the microvesicles detected using multicolor, it is possible to separate the cells from which they originate into microvesicle populations and characterize them. Through this step, it is possible to confirm that the microvesicles are isolated in the blood.

Although microvesicles in blood can be separated and characterized according to the present invention, these characterized fractions can also be further separated using a cell sorter. It is also possible to analyze the specific contents (nucleic acids, metabolites, proteins, lipids) from these separated fractions and apply them clinically. The separated fractions may be further used for frequency analysis of each microvesicle population and expression level analysis of target molecules.

In addition to the above processes as a measurement technology that can be separated and observed by microvesicle, for example, Nanosight (Nanosite, Malvern Panalytical) is NTA (NANO TRACKING AnalySisis). ) Technology and FFF (FIELD FLOW FRACTIONATION) ) techniques can also be used in combination. With NTA technology, the Brownian motion of nanoparticles in liquid can be observed in real time on a PC screen, and with FFF technology, separation is performed in a thin flow channel; Due to the special geometry, this flow forms a laminar flow with a radial cross section, and by perpendicular to this laminar flow, a separating force is generated, which separates small particles such as microvesicles by size. Therefore, more accurate separation and analysis are expected. By combining such separation/observation methods with surface antigens that characterize individual microvesicles, microvesicles can be characterized, increasing organ specificity and disease specificity, and can be used for clinical applications.

In the microvesicle separation method that can be used in the present invention, a method that combines pretreatment steps that can remove impurities and concentrate microvesicles can be used. Combining such pretreatment steps is preferable because it not only facilitates setting of conditions in the separation step using a flow cytometer, but also improves separation speed and sensitivity. The pretreatment step characterized by centrifugation can be carried out, for example, according to the following steps.

A blood sample collected in a blood collection tube is separated into plasma or serum components by low-speed centrifugation (pretreatment step 1). At this time, conditions such as low-speed centrifugation can be carried out according to known methods, and a supernatant obtained by centrifugation at 2,000 x g for 20 minutes at room temperature can be used.

Next, the plasma and serum obtained in the pretreatment step 1 are further centrifuged at low speed (pretreatment step 2). This centrifugation step is preferable because platelets, apoptotic blisters, and the like can be precipitated. As in pretreatment step 1, the supernatant obtained by centrifugation at 2,000×g for 20 minutes at room temperature can be collected and used.

The supernatant obtained in pretreatment step 2 is centrifuged at high speed to precipitate the microvesicle fraction (pretreatment step 3). As conditions for this step, for example, a precipitate obtained by centrifugation at 20,000 x g for 30 minutes at room temperature can be used.

For example, a buffer such as PBS is added to the precipitate fraction obtained in pretreatment step 3 to suspend it, and further high-speed centrifugation is performed (pretreatment step 4). This high-speed centrifugation is preferable because the microvesicle fraction can be concentrated and impurities can be removed. As conditions for this step, a precipitate obtained by centrifugation at 20,000 xg for 30 minutes at room temperature can be used. Pretreatment step 4 may be repeated as necessary. By performing centrifugation at high speed multiple times, impurities can be removed and the purity of the microvesicle fraction can be increased, which is preferable.

In addition to the above pretreatment steps, other pretreatment methods that can be used as one aspect of the present invention may be used. For example, the desired microvesicle fraction can be extracted by combining membrane filters with various pore sizes used for filter sterilization of aqueous solutions and solutions containing proteins. In this case, it is preferable to use a method corresponding to micro-filtration using pores with a diameter of 0.1 to 10 μm.

Other pretreatment methods include equilibrium density gradient centrifugation, in which the sample is centrifugally fractionated together with a density gradient solute, and antibodies specific to the surface antigen of the micromembrane fraction are used to transfer the micromembrane fraction to various carriers. Immunological capture method in which the membrane components of the micromembrane fraction are collected using immunological capture methods, methods in which size exclusion chromatography (gel filtration method) is used to collect fractions that elute faster than soluble proteins, and metal It may be carried out in combination with a phospholipid affinity method using a carrier that binds in the presence of ions, or a polymer precipitation method in which a high molecular weight polymer and a micromembrane fraction are mixed and the desired micromembrane fraction is precipitated. A person skilled in the art can appropriately select and combine these methods depending on the required tissue, cells, and purpose. By combining multiple pretreatment steps, it is possible to perform separation using a flow cytometer with high sensitivity and precision.

In a different embodiment of the invention, it can also be used as a method for monitoring disease progression in a subject, and for monitoring disease recurrence in an individual. In addition to the step of separating microvesicles from a blood sample, these methods include a profiling step of analyzing substances contained in the microvesicles. By analyzing the profile of a subject with a particular medical condition, it can be used, for example, to estimate the presence of a particular disease. For example, by appropriately setting the sample collection period for isolating microvesicles depending on the target disease detection, etc., it is possible to obtain a more detailed profile, observe the condition, and assist in diagnosis. It becomes possible. It can also be used as a method of monitoring disease status after drug administration.

According to the present invention, microvesicles can be effectively used as clinical test materials, so microvesicles present in blood can be easily and specifically extracted and observed, and surface antigens present on membranes can be detected. It can be used to characterize individual microvesicles (what cells, tissues, and organs they originate from). As a result, it is expected that the value of the test will improve as a test that can be used for diagnosis and testing that focuses on specific organs, tissues, and cells that match specific diseases. Furthermore, by analyzing the fractions obtained according to the present invention, it becomes possible to estimate diseases, drug administration effects, or other medical conditions in a subject.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but these are not intended to limit the scope of the present invention.

《Example 1: Concentration of microvesicles from human blood》
5 mL to 7 mL of blood was collected from a healthy person into a TERUMO Venoject II vacuum blood collection tube EDTA-Na (VENOJect II (registered trademark), Terumo). Blood samples were centrifuged at 2,330 x g for 10 minutes at 20°C to obtain plasma, followed by centrifugation at about 2,000 x g for 10 minutes at about 20°C to remove intraplasma lipids and cellular debris. , platelets were removed, and the supernatant was used for the following experiments. The experiment from this blood collection was conducted under the approval of the Kyushu University Medical Department Clinical Research Ethics Review Committee (permit number 29-340).

600 μL of the resulting plasma was centrifuged at 18,900×g for 30 minutes at 20°C. The supernatant was removed from the centrifuged product and the microvesicle fraction was concentrated in a pellet. The pellet was diluted using 600 μL of phosphate buffered saline (PBS). The diluted pellet was centrifuged at 18,900 xg for 30 minutes at 20°C. The microvesicle fraction was concentrated in a pellet from the centrifuged product.

《Example 2: Observation of plasma microvesicles using a flow cytometer》
A. Microvesicle observation using a flow cytometer when plasma is directly stained (comparison data with pre-treated concentrated fraction)
FITC-Annexin5 (Becton Dickinson Biosciences), PEanti-human CD235a (Biolegend), PerCP anti-human CD61 (Biolegen) were added to 60 μL of plasma obtained by centrifugation (2,000 Add 1 μL of each of It was left standing at room temperature for 30 minutes. This staining solution was suspended in 750 μL of 10 mmol/L Hepes (pH 7.4), 0.14 mol/L NaCl, and 2.5 mmol/L CaCl ₂ and used as a sample. The parameters measured by the flow cytometer are described in section C below. FIG. 1 shows an observed image in which the measurement data is expanded with the horizontal axis: side scattered light (SSC) intensity and the vertical axis: FITC (Annexin 5) intensity. In this figure, a region with high fluorescence intensity obtained by separating from the noise-derived population at the bottom was extracted, and this region was analyzed as an Annexin5-positive population. Regarding the Annexin5 positive fraction, the results observed by directly staining plasma and those observed by staining the pretreated concentrated fraction obtained in Example 1 above showed similar results.

B. Immunofluorescence staining of microvesicles concentrated from plasma 60 μL of PBS was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and the microvesicle fraction was dispersed in the solution by vortexing. To this solution, FITC-Annexin5 (Becton Dickinson Biosciences), Brilliant Violet510anti-human CD5 (Biolegen), PerCPanti-human CD15 (Biolegen) were added. d), APC/Cy7anti-human CD41 (Biolegend), PE/Cy7anti-human CD45 (Biolegend), PEanti-human CD59 (Biolegend), Brilliant Violet421anti-human CD105 (Biolegend), PEanti-human CD146 (Biolegend), PE/Cy7anti-huma n CD235a (Biolegend), PerCP anti-human CD61 (Biolegend), APC-labeled mouse IgG (purchased 1 μL of each of Normal Mouse IgG (Wako) labeled according to the standard protocol attached to the Mix-n-Stain APC Antibody Labeling kit (Biotium) was added, and the mixture was allowed to stand at room temperature for 30 minutes.

C. Observation of microvesicles in plasma using a flow cytometer Measurements were performed using a BD FACSVerse™ (Becton Dickinson and Company) as a flow cytometer. The measurement procedure and parameter settings are as follows. The samples used were those obtained by suspending the various fluorescently stained fractions in Example 2B in 750 μL of 10 mmol/L Hepes (pH 7.4), 0.14 mol/L NaCl, and 2.5 mmol/L CaCl ₂ . The flow rate was set to 12 μL/min, the voltage of forward scattered light was set to 381, the voltage of side scattered light was set to 340, and the respective thresholds were set to 200. The wavelength and voltage of the excitation light (Ex.) and fluorescence detection filter (Em.) for each fluorescent substance are determined by FITC:Ex. 488 nm, Em. 527/32 nm, voltage 442, PE: Ex. 488 nm, Em. 586/42 nm, voltage 411, PerCP: Ex. 488 nm, Em. 700/54 nm, voltage 556, PE/Cy7: Ex. 488 nm, Em. 783/56 nm, voltage 564.3, APC: Ex. 640nm, Em. 660/10 nm, voltage 538.2, APC/Cy7: Ex. 640nm, Em. 783/56 nm, voltage 584.8, Brilliant Violet421:Ex. 405 nm, Em. 448/45 nm, voltage 538.2, Brilliant Violet510: Ex. 405 nm, Em. The wavelength was 528/45 nm and the voltage was 540.

D. Size verification measurement using polystyrene beads in the set observation area With the flow cytometer parameters set above, use polystyrene beads (SPHEROTM Nano Polystyrene Size Standard Kit, Spherotech) with a uniform size to estimate the particle size in the observation area. Measurements were taken. The polystyrene beads used had particle diameters of 0.22 μm, 0.45 μm, 0.88 μm, and 1.35 μm.

E. Interpretation of Microvesicle Measurement Results with a Flow Cytometer Figure 2 shows an observation image in which the measurement data is expanded with the horizontal axis: side scattered light (SSC) intensity and the vertical axis: FITC (Annexin 5) intensity. In this figure, a region with high fluorescence intensity obtained by separating from the noise-derived population at the bottom was extracted, and this region was analyzed as an Annexin5-positive population. In addition, there is a proportional relationship between the intensity of side scattered light and the size of polystyrene beads of 0.22 to 1.35 μm in the observation region, and in particular, there is a microvesicle fraction between polystyrene beads with diameters of 0.22 μm and 0.45 μm. It was observed that the median values of

《Example 3: Electron microscope observation of microvesicle fraction concentrated from blood》
A. Immobilization of microvesicles in plasma 100 μL of PBS was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and a fixative solution (0.1 mol/L containing 4% paraformaldehyde and 4% glutaraldehyde) was added to the pellet. 100 μL of phosphate buffer (pH 7.4) was added, stirred, and left at 4° C. for 1 hour.

B. Negative Staining Method The fixed sample was adsorbed onto grid-coated formvar film and stained with 2% phosphotungstic acid (pH 7.0) for 30 seconds.

C. Observation with a transmission electron microscope The above grid was observed with a transmission electron microscope (JEM-1400; JEOL Ltd.). Acceleration voltage was set at 100 kV. A digital image (3296×2472 pixels) was acquired with a CCD camera (EM-14830RUBY2; JEOL Ltd.). The observed image is shown in Figure 3. A micromembrane fraction having the size of a microvesicle and containing a membrane structure and internal structures (organelles, etc.) inside the membrane structure was detected.

《Example 4: Particle measurement of extracted fraction using nanoparticle analysis system》
100 μL of PBS was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and the pellet was further diluted 3 times with PBS and measured using Nanosight (Malvern Panalytical). Table 1 shows the observation conditions of the device. Figure 4 shows a particle size distribution histogram obtained by the nanoparticle analysis system. In this concentration operation, the fraction with a diameter of 1 μm or more is hardly included, and the fraction with a diameter of 600 μm or less is concentrated, and the particle size mainly contains a homogeneous group with a particle size of about 200 to 300 nm. It was confirmed that the fraction contained a diameter (~100 nm).

《Example 5: Proteins detected in concentrated microvesicle fraction by shotgun proteomics analysis》
A. Extraction of protein fraction from concentrated microvesicle fraction obtained by blood pretreatment In order to efficiently extract especially membrane proteins from the concentrated microvesicle fraction in Example 1, PTS (Phase Transfer Surfactant) was used. A protein solubilization method using a solubilizing agent) was performed. MPEX PTS Reagents for MS (GL Science Inc.) was used as a reagent kit using this principle. 250 μL of the kit reagent B was added to the pellet obtained by concentrating the microvesicle fraction in Example 1, and a cycle consisting of an operation of 1 minute 30 seconds and an interval of 30 seconds using an ultrasonic homogenizer (Bioruptor: Sonic Bio Inc.) was performed. was sonicated (power: MAX) 10 times at 10°C. After crushing, the mixture was concentrated using a centrifugal filter (Amicon ultra 3K, Merck) twice at 14,000 g for 15 min. Protein in this fraction was quantified by BCA assay (using Pierce ^™ BCA Protein Assay kit (Thermo Fisher Scientific Inc.)).

B. Enzyme Digestion of Protein Fraction The protein fraction was quantified by BCA assay and dissolved in the reagent B to a concentration of 30 μg/20 μL, which was used as a sample for membrane protein digestion. 1 μL of 100 mmol/L dithiothreitol (DTT) was added (for reductive cleavage of disulfide bonds) and incubated for 30 minutes at room temperature. To this was added 1 μL of 550 mmol/L iodoacetamide (for carbamidomethylation of Cys), and the mixture was incubated at room temperature for 30 minutes in the dark. 77 μL of reagent A of the kit was added, and 1.5 μL of trypsin (TPCK-Trypsin (Thermo Fisher Scientific Inc.: Prod #20233)) was added. Protein digestion was performed by incubating overnight at room temperature. 150 μL of Reagent C and 1.5 μL of Reagent D were added. After the addition, the mixture was vortexed for 1 minute and centrifuged at 25° C. and 15,600×g for 2 minutes to separate the two phases. Unnecessary solubilizing agent was present in the upper phase and was removed by suction with a pipette. In order to remove the remaining Reagent C, after performing centrifugal concentration, 50 μL of 5% acetonitrile, 0.1% trifluoroacetic acid (TFA) was added and vortexed.

C. Desalting and Concentration of Peptides GL-Tip SDB (GL Science Inc.) was used to desalt and concentrate the enzymatically digested peptides described above. The chip was first conditioned by adding 20 μL of 80% acetonitrile and 0.1% TFA aqueous solution and centrifuging at 3,000×g for 2 minutes at room temperature. 20 μL of 5% acetonitrile and 0.1% TFA aqueous solution was added thereto, and the column was equilibrated by centrifuging at 3,000×g at room temperature for 2 minutes. To this, the entire amount of the sample obtained in the procedure of Example 5B was added, and the peptide was adsorbed onto the column by centrifuging at 3,000 xg at room temperature for 5 minutes. Furthermore, 20 μL of 5% acetonitrile, 0.1% TFA aqueous solution was added, and the column was washed by centrifuging at 3,000 × g for 2 minutes at room temperature, and then 50 μL of 80% acetonitrile, 0.1% TFA aqueous solution was added. Peptides were eluted by centrifugation at 3,000 xg for 2 minutes at room temperature.

D. Mass spectrometry measurement The above sample was measured using a Fourier transform Orbitrap mass spectrometer (Q-Exactive: Thermo Fisher Scientific Inc.) connected to a nano-LC system (EASY-nLC1000: Thermo Fisher Scientific Inc.). LC-MS/MS An analysis was performed. 2 μL of the sample for mass spectrometry measurement prepared above was subjected to measurement. The trap column was Acclaim ^RT PepMap100 (Thermo Fisher Scientific Inc.: C18, packing diameter 3 μm, inner diameter 75 μm, column length 2 cm), and the analysis column was Acclaim ^RT PepMap RSLC (Thermo Fisher Scientific Inc.: C18, filler diameter 2 μm , inner diameter 50 μm, column length 15 cm). Mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid/acetonitrile, flow rate is 200 nL/min, gradient is 0-40% mobile phase B/200 minutes, 40-100% mobile phase B /10 minutes, 100% mobile phase B/10 minutes.

The scanning conditions for MS measurement were Full MS/dd-MS2 mode, and after scanning Full MS, an MS/MS spectrum of a signal with high intensity was obtained. Table 2 shows the setting conditions.

E. Data analysis The obtained data were compiled into the human protein database Homo_sapiens_Uniprot_201210. A search was performed on ``fast'' using the SequestHT algorithm. Table 3 shows the search conditions in SequestHT.

We picked up items with a Score of 1.0 or higher from the search results. Table 4 shows a list of proteins detected by shotgun proteomic analysis of the extracted microvesicle fraction. In addition, Figure 5 shows the results of protein enrichment analysis using metascape (Tripathi et al., Cell Host & Microbe (2015), 18: 723-735) from proteins detected by shotgun proteomics analysis. The results of network analysis for each cluster are shown in FIG. 6. Many of the proteins shown in Table 4 are categorized as cell membranes or intracellular, and include those that have functions in blood coagulation, immune response, and various signal transmission (Figures 5 and 6). It has the potential to be used as a surface marker to characterize microvesicles, and its contents as a biomarker for clinical testing and diagnosis.

《Example 6: Characterization of plasma microvesicles using a flow cytometer》
A. Gate out of aggregated microvesicles by pulse treatment (exclusion from observation image)
The concentrated microvesicle fraction in Example 1 was stained with each surface antigen and measured under the flow cytometer observation conditions of Example 2. At this time, an image in which multiple microvesicles appeared to aggregate together was observed (a group in which all of the various stained surface antigens were positive). In order to remove this from the observed image, we created an observation image (Figure 7) developed by the pulse width (vertical axis) and pulse area (horizontal axis) in side scattered light, forward scattered light (horizontal axis), and side scattered light. An observation image (Fig. 8) developed by the area of (vertical axis) was prepared, and groups that deviated significantly from each were excluded from the observation image.

B. Gate out of IgG captured fraction (exclusion from observation image)
After the operation in Example 6A, the APC-positive population, that is, the population that captures IgG, was extracted from the developed diagram (FIG. 9) of side scattered light (horizontal axis) and APC-labeled mouse IgG (vertical axis), and excluded from the observed image. At the same time, the APC-negative population was subjected to the following characterization. Table 5 shows the expression patterns of surface antigens used (or available for use) in microvesicle characterization in various cells.

C. Characterization of red blood cell-derived microvesicles After the operation in Example 6B, an Annexin5-positive population was extracted from the side scattered light and the Annexin5 development diagram, a CD45-negative population was extracted from this population, and a CD235a-positive population was further extracted from this population. In addition, a CD59-positive population was selected (FIG. 10). Also included is a table showing the surface antigens used for characterization and whether they are present (○) or absent (x) in various cells. This population ("Erythrocyte" column in the table of FIG. 10) was defined as red blood cell-derived microvesicles, and the proportion of this population in the Annexin5-positive population was 32%.

D. Characterization of macrophage/monocyte/granulocyte-derived microvesicles After the operation of Example 6B, an Annexin5-positive population was extracted from the side scattered light and the Annexin5 development diagram, and a CD235a-negative population was extracted from this population. Furthermore, a CD45-positive and CD15-positive population was selected from this population (FIG. 11). Also included is a table showing the surface antigens used for characterization and whether they are present (○) or absent (x) in various cells. This population ("Macrophage/Monocyte" and "Granulocyte" columns in the table of Figure 11) was defined as macrophage/monocyte/granulocyte-derived microvesicles, and the proportion of this population in the Annexin5-positive population was 2.6%. Ta.

E. Characterization of T cell/B cell-derived microvesicles After the operation in Example 6B, an Annexin5 positive population was extracted from the side scattered light and the Annexin5 development diagram, a CD235a negative population was extracted from this population, and a CD235a negative population was extracted from this population. A CD45-positive and CD5-positive population was selected from the population (FIG. 12). Also included is a table showing the surface antigens used for characterization and whether they are present (○) or absent (x) in various cells. This population ("T Cell" and "B Cell" columns in the table of FIG. 12) was defined as T cell/B cell-derived microvesicles, and the proportion of this population in the Annexin5-positive population was 0.4%.

F. Characterization of platelet-derived microvesicles After the operation in Example 6B, an Annexin5-positive population is extracted from the side scattering light and the Annexin5 development diagram, a CD45-negative and CD5-negative population is extracted from this population, and a population that is CD45-negative and CD5-negative is further extracted from this population. A CD41-positive and CD61-positive population was selected from the following (FIG. 13). Also included is a table showing the surface antigens used for characterization and whether they are present (○) or absent (x) in various cells. This population ("Platelet" column in the table of FIG. 13) was defined as platelet-derived microvesicles, and the proportion of this population in the Annexin5-positive population was 62%.

G. Characterization of Microvesicles Derived from Vascular Endothelial Cells After the operation in Example 6B, an Annexin5-positive population was extracted from the side scattering light and a developed diagram of Annexin5, and a CD105-positive and CD146-positive population was selected from this population (FIG. 14). . Also included is a table showing the surface antigens used for characterization and whether they are present (○) or absent (x) in various cells. This population ("Endothelial Cell" column in the table of FIG. 14) was defined as vascular endothelial cell-derived microvesicles, and the proportion of this population in the Annexin5-positive population was 0.3%.

H. 5 Classifications of Plasma Microvesicles In the above, an example of a diagram superimposed on a developed diagram of side scattered light and Annexin 5 after characterizing the microvesicles to the cell populations from which they are derived is shown in FIG.

I. Characterization of each cell-derived microvesicle by Annexin5 positivity and negativity After the operation of Example 6B, Annexin5 positive and negative populations were extracted from the side scattered light and the Annexin5 development diagram, and from this population, the above C, D, E, Microvesicle characterization from the derived cells using the CD antigens shown in F and G was also performed (FIG. 16). When comparing the proportion of Annexin5 positive and negative cells for each derived vesicle, microvesicles derived from red blood cells, macrophages/monocytes/granulocytes, and vascular endothelial cells had a large Annexin5 positive population, while microvesicles derived from T cells/B cells had a high Annexin5 positive population. There were many negative groups (Figure 17). The comparison test used the Wilcoxon signed rank test (* p < 0.05, ** p < 0.01).
Regarding conventionally used microvesicle markers (for example, Annexin 5), the above results revealed that there are many Annexin 5-negative microvesicles, and the proportion thereof varies greatly depending on the cell of origin. In other words, it has been discovered that the behavior of substances in microvesicles derived from each cell may be different from the conventional behavior. This means that the exact nature of microvesicles may be overlooked by characterization using conventionally used methods. It has been suggested that by using the present invention, it may be possible to obtain clinical test materials for identifying disease sites and obtaining results that match diagnostic purposes.

Based on the above results, it is possible to detect microvesicles in blood by directly observing the microvesicles from plasma or by observing the concentrated microvesicle fraction from plasma. It has been shown that it can be customized. By using the present invention, it has become possible to identify the cells and organs from which individual microvesicles present in the blood originate, and to measure their increase/decrease and content rate.

The pretreatment method for concentrating microvesicles according to the present invention enables efficient extraction of microvesicles without any particularly complicated operations. By implementing this pretreatment method, it is possible to concentrate candidate substances (a list of proteins is exemplified in the example) contained in microvesicles to become biomarkers, and the concentration is higher than when directly measuring plasma or serum. It may be possible to perform sensitive measurements.

Furthermore, according to the present invention, it is possible to easily characterize individual microvesicles in blood using surface antigens of microvesicles, and to measure/quantitate what cells and organs the microvesicles are derived from. / Can be analyzed. Micromembrane fractions such as microvesicles contain transmitters necessary for communication between cells and organs, and are expected to be used clinically as liquid biopsies. It is thought that there are clinical and physiological implications as an effect.

These points indicate that the observation of microvesicles according to the present invention can be applied to testing methods for determining specific diseases and patient conditions, and can be used to manage the transition of individuals from a healthy state to a pre-diseased (pre-disease) state on a daily basis. Clinical applications as preventive and predictive markers are also possible. If there is a system that can measure smaller particles together with surface antigens with performance over a wide dynamic range, the clinical application value of micromembrane fractions, including exosomes and microvesicles, can be increased.

Claims (9)

A method for separating microvesicles derived from a target organ, target tissue, or target cell in a blood sample, the method comprising the following steps;
(Step A) A blood sample or a processed product thereof: (1) a labeled staining reagent that can specifically bind to the surface antigen of microvesicles derived from a target organ, target tissue, or target cell; and (2) a label. a step of adding the IgG in this order, in the reverse order, or at the same time;
(Step B) a step of subjecting the reaction product obtained in the step A to measurement with a flow cytometer;
(Step C) Analyze the data acquired in step B, and observe images of (1) aggregated microvesicles and (2) particles that react with labeled IgG in this order, in the reverse order, or simultaneously. a process to be excluded from;
(Step D) A step of detecting the microvesicles stained with the staining reagent added in the step A and characterizing the microvesicle population using the surface antigen. The method according to claim 1, wherein the observation range in the step B is either 1 μm or less, or 200 nm or more and 1 μm or less. 3. The method according to claim 1, further comprising the step of setting parameters of the flow cytometer in order to converge particles of a desired diameter in the observation area of the flow cytometer. The method according to any one of claims 1 to 3, wherein polystyrene beads having a particle size of 0.22 to 1.35 μm are used to estimate the particle size in the observation area. 5. The method according to claim 1, wherein the processed blood sample is a sample separated by centrifugation. The method according to any one of claims 1 to 5, wherein the diameter of the microvesicles to be separated is 1 μm or less. The method according to any one of claims 1 to 6, wherein the separated microvesicles contain the proteins listed in Table 4. A method for searching for substances specifically contained in blood-derived microvesicles using the fraction obtained by the method according to any one of claims 1 to 7. A method for assisting in the detection of a disease, drug administration effect or other medical condition in a subject using the fraction obtained by the method according to any one of claims 1 to 7.