Methods for analyzing biomolecules present in vesicles

Abstract

Provided is a method for analyzing biomolecules present in vesicles, said method involving: bringing vesicles and trapping molecules immobilized in a support body into contact; bringing the vesicles and first detection molecules labeled with first metal particles into contact; bringing the vesicles and second detection molecules labeled with second metal particles into contact; and quantifying the first metal particles and the second metal particles bound to the vesicles by means of inductively coupled plasma (ICP) mass spectrometry or ICP emission spectroscopy.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing biomolecules present in vesicles. [Background technology]

[0002] Due to the small size of extracellular vesicles, it is difficult to directly detect and quantify surface antigens using a flow cytometer or other instrument. Therefore, methods have been developed to detect and quantify surface antigens after capturing vesicles on beads to which antibodies or other agents have been immobilized. Patent Document 1 discloses a method in which exosomes are reacted with beads bound to antibodies that recognize extracellular vesicle surface markers, followed by reaction of these with an APC-labeled tetraspanin antibody, and then subjected to a flow cytometer to detect and quantify exosome surface markers. Patent Documents 2 and 3 disclose methods for detecting targets, such as intracellular molecules or viruses, by metal labeling and detecting the metal element using inductively coupled plasma mass spectrometry (ICP-MS). Patent Document 4 discloses a technique for simultaneously quantifying multiple surface antigens on exosomes using mass spectrometry. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] European Patent Application Publication No. 3093664 [Patent Document 2] International Publication No. 2016 / 109603 [Patent Document 3] International Publication No. 2016 / 182402 [Patent Document 4] International Publication No. 2020 / 235424 Summary of the Invention [Problem to be solved by the invention]

[0004] When vesicles are immobilized on a capture agent and surface antigens on the vesicles are detected using antibodies, it has been difficult to simultaneously quantify multiple surface antigens with good sensitivity using the methods described in Patent Documents 1 to 4. An object of the present invention is to provide an analytical method that can simultaneously quantify multiple biomolecules present on vesicles with good sensitivity. [Means for solving the problem]

[0005] The present invention provides contacting a sample containing vesicles with capture molecules immobilized on a support; contacting the vesicles with a first detection molecule labeled with a first metal particle; contacting the vesicles with second detection molecules labeled with second metal particles; quantitating the first metal particles and the second metal particles bound to the vesicles by inductively coupled plasma (ICP) mass spectrometry or ICP optical emission spectrometry; The present invention relates to a method for analyzing biomolecules present in vesicles comprising: [Effects of the Invention]

[0006] According to the present invention, multiple biomolecules present in vesicles can be simultaneously quantified with good sensitivity. [Brief explanation of the drawings]

[0007] [Figure 1] Figure 1 is a graph showing the amount of CD9 expression on the surface of exosomes quantified by the amount of gold ions in Experiment 1. [Figure 2] Figure 2 is a graph showing the quantification of the CD81 expression level on the exosome surface based on the amount of palladium ions in Experiment 1. [Figure 3] Figure 3 is a graph plotting the amount of three types of metal ions (vertical axis) against the number of exosome particles (horizontal axis) in Experiment 3. [Figure 4] FIG. 4 is a graph plotting the amounts of three metal ions (vertical axis) against the culture time (horizontal axis) of cultured cells in Experiment 4. [Figure 5]FIG. 5 is a graph plotting the amounts of three metal ions (vertical axis) against the amount of serum used (horizontal axis) in Experiment 5. DETAILED DESCRIPTION OF THE INVENTION

[0008] [Method for analyzing biomolecules present in vesicles] A method for analyzing biomolecules present in vesicles according to one aspect of the present invention includes: contacting the vesicles with capture molecules immobilized on a support; contacting the vesicles with a first detection molecule labeled with a first metal particle; contacting the vesicles with second detection molecules labeled with second metal particles; and quantifying the first and second metal particles bound to the vesicles by inductively coupled plasma (ICP) mass spectrometry or ICP optical emission spectrometry.

[0009] According to the method of the present invention, it is possible to simultaneously quantify multiple biomolecules present in vesicles. If the profile of multiple biomolecules present in vesicles is clarified, it is also possible to identify the type of vesicle. According to the method of the present invention, the binding of detection molecules to biomolecules is detected and quantified by mass spectrometry, so biomolecules present in vesicles can be analyzed with high sensitivity. Furthermore, according to the method of the present invention, a wide dynamic range (1×10 6 ~1×10 10 ) amount of vesicles, the biomolecules present in the vesicles can be analyzed.

[0010] As used herein, vesicles have a membrane-enclosed, sac-like structure. Examples of vesicles include extracellular vesicles and enveloped viruses. Extracellular vesicles are small vesicles measuring tens to hundreds of nanometers in size, surrounded by a lipid bilayer membrane, and containing signaling molecules such as proteins and nucleic acids. Extracellular vesicles are known to be released from cells and transmit signals to other cells. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. The size of exosomes is, for example, approximately 50 nm to 150 nm in diameter, the size of microvesicles is, for example, approximately 100 nm to 1 μm in diameter, and the size of apoptotic bodies is, for example, approximately 1 μm to 5 μm in diameter. In the method of the present invention, the vesicles are preferably exosomes. Biomolecules contained in exosomes can be used for the detection and diagnosis of diseases, including cancer and lifestyle-related diseases. Examples of enveloped viruses include coronaviruses, influenza viruses, rubella viruses, hepatitis C viruses, measles viruses, hepatitis B viruses, human immunodeficiency viruses, and herpes simplex viruses. The enveloped virus may be a recombinant virus.

[0011] The biomolecules present in the vesicles may be any molecules including proteins, nucleic acids, sugar chains, etc. The proteins present in the vesicles may be membrane proteins, cell adhesion molecules, receptor proteins, proteins involved in membrane fusion or scission, major histocompatibility complex (MHC) molecules, heat shock proteins, disease-specific marker proteins, etc. The proteins present in the vesicles may be glycoproteins. The nucleic acids present in the vesicles may be DNA or RNA, and the RNA may include messenger RNA, microRNA, non-coding RNA, etc. The biomolecules present in the vesicles may be present inside the vesicles, but are preferably present on the surface of the vesicles.

[0012] <Step of contacting vesicles with capture molecules immobilized on a support> The process of contacting vesicles with capture molecules immobilized on a support is described below. This process captures vesicles on the support. Capturing vesicles on the support allows for the vesicles to be concentrated, improving detection sensitivity. Furthermore, after contacting the capture molecules with the vesicles, washing the support removes nonspecifically bound vesicles, thereby reducing background noise. According to the present invention, even when the number of vesicles contained in a sample is small, for example, 1×10 10 Less than (e.g., 1 × 10 6 More than 1×10 10 Even if the density of the vesicles is less than 1 × 10, the biomolecules on the vesicles can be quantified. In one aspect of this embodiment, the method for analyzing biomolecules present in vesicles can also be understood as being capable of quantifying the vesicle density (the number of vesicles per unit volume). That is, the method for analyzing biomolecules present in vesicles can be used to quantify the vesicle density even if the density of the vesicles is less than 1 × 10. 10 / 100 μl or less (e.g., 1 × 10 6 / 100μl or more 1×10 10 Even if the volume of the vesicles is less than 100 μl, it is possible to quantify biomolecules on the vesicles. The capture molecule is preferably a molecule that binds to a biomolecule known to be present in the vesicles. The capture molecule may be a protein (antibody, lectin, etc.) or a nucleic acid molecule (aptamer, nucleic acid molecule having a complementary sequence, etc.). The capture molecule may be a molecule that binds to a protein expressed on the vesicle surface, such as an exosome marker, a microvesicle marker, an apoptotic body marker, or other membrane protein. The capture molecule may also be a molecule that binds to a disease-specific marker. The capture molecule is preferably a molecule that binds to a marker selected from the group consisting of an extracellular vesicle marker and a disease-specific marker.

[0013] When the capture molecule is a molecule that binds to an exosome marker and the detection molecule is a molecule that binds to any biomolecule, exosomes in each sample can be captured on a support, and the expression of any biomolecule present in the exosomes can be quantified.When the capture molecule is a molecule that binds to a marker for extracellular vesicles, such as a microvesicle marker or an apoptotic body marker, and the detection molecule is a molecule that binds to any biomolecule, the expression of any biomolecule present in extracellular vesicles, such as a microvesicle marker or an apoptotic body marker, can be quantified.

[0014] When the capture molecule is a molecule that binds to a disease-specific marker, the vesicles that have the disease-specific marker are captured on the support.When at least one of the detection molecules is a molecule that binds to the marker of each vesicle, the amount of disease-specific marker present in each vesicle can be quantified.When the capture molecule is a molecule that binds to a disease-specific marker, the accuracy of detecting the disease-specific marker on the vesicle may be improved compared to when the molecule that binds to the marker of the vesicle is used as the capture molecule.

[0015] Exosome markers include Alix, Tsg101, tetraspanins, flotillin, etc., and tetraspanins include CD9, CD63, CD81, etc. Microvesicle markers include integrins, selectins, CD40, etc. Apoptotic body markers include phosphatidylserine, annexin V, etc. Disease-specific markers include carcinoma embryonic antigen (CEA), prostate specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), etc. The capture molecule may be an antibody that recognizes these proteins. When capturing exosomes more selectively, the capture molecule is preferably selected from the group consisting of anti-CD9 antibodies, anti-CD63 antibodies, and anti-CD81 antibodies.

[0016] The support may be a multiwell plate or beads. Using a multiwell plate (e.g., a 96-well plate) as the support can improve analytical throughput. The method for immobilizing the capture molecule on the support is not particularly limited, and immobilization may be achieved by chemical bonding, hydrogen bonding, ionic bonding, complex formation, hydrophobic interaction, van der Waals interaction, electrostatic interaction, stereoselective interaction, or the like. When the capture molecule is an antibody, the capture molecule can also be immobilized on the support by using a support on which a linker (e.g., Protein A, Protein G) that site-specifically binds to the Fc domain of the antibody is immobilized.

[0017] For example, when immobilizing the capturing molecules by adding an aqueous solution containing the capturing molecules to a multi-well plate, the concentration of the capturing molecules in the aqueous solution may be 1 to 10 μg / mL, or may be 0.1 to 1 μg / mL.

[0018] The aqueous solution in which the capture molecules are dissolved is preferably a buffer solution. Examples of the buffer solution include PBS, HEPES buffer, acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, and Tris buffer. The aqueous solution may contain a salt. The concentration of the salt contained in the aqueous solution is preferably 150 mM or less, more preferably 10 mM to 150 mM, and even more preferably 15 mM to 100 mM. Examples of the salt include sodium chloride (NaCl), potassium chloride (KCl), and calcium chloride (CaCl2). The pH of the aqueous solution is not particularly limited as long as the capture molecules can be stably present, but is preferably, for example, 6 to 10.

[0019] In one aspect of this embodiment, the capturing molecules are preferably brought into contact with the multiwell plate (support) in an environment of 20° C. or higher and 30° C. or lower. In other words, the temperature of the aqueous solution is preferably 20° C. or higher and 30° C. or lower.

[0020] In one aspect of this embodiment, the time for which the capturing molecules are brought into contact with the support may be, for example, 0.5 hours or more and 3 hours or less, or 1 hour or more and 2 hours or less.

[0021] In another aspect of this embodiment, the support on which the capturing molecules are immobilized may be washed with PBS or the like to remove excess capturing molecules. The support on which the capturing molecules are immobilized may also be subjected to a blocking treatment with a blocking agent. Examples of blocking agents include BSA, Lipidure BL802 (manufactured by NOF Corporation), and casein. The concentration of the blocking agent is preferably 1% by weight or more and 10% by weight or less, and more preferably 2% by weight or more and 5% by weight or less.

[0022] When the support is a multi-well plate, a sample containing vesicles can be added to wells on which capture molecules are immobilized, bringing the capture molecules and vesicles into contact. When the support is beads, a sample containing vesicles can be added to beads on which capture molecules are immobilized or a suspension containing the beads, and stirred to bring the capture molecules and vesicles into contact. When the support is a packing material provided in a column, a sample containing vesicles can be poured into the column. After bringing the capture molecules and vesicles into contact, the support can be washed. Unbound vesicles and non-specifically bound vesicles can be removed by washing.

[0023] Two or more types of capture molecules may be immobilized on one support (e.g., one well of a multiwell plate), or one type of capture molecule may be immobilized on one support. In one embodiment, one type of capture molecule may be immobilized on one support and another type of capture molecule may be immobilized on another support, and the same sample may be contacted with each capture molecule to capture vesicles on each support.

[0024] In this embodiment, the amount of the capturing molecules immobilized on the support is preferably 0.01 to 2 μg, more preferably 0.01 to 1 μg, even more preferably 0.01 to 0.5 μg, and even more preferably 0.01 to 0.1 μg.

[0025] Vesicles may be derived from cultured cells, cultured tissues, or their culture supernatants, but are preferably derived from a living organism. A sample derived from a living organism (biological sample) may be blood obtained from a subject, or may be biological tissue, urine, feces, sweat, saliva, lymph, amniotic fluid, breast milk, pleural effusion, ascites, cerebrospinal fluid, tears, etc. Blood may be whole blood, but is preferably plasma or serum. The sample may be diluted with physiological saline, buffer solution, etc. Biological samples may be collected by any known method appropriate for the type of biological sample. The subject may be a human or a non-human animal. When vesicles are derived from a living organism, the type and amount of biomolecules present in the vesicles in the living organism can be measured.

[0026] Vesicle-containing samples may be appropriately purified or pretreated, and may be subjected to procedures such as filtration, centrifugation, ultracentrifugation, affinity separation, dialysis, and precipitation. Vesicle-containing samples may be purified vesicle fractions or purified exosome fractions. When vesicle-containing samples are purified exosome fractions, contaminants in the sample can be reduced, improving measurement accuracy. Exosome purification can be performed using known methods such as commercially available purification kits, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and filtration. On the other hand, the method of the present invention makes it possible to selectively capture and concentrate vesicles from vesicle-containing samples and quantify biomolecules contained in the vesicles, without the need for vesicle purification such as ultracentrifugation.

[0027] <Step of Contacting First Detection Molecules Labeled with First Metal Particles with Vesicles and Step of Contacting Second Detection Molecules Labeled with Second Metal Particles with Vesicles> The following describes the steps of contacting vesicles with first detection molecules labeled with first metal particles and second detection molecules labeled with second metal particles. These steps bind the first and second metal particles to the vesicles. The steps of contacting vesicles with the first detection molecules and second detection molecules may be performed sequentially or simultaneously. Specifically, the first and second detection molecules may be contacted sequentially with the vesicles on the support, or the first and second detection molecules may be premixed and then contacted with the vesicles on the support. After contacting the detection molecules with the vesicles, the support may be washed. Excess detection molecules and nonspecifically bound detection molecules can be removed by washing.

[0028] In the present invention, at least two types of detection molecules are used. Three or more, four or more, five or more, or ten or more types of detection molecules may be used, or 50 or fewer types may be used. When three or more types of detection molecules are used, the third or subsequent detection molecules may be contacted with the vesicles simultaneously with the first or second detection molecules, or may be contacted with the vesicles in a step separate from the step of contacting the first or second detection molecules with the vesicles.

[0029] In the present invention, the step of contacting the detection molecule with the vesicles and the step of contacting the capture molecule immobilized on the support with the vesicles may be performed in either order. When the step of contacting the capture molecule with the vesicles is performed first, for example, a sample containing vesicles may be added to the support, and a solution containing the detection molecule may be added to the vesicles captured on the support. When the step of contacting the vesicles with the detection molecule is performed first, for example, a sample containing vesicles may be mixed with the detection molecule, and the resulting solution may be added to the support. The first and second detection molecules may be contacted with vesicles on one support, or the first and second detection molecules may be contacted with vesicles on multiple supports, respectively.

[0030] In this specification, the first and second detection molecules may be collectively referred to as detection molecules. The same applies when three or more detection molecules are used. The detection molecule may be a molecule that binds to any biomolecule, including proteins, nucleic acids, glycans, etc. The detection molecule may include, for example, a molecule that binds to a biomolecule known to be present in vesicles, or a molecule that binds to a biomolecule that is not commonly present in vesicles or a biomolecule that is not normally present in vesicles. When the biomolecule is a protein, the biomolecule may be a membrane protein, a cell adhesion molecule, a receptor protein, a protein involved in membrane fusion or scission, a major histocompatibility complex (MHC) molecule, a heat shock protein, a disease-specific marker protein, etc. The biomolecule may be a glycoprotein or a glycan. When the biomolecule is a nucleic acid, the biomolecule may be DNA or RNA, and the RNA may include messenger RNA, microRNA, non-coding RNA, etc.

[0031] The detection molecules may be proteins (antibodies, lectins, etc.) or nucleic acid molecules (aptamers, nucleic acid molecules with complementary sequences, etc.). At least one of the detection molecules may be a molecule that binds to a biomolecule contained in the vesicles, such as an extracellular vesicle marker (e.g., an exosome marker, a microvesicle marker, an apoptotic body marker), other membrane proteins, or glycans. At least one of the detection molecules may be a molecule that binds to a disease-specific marker. At least one of the detection molecules (at least one selected from the first detection molecule and the second detection molecule) may be a molecule that binds to a marker selected from the group consisting of an extracellular vesicle marker and a disease-specific marker. When at least one of the detection molecules is a molecule that binds to an exosome marker, the type of vesicles captured on the support can be identified, for example, whether they are exosomes. For example, whether a vesicle is an exosome can be determined by confirming the presence or absence of multiple exosome markers. Multiple exosome markers on the vesicles may be detected using a combination of a capture molecule and a detection antibody, or a combination of at least two detection antibodies. When at least one of the detection molecules is a molecule that binds to a disease-specific marker, the disease-specific marker present in the vesicles captured on the support can be quantified.

[0032] The first detection molecule and the second detection molecule are different molecules. The first detection molecule and the second detection molecule may be molecules that bind to the same biomolecule or may be molecules that bind to different biomolecules. One of the detection molecules may be the same molecule as the capture molecule. At least one of the detection molecules may bind to the same molecule as the molecule to which the capture molecule binds. At least one of the detection molecules may bind to a molecule different from the molecule to which the capture antibody binds. In one aspect of this embodiment, the detection molecule may be a molecule different from the capture molecule.

[0033] For example, when the capture molecule is an antibody against a surface antigen of the extracellular vesicle, at least one of the detection molecules may be an antibody against a surface antigen of the extracellular vesicle, and at least one of the detection molecules may be an antibody against a disease-specific marker protein. For example, when the capture molecule is an antibody against a disease-specific marker protein, at least one of the detection molecules may be an antibody against a surface antigen of the extracellular vesicle.

[0034] The multiple detection molecules are each labeled with a different metal particle. That is, the first metal particle and the second metal particle each contain a different metal element. The amount of each metal can be measured simultaneously, and the amount of detection molecules bound to the metal particles can be evaluated. Metal elements contained in the metal particles include gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), ruthenium (Ru), aluminum (Al), copper (Cu), tellurium (Te), bismuth (Bi), lead (Pb), iron (Fe), cerium (Ce), molybdenum (Mo), niobium (Nb), tungsten (W), antimony (Sb), tin (Sn), vanadium (V), manganese (Mn), nickel ( Examples of suitable metals include nickel (Ni), cobalt (Co), zinc (Zn), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y), and titanium (Ti). From the standpoint of material stability and ease of synthesis, at least one of the detection molecules preferably contains a metal element selected from gold, platinum, iridium, palladium, silver, and copper, and is preferably composed of any of these metal elements. In one aspect of this embodiment, the first metal particle and the second metal particle may each be a simple metal or an alloy of two or more metal elements.

[0035] For example, if the first metal particle is an alloy made of metal element A and metal element B, and the second metal particle is an alloy made of metal element A and metal element C, the amount of metal element A corresponds to the total amount of the first detection molecule and the second detection molecule, and the amounts of metal element B and metal element C correspond to the amounts of the first detection molecule and the second detection molecule, respectively. As will be described later, in the analysis method according to this embodiment, the first metal particle and the second metal particle are simultaneously quantified by ICP mass spectrometry or the like, making it possible to simultaneously estimate the total amount of the first detection molecule and the second detection molecule, the amount of the first detection molecule, and the amount of the second detection molecule, and further to correct the amount of the first metal particle and the amount of the second metal particle using the total amount of the first detection molecule and the second detection molecule.

[0036] The metal particles may be metal nanoparticles, metal nanorods, or metal nanoplates. The metal particles are preferably small so as not to inhibit the binding between the vesicles and the detection molecules. The particle diameter (the length of the largest part of the particle) of the metal particles is typically 10 nm or more and 500 nm or less, preferably 200 nm or less, and more preferably 100 nm or less. When the metal particles are approximately spherical, the diameter may be 500 nm or less, 200 nm or less, or 100 nm or less. The lower limit of the diameter may be 10 nm or more. When the metal particles are metal nanorods, the length of their minor axis may be 100 nm or less, 50 nm or less, or 10 nm or less, and the length of their major axis may be 500 nm or less, 200 nm or less, or 100 nm or less. The lower limit of the length of the minor axis may be 2 nm or more. The lower limit of the length of the major axis may be 5 nm or more. When the metal particles are metal nanoplates, their thickness may be 100 nm or less, 50 nm or less, or 30 nm or less, and the maximum planar length may be 500 nm or less, 200 nm or less, or 100 nm or less. The lower limit of the thickness may be 4 nm or more. The lower limit of the maximum planar length may be 10 nm or more. The size of the metal particles may be measured, for example, by observation with a scanning electron microscope (SEM), a scanning transmission electron microscope (STEM), or a transmission electron microscope (TEM). An example of an apparatus for measurement is the SU-70 ultra-high resolution analytical scanning electron microscope (manufactured by Hitachi, Ltd.). The maximum diameter may also be measured using a dynamic light scattering particle size analyzer (DLS). An example of an apparatus for measurement is the ELSZ-2000ZS Zeta Potential / Particle Size / Molecular Weight Measurement System (manufactured by Otsuka Electronics Co., Ltd.). Other maximum diameter measurement devices include the SALD-7500nano laser diffraction particle size distribution analyzer (Shimadzu Corporation) and the NanoSight LM10 nanoparticle analysis system (Malvern Panalytical). When measuring the maximum diameter of anisotropic gold nanoparticles such as metal nanorods and metal nanoplates from SEM, STEM, or TEM photographs, the maximum diameter of any 125 anisotropic gold nanoparticles may be measured, and 100 data points (excluding the top and bottom 10%) may be prepared from the total of 125 data points, and the maximum diameter may be calculated by averaging these data points.

[0037] In this embodiment, the surfaces of the metal particles may be coated with a surfactant. This provides stability to the metal particles in the aqueous solution. In one aspect of this embodiment, the aqueous solution may contain a dispersant to improve the stability of the metal particles in the aqueous solution. Examples of dispersants include sodium citrate.

[0038] In this embodiment, the method for labeling the detection molecule with the metal particle is not particularly limited, and any known method may be used. For example, a chemical modification method using a functional group such as maleimide, a physical adsorption method, etc. In addition, the detection molecule labeled with the metal particle may be purchased as a commercially available product.

[0039] In this embodiment, the first and second detection molecules are preferably contacted with the vesicles in an aqueous solution containing a salt. The concentration of the salt contained in the aqueous solution is preferably 150 mM or less, more preferably 15 mM to 150 mM, even more preferably 20 mM to 150 mM, and even more preferably 20 mM to 100 mM. Examples of the salt include sodium chloride (NaCl), potassium chloride (KCl), and calcium chloride (CaCl). In this case, the concentration of the detection molecule is preferably such that, when 100 μL of the solution is measured using a 96-well plate with a plate reader (Infinite M-200 pro, TECAN), the absorbance at the maximum absorption wavelength is 0.01 to 2, more preferably 0.5 to 1.5. If the vesicle does not have a maximum absorption wavelength, the absorbance at an absorption wavelength of 550 nm is used.

[0040] In one aspect of this embodiment, the aqueous solution containing the salt is preferably a buffer solution. Examples of the buffer solution include PBS, HEPES buffer, acetate buffer, acetate-phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, and Tris buffer. The pH of the aqueous solution containing the salt is not particularly limited as long as the detection molecule can be stably present therein, but is preferably between 6 and 10.

[0041] To stabilize the detection molecule, the salt-containing aqueous solution may further contain a blocking agent. Examples of blocking agents include BSA, Lipidure BL802 (NOF Corporation), and casein. The concentration of the blocking agent is preferably 1% by weight or more and 10% by weight or less, and more preferably 2% by weight or more and 5% by weight or less.

[0042] In another aspect of this embodiment, the first detection molecule and the second detection molecule are preferably contacted with the vesicles in an environment of 20° C. or higher and 30° C. or lower. In other words, the temperature of the salt-containing aqueous solution is preferably 20° C. or higher and 30° C. or lower.

[0043] In this embodiment, the time for which the detection molecule is brought into contact with the vesicles is not particularly limited as long as the interaction between the detection molecule and the vesicles reaches an equilibrium state, but may be, for example, 0.5 hours or more and 3 hours or less, or 1 hour or more and 2 hours or less.

[0044] <Step of quantifying the first metal particles and the second metal particles bound to the vesicles by inductively coupled plasma mass spectrometry or ICP atomic emission spectrometry> The following describes a process for quantifying the first and second metal particles bound to vesicles using inductively coupled plasma mass spectrometry (ICP-MS) or ICP atomic emission spectrometry (ICP-AES / ICP-OES). The metal particles bound to vesicles via the detection molecules are first dissolved and extracted using an acid such as aqua regia (a mixture of concentrated nitric acid and concentrated hydrochloric acid), nitric acid, hydrochloric acid, or sulfuric acid. After the metal particles are separated from the support using a stripping solution, the metal particles contained in the stripping solution may be dissolved using an acid. The concentration of the aqua regia (concentrated hydrochloric acid:concentrated nitric acid = 3:1) may be 50% or higher. The temperature for dissolving the metal particles may be, for example, 25°C or higher, and the reaction time may be 10 minutes to 24 hours. From the perspective of highly sensitive detection of metal particles, it is preferable to dissolve the metal particles using aqua regia.

[0045] When the first detection molecule and the second detection molecule are bound to one support, a solution containing multiple metal elements can be obtained by acid treatment. When the first detection molecule is bound to one support and the second detection molecule is bound to another support, the solutions containing multiple metal elements obtained by acid treatment can be mixed to obtain a solution containing multiple metal elements.

[0046] A solution containing multiple metal elements is introduced into an ICP-MS or ICP-AES instrument. In the case of ICP-MS, the metal elements in the solution are ionized by inductively coupled plasma and separated by an electric or magnetic field, and the concentration of each element is quantified. In the case of ICP-AES, the concentration of each element is quantified from the wavelength and intensity of light emitted from the elements excited by the plasma. The inductively coupled plasma can be argon gas plasma.

[0047] The amount of quantified metal ions correlates with the amount of biomolecules present in the vesicles. ICP-MS or ICP-AES can simultaneously quantify the amounts of multiple metal ions, allowing for highly sensitive simultaneous quantification of multiple biomolecules present in the vesicles.

[0048] An example of an ICP-MS device is ICPMS-2030 (manufactured by Shimadzu Corporation), etc. An example of an ICP-AES device is ICPE-2030 (manufactured by Shimadzu Corporation).

[0049] [Diagnostic aid method] A diagnostic assistance method according to one embodiment of the present invention includes a step of providing information for diagnosing a disease based on the type and amount of biomolecules present in vesicles obtained by the above-described analytical method.

[0050] The diagnostic assistance method according to the present invention makes it possible to easily provide information for diagnosing a disease. The diagnostic assistance method according to the present invention does not include a judgment step by a doctor and does not fall under the category of a method for diagnosing a human. The diagnostic assistance method according to the present invention can provide information for diagnosing a disease in a non-invasive manner.

[0051] The diagnostic assistance method of the present invention may include a method for analyzing biomolecules present in the above-mentioned vesicles and a step of providing information for diagnosing a disease based on the type and amount of biomolecules present in the vesicles obtained by the above-mentioned analysis method.

[0052] Specific biomolecules present in vesicles obtained from biological samples, such as extracellular vesicle markers, disease-specific markers, and / or combinations thereof, can be used as diagnostic markers for diseases. Disease-specific markers can be biomolecules such as nucleic acids (e.g., miRNA), lipids, and proteins. Diseases include cancers such as lung cancer, colon cancer, pancreatic cancer, cervical cancer, breast cancer, and glioblastoma, as well as lifestyle-related diseases, neuropsychiatric disorders, immune / allergic disorders, and infectious diseases. [Example]

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0054] (Experiment 1) The culture supernatant of cultured cells (MCF7) was filtered through a 0.22 μm filter, and the filtrate was ultracentrifuged (100,000 g, 70 min) to prepare an exosome fraction. The obtained exosome fraction was suspended in PBS and diluted to 1.0 × 10 10 , 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 Samples containing either 0 or 0 (blank) exosome particles were prepared. Particle counts were detected using NanoSight (Japan Quantum Design Co., Ltd.). Detection molecules were prepared by metal-labeling anti-CD9 antibodies with gold (Au) particles (40 nm diameter) (Au-CD9 Ab) and anti-CD81 antibodies with palladium (Pd) particles (100 nm diameter) (Pd-CD81 Ab).

[0055] A sample containing exosomes was added to each well on which anti-CD9 antibody had been immobilized, and the exosomes were captured on the support. Au-CD9 Ab solution and Pd-CD81 Ab solution were added to each well, respectively, and the surface of the captured exosomes was stained with the antibody. Excess antibody was removed by washing. Aqua regia was added to each well to elute the metal particles bound to the exosomes via the antibody. The solution containing the metal particles was subjected to ICP-MS (ICPMS-2030, Shimadzu Corporation) to quantify Au and Pd ions. This allows for quantification of surface antigens on exosomes.

[0056] The amount of Au ions was quantified for each exosome particle number, and when the amount of Au ions was plotted against the number of exosome particles, it was confirmed that the amount of Au ions increased in a highly correlated manner with the number of exosome particles (Figure 1). Similarly, when the amount of Pd ions was quantified for each exosome particle number, and when the amount of Pd ions was plotted against the number of exosome particles, it was confirmed that the amount of Pd ions increased in a highly correlated manner with the number of exosome particles (Figure 2). 10 10 Surface antigens could be quantified with high sensitivity in exosomes with particle numbers less than 10 ...

[0057] This method demonstrates that biomolecules in vesicles can be quantified using various antibodies (detection molecules) labeled with various metal particles, suggesting that the method of the present invention can simultaneously quantify any biomolecules in vesicles in a sample.

[0058] (Experiment 2) The culture supernatant of cultured cells (MCF7) was filtered through a 0.22 μm filter, and the filtrate was ultracentrifuged (100,000 g, 70 min) to prepare an exosome fraction. The obtained exosome fraction was suspended in PBS and diluted to 1.0 × 10 10 , 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 Samples containing 0 (blank) or 100 μl of exosome particles were prepared. The particle counts were measured using a NanoSight (Quantum Design Japan Co., Ltd.). The following detection molecules were prepared: anti-CD9 antibody labeled with gold (Au) particles (40 nm diameter) (Au-CD9 Ab) (concentration: absorbance at 530 nm of 0.1); anti-CD63 antibody labeled with platinum (Pt) particles (30 nm diameter) (Pt-CD63 Ab) (concentration: absorbance at 500 nm of 0.1); and anti-CD81 antibody labeled with palladium (Pd) particles (100 nm diameter) (Pd-CD81 Ab) (concentration: absorbance at 500 nm of 0.1).

[0059] Next, anti-CD9 antibodies (capture molecules) were immobilized on a 96-well plate as follows: First, 100 μl of a solution containing anti-CD9 antibodies (concentration: absorbance at 530 nm of 0.1) was added per well and incubated at 4°C for 12 hours. Next, 200 μl of a blocking agent was added per well and incubated at room temperature for 2 hours. Then, 100 μl of a sample containing exosomes was added to each well with the immobilized anti-CD9 antibodies, and the exosomes were captured on the support. Then, 100 μl of an equal mixture of Au-CD9 Ab solution, Pt-CD63 Ab solution, and Pd-CD81 Ab solution was added to each well, and the surface of the captured exosomes was stained with antibodies. Each of the metal-conjugated antibodies was suspended in an aqueous solution containing 10 mM HEPES (pH 7.5), 1% BSA, 150 mM NaCl, and 0.05% Lipidure BL802 (referred to as "150 mM NaCl") or 10 mM HEPES (pH 7.5), 1% BSA, 20 mM NaCl, and 0.05% Lipidure BL802 (referred to as "20 mM NaCl"). After reaction with the metal-conjugated antibody, excess antibody was removed by washing with a 10 mM HEPES (pH 7.5), 20 mM NaCl aqueous solution. 300 μl of aqua regia was added to each well to elute the metal particles bound to exosomes via the antibody. The solution containing the metal particles was diluted 18 times with water and subjected to ICP-MS (ICPMS-2030, Shimadzu Corporation) to quantify Au ions, Pt ions, and Pd ions. This allowed the quantification of surface antigens on exosomes.

[0060] The amount of Au, Pt, and Pd ions was quantified for each exosome particle number, and the ratio of the amount of detected ions for each exosome particle number to the amount of detected ions for the blank (S / N ratio) was calculated (Table 1). 10 , and 10 9In the analysis of exosome particle numbers, the S / N ratio was improved when the antibody reaction was performed in a 20 mM NaCl aqueous solution compared to when the antibody reaction was performed in a 150 mM NaCl aqueous solution (Table 1). Salt is typically used to stabilize antibody reactions and metal particle dispersion. This indicates that antibody reactions in aqueous solutions with low salt concentrations can yield better data in mass spectrometry of exosomes.

[0061] [Table 1]

[0062] (Experiment 3) The culture supernatant of cultured cells (MCF7) was filtered through a 0.22 μm filter, and the filtrate was ultracentrifuged (100,000 g, 70 min) to prepare an exosome fraction. The obtained exosome fraction was suspended in PBS and diluted to 1.0 × 10 10 , 1.0×10 9 , 1.0×10 8 , 1.0×10 7 , 1.0×10 6 Samples containing 0 (blank) or 100 μl of exosome particles were prepared. The particle counts were measured using a NanoSight (Quantum Design Japan Co., Ltd.). The following detection molecules were prepared: anti-CD9 antibody labeled with gold (Au) particles (40 nm diameter) (Au-CD9 Ab) (concentration: absorbance at 530 nm of 0.1); anti-CD63 antibody labeled with platinum (Pt) particles (30 nm diameter) (Pt-CD63 Ab) (concentration: absorbance at 500 nm of 0.1); and anti-CD81 antibody labeled with palladium (Pd) particles (100 nm diameter) (Pd-CD81 Ab) (concentration: absorbance at 500 nm of 0.1).

[0063] Next, anti-CD9 antibodies (capture molecules) were immobilized on a 96-well plate using the same procedure as in Experiment 2. Then, 100 μl of the exosome-containing sample was added to each well, allowing the exosomes to be captured on the support. An equal mixture of Au-CD9 Ab solution, Pt-CD63 Ab solution, and Pd-CD81 Ab solution (100 μl each) was added to each well, and the surface of the captured exosomes was stained with the antibodies. Each of the aforementioned metal-conjugated antibodies was suspended in an aqueous solution containing 10 mM HEPES (pH 7.5), 1% BSA, 20 mM NaCl, and 0.05% Lipidure BL802. After reaction with the metal-conjugated antibodies, excess antibodies were removed by washing with a 10 mM HEPES (pH 7.5), 20 mM NaCl aqueous solution. 300 μl of aqua regia was added to each well to elute the metal particles bound to the exosomes via the antibodies. The solution containing the metal particles was diluted 18 times with water and subjected to ICP-MS (ICPMS-2030, Shimadzu Corporation) to quantify Au, Pt, and Pd ions. This allowed the quantification of surface antigens on exosomes.

[0064] The amounts of Au, Pt, and Pd ions were quantified for each exosome particle number, and the amounts of the three ions were plotted against the number of exosome particles. It was confirmed that the amount of all three metals increased in an exosome particle number-dependent manner with a high correlation (coefficient of determination R2 > 0.99) (Figure 3).

[0065] (Experiment 4) MCF7 cells were cultured for 48 hours. Culture supernatants were collected at 0, 12, 24, 36, and 48 hours after the start of culture and filtered through a 0.22 μm filter. The following detection molecules were prepared: anti-CD9 antibody labeled with gold (Au) particles (40 nm diameter) (Au-CD9 Ab) (concentration: absorbance at 530 nm: 0.1); anti-CD63 antibody labeled with platinum (Pt) particles (30 nm diameter) (Pt-CD63 Ab) (concentration: absorbance at 500 nm: 0.1); and anti-CD81 antibody labeled with palladium (Pd) particles (100 nm diameter) (Pd-CD81 Ab) (concentration: absorbance at 500 nm: 0.1).

[0066] Next, anti-CD9 antibodies (capture molecules) were immobilized on a 96-well plate using the same procedure as in Experiment 2. Then, 100 μl of the aforementioned culture supernatant sample was added to each well containing the immobilized anti-CD9 antibody, allowing exosomes to be captured on the support. An equal mixture of Au-CD9 Ab, Pt-CD63 Ab, and Pd-CD81 Ab was added to each well, and the surface of the captured exosomes was stained with antibodies. Each of the aforementioned metal-conjugated antibodies was suspended in an aqueous solution containing 10 mM HEPES (pH 7.5), 1% BSA, 20 mM NaCl, and 0.05% Lipidure BL802. After reaction with the metal-conjugated antibodies, excess antibody was removed by washing. 300 μl of aqua regia was added to each well to elute the metal particles bound to exosomes via the antibodies. The solution containing the metal particles was subjected to ICP-MS (ICPMS-2030, Shimadzu Corporation) to quantify Au, Pt, and Pd ions, thereby quantifying the surface antigens on exosomes.

[0067] The culture supernatant collected at each culture time was pretreated as described above, and the amounts of Au, Pt, and Pd ions in the samples were quantified. When the amounts of the three ions for each sample were plotted, it was confirmed that the amounts of all three metals increased in a culture time-dependent manner (Figure 4). This suggests that this method can be used to monitor the amount of exosome secretion during cell culture.

[0068] (Experiment 5) Serum samples (10 μL, 50 μL, and 100 μL) were analyzed with Tween-20 added to a final concentration of 0.05%. The following detection molecules were prepared: anti-CD9 antibody labeled with gold (Au) particles (40 nm diameter) (Au-CD9 Ab) (concentration: absorbance at 530 nm: 0.1); anti-CD63 antibody labeled with platinum (Pt) particles (30 nm diameter) (Pt-CD63 Ab) (concentration: absorbance at 500 nm: 0.1); and anti-CD81 antibody labeled with palladium (Pd) particles (100 nm diameter) (Pd-CD81 Ab) (concentration: absorbance at 500 nm: 0.1).

[0069] Next, anti-CD9 antibodies (capture molecules) were immobilized on a 96-well plate using the same procedure as in Experiment 2. Then, 10 μL, 50 μL, or 100 μL of the serum samples described above were added to each well containing the immobilized anti-CD9 antibodies, allowing exosomes to be captured on the support. An equal mixture of Au-CD9 Ab solution, Pt-CD63 Ab solution, and Pd-CD81 Ab solution (100 μL) was added to each well, and the surface of the captured exosomes was stained with antibodies. Each of the metal-conjugated antibodies was suspended in an aqueous solution containing 10 mM HEPES (pH 7.5), 1% BSA, 20 mM NaCl, and 0.05% Lipidure BL802. After reaction with the metal-conjugated antibodies, excess antibody was removed by washing with a 10 mM HEPES (pH 7.5), 20 mM NaCl aqueous solution. 300 μL of aqua regia was added to each well to elute the metal particles bound to exosomes via antibodies. The solution containing the metal particles was diluted 18-fold with water and subjected to ICP-MS (ICPMS-2030, Shimadzu Corporation) to quantify Au, Pt, and Pd ions. This allowed the quantification of surface antigens on exosomes.

[0070] The amounts of Au, Pt, and Pd ions in samples of each serum volume were quantified after pretreatment as described above, and the amounts of the three ions for each sample were plotted. It was confirmed that the amounts of all three metals increased depending on the analytical sample volume (Figure 5). This suggests that this method can be applied to serum samples of at least 10 μL or more.

[0071] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0072] (Section 1) A method for analyzing biomolecules present in vesicles according to one embodiment includes the steps of: contacting the vesicles with capture molecules immobilized on a support; contacting the vesicles with a first detection molecule labeled with a first metal particle; contacting the vesicles with second detection molecules labeled with second metal particles; quantitating the first and second metal particles bound to the vesicles by inductively coupled plasma (ICP) mass spectrometry or ICP optical emission spectrometry; Includes.

[0073] According to the analytical method described in paragraph 1, multiple biomolecules present in vesicles can be simultaneously quantified.

[0074] (Section 2) In the analytical method described in item 1, the first metal particles and the second metal particles each contain a different metal element.

[0075] According to the analytical method described in paragraph 2, the amount of each metal can be measured.

[0076] (Section 3) In the analytical method described in paragraph 1 or 2, the first detection molecule and the second detection molecule are each contacted with the vesicle in an aqueous solution containing a salt. According to the analytical method described in paragraph 3, multiple biomolecules present in the vesicles can be accurately quantified.

[0077] (Section 4) In the analytical method according to any one of items 1 to 3, the capture molecule is a molecule that binds to a marker selected from the group consisting of an extracellular vesicle marker and a disease-specific marker.

[0078] According to the analytical method described in paragraph 4, when the capture molecule binds to the extracellular vesicle marker, the extracellular vesicles can be captured on the support. When the capture molecule binds to the disease-specific marker, vesicles containing the disease-specific marker are captured on the support.

[0079] (Section 5) In the analytical method according to item 4, the capture molecule is selected from the group consisting of an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody.

[0080] According to the analytical method described in item 5, exosomes can be captured on a support.

[0081] (Section 6) In the analysis method according to any one of items 1 to 5, at least one selected from the first detection molecule and the second detection molecule is a molecule that binds to a biomolecule contained in a vesicle.

[0082] The analytical method described in paragraph 6 allows for the detection and quantification of any molecule contained in the vesicles.

[0083] (Section 7) In the analysis method described in item 6, at least one selected from the first detection molecule and the second detection molecule is a molecule that binds to a marker selected from the group consisting of an extracellular vesicle marker and a disease-specific marker.

[0084] The analytical method described in item 7 allows for identification of the type of vesicles, and also allows for quantification of the expression of disease-specific markers in vesicles.

[0085] (Section 8) In the analytical method described in any one of items 1 to 7, at least one selected from the first metal particles and the second metal particles contains a metal element selected from the group consisting of gold, platinum, iridium, palladium, silver, and copper.

[0086] The metal elements described in item 8 are excellent in stability and ease of synthesis.

[0087] (Section 9) In the analysis method according to any one of items 1 to 8, the particle diameters of the first metal particles and the second metal particles are 500 nm or less.

[0088] According to the analytical method described in item 9, the binding between the vesicles and the detection molecules is less likely to be inhibited.

[0089] (Section 10) In the analysis method according to any one of items 1 to 9, the vesicles are derived from a living organism.

[0090] According to the analytical method described in item 10, the types and amounts of biomolecules present in vesicles derived from a living organism can be measured.

[0091] (Section 11) In the analysis method according to any one of items 1 to 10, the vesicles are exosomes.

[0092] According to the analytical method described in item 11, it is possible to quantify the biomolecules contained in exosomes, and the method can be used to detect and diagnose various diseases, including cancer and lifestyle-related diseases.

[0093] (Section 12) In the analytical method according to any one of items 1 to 11, the support is a multiwell plate.

[0094] According to the analytical method described in paragraph 12, the analytical throughput can be improved.

[0095] (Section 13) The analytical method according to any one of items 1 to 12, further comprising dissolving at least one of the first metal particles and the second metal particles bound to the vesicles with aqua regia.

[0096] According to the analytical method described in item 13, metal particles can be detected with high sensitivity, and the detection sensitivity of biomolecules contained in vesicles can be improved.

[0097] (Section 14) A diagnostic assistance method according to one embodiment includes providing information for diagnosing a disease based on the types and amounts of biomolecules present in vesicles obtained by the analytical methods described in paragraphs 1 to 13.

[0098] According to the diagnostic assistance method described in item 14, information for diagnosing a disease can be provided in a non-invasive manner.

Claims

1. contacting the vesicles with capture molecules immobilized on a support; contacting the vesicles with a first detection molecule labeled with a first metal particle; contacting the vesicles with second detection molecules labeled with second metal particles; quantitating the first and second metal particles bound to the vesicles by inductively coupled plasma (ICP) mass spectrometry or ICP optical emission spectrometry; A method for analyzing biomolecules present in vesicles comprising: contacting the first detection molecule with the vesicles and contacting the second detection molecule with the vesicles may be performed sequentially or simultaneously; each of the first and second detection molecules is contacted with the vesicle in an aqueous solution containing a salt; The method, wherein the concentration of the salt contained in the aqueous solution is 150 mM or less.

2. The method of claim 1 , wherein the first metal particles and the second metal particles each comprise a different metal element.

3. A method described in claim 1 or 2, wherein the concentration of the salt contained in the aqueous solution is 20 mM or more and 100 mM or less.

4. The method of claim 1 or 2, wherein the capture molecule is a molecule that binds to a marker selected from the group consisting of an extracellular vesicle marker and a disease-specific marker.

5. The method of claim 4, wherein the capture molecule is selected from the group consisting of an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody.

6. The method of claim 1 or 2, wherein at least one selected from the first detection molecule and the second detection molecule is a molecule that binds to a biomolecule contained in the vesicle.

7. The method of claim 6, wherein at least one selected from the first detection molecule and the second detection molecule is a molecule that binds to a marker selected from the group consisting of an extracellular vesicle marker and a disease-specific marker.

8. 3. The method according to claim 1, wherein at least one selected from the first metal particles and the second metal particles comprises a metal element selected from the group consisting of gold, platinum, iridium, palladium, silver, and copper.

9. The method according to claim 1 or 2, wherein the particle diameter of the first metal particles and the second metal particles is 500 nm or less.

10. The method of claim 1 or 2, wherein the vesicles are derived from a living organism.

11. 3. The method of claim 1 or 2, wherein the vesicles are exosomes.

12. The method of claim 1 or 2, wherein the support is a multi-well plate.

13. 3. The method of claim 1, further comprising dissolving at least one of the first metal particles and the second metal particles bound to the vesicles with aqua regia.

14. A diagnostic aid method comprising providing information for diagnosing a disease based on the type and amount of biomolecules present in vesicles obtained by the method of claim 1 or 2.

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