Sensitization particle, target substance detection reagent, target substance detection kit, and target substance detection method

Abstract

To provide a sensitization particle that excels in dispersibility.SOLUTION: A sensitization particle according to the present invention includes, in at least a part of a surface of a core particle, a ligand and a blocking protein fixed by chemical bonding, and a blocking protein fixed by physical bonding. The surface unevenness of the sensitization particle measured using an atomic force microscope under a prescribed condition satisfies a condition that an average value of heights of protrusions is 10.0 nm or less and / or a ratio of particles having protrusions in height of 10 nm or greater is 40% or less.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to sensitized particles, a target substance detection reagent, a target substance detection kit, and a method for detecting a target substance. [Background technology]

[0002] A latex immunoagglutination method is known as a method for measuring a target substance in a sample. The latex immunoagglutination method is a measurement method in which, for example, latex particles carrying antibodies against a target substance are used, and the degree of agglutination (turbidity) of the latex particles caused by binding of the target substance, an antigen, to the antibody-carrying latex particles is detected by optical means or the like. Known examples of this type of technology include the technology described in Patent Document 1. Patent Document 1 describes a reagent for measuring a target substance in a sample using a latex immunoagglutination method, which includes latex particles carrying an antibody that specifically recognizes the target substance in the sample (claim 7 of Patent Document 1). Furthermore, Patent Document 1 describes that after reacting an antibody with latex particles, a blocking treatment is carried out using bovine serum albumin (BSA) (paragraph 0029 of Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2021-162593 Summary of the Invention [Problem to be solved by the invention]

[0004] In the blocking treatment and washing treatment described in Patent Document 1, it is known that blocking proteins such as BSA are physically adsorbed onto the surface of latex particles. However, as a result of investigations by the present inventors, it was found that there is room for improvement in terms of dispersibility of the sensitized latex particles after the blocking treatment described in Patent Document 1. [Means for solving the problem]

[0005] After further investigation, the inventors discovered that by introducing a blocking protein that chemically bonds to the surface of core particles such as latex particles, it is possible to appropriately control the surface irregularities of sensitized particles to which ligands are chemically bonded, thereby improving the dispersibility in the dispersion medium before the ligand and target substance bond, and thus completed the present invention.

[0006] According to one aspect of the present invention, there are provided the following sensitized particles, target substance detection reagent, target substance detection kit, and target substance detection method. 1. Sensitized particles having a ligand and a blocking protein immobilized by chemical bonds and a blocking protein immobilized by physical bonds on at least a portion of the surface of a core particle, Sensitized particles, wherein the surface irregularities of the sensitized particles measured using an atomic force microscope according to the measurement procedure below have an average height of convex portions of 10.0 nm or less, and / or the proportion of particles having convex portions with a height of 10 nm or more is 40% or less. <Surface roughness measurement procedure> Using an atomic force microscope under the following (observation conditions), four images are obtained for four particles contained in the sample obtained under the following (sample preparation). A one-dimensional profile is obtained in a direction parallel to the X-axis so as to pass through the brightest areas of each image, and the height of the bright areas is measured. The average value of the heights of the bright areas at four points is calculated as the average height of the convex portions. Furthermore, the number of particles having a convex portion with a height of 10 nm or more included in the one-dimensional profile is counted, and the percentage (%) of such particles in the total particles is calculated as the particle percentage. (Observation conditions) Cantilever (material: Si, spring constant: 0.1 N / m, tip curvature radius: 8 nm) Measurement atmosphere (23°C, HEPES aqueous solution (10 mM, pH 7.5)) ·Resolution: XY axis direction: 0.09nm, Z axis direction: 0.04nm (Sample preparation) 10 μL of a silane coupling agent having a primary amino group and 490 μL of methanol are placed in a plastic container with a cap and stirred with a vortex to prepare a surface treatment agent. The surface treatment agent is dropped onto the surface of a silicon wafer and left to stand for 3 minutes, after which the droplets are blown away with a blower and the wafer is left to stand in an oven at 120°C to perform the surface treatment on the silicon wafer surface. The dispersion containing the sensitized particles is then dropped onto the surface of the surface-treated silicon wafer and allowed to stand for 10 minutes.The surface of the silicon wafer is then washed with pure water, and the sensitized particles on the surface are stored in a HEPES aqueous solution (10 mM, pH 7.5). 2. Sensitized particles according to 1., The sensitized particle, wherein the diameter of the convex portions of the surface irregularities that are 10 nm or more in height is 35 nm or less. 3. Sensitized particles according to 1. or 2., Sensitized particles, in which the average distance from the particle center to the bright area in the image is 35 nm or less. 4. Sensitized particles according to any one of 1. to 3., Sensitized particles, wherein the average particle diameter of the particle group of the sensitized particles is 0.01 μm or more and 1.0 μm or less. 5. Sensitized particles according to any one of 1. to 4., The sensitized particle, wherein the core particle is an organic particle or an inorganic particle. 6. Sensitized particles according to any one of 1. to 5., The sensitized particle, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid. 7. Sensitized particles according to any one of 1. to 6., Sensitized particles used to detect target substances using agglutination methods. 8. A target substance detection reagent comprising the sensitized particles described in any one of 1. to 7. and a dispersion medium. 9. A target substance detection kit comprising a reagent container containing the target substance detection reagent described in 8. 10. A method for detecting a target substance, comprising the step of mixing a target substance with the target substance detection reagent described in 8. [Effects of the Invention]

[0007] According to the present invention, there are provided sensitized particles with excellent dispersibility, a target substance detection reagent, a target substance detection kit, and a target substance detection method. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a cross-sectional view schematically illustrating an example of the configuration of a sensitized particle according to the present embodiment. [Figure 2] 1 shows an AMF image of Example 1. [Figure 3] 1 shows a one-dimensional profile of Example 1. [Figure 4] 1 shows an AMF image of Example 2. [Figure 5] 1 shows a one-dimensional profile of Example 2. [Figure 6] 1 shows an AMF image of Comparative Example 1. [Figure 7] 1 shows a one-dimensional profile of Comparative Example 1. [Figure 8] 1 shows an AMF image of Comparative Example 2. [Figure 9] 1 shows a one-dimensional profile of Comparative Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all drawings, similar components are designated by similar reference numerals, and descriptions thereof will be omitted as appropriate. Furthermore, the drawings are schematic diagrams and do not correspond to actual dimensional proportions.

[0010] The sensitized particles of this embodiment will be outlined below.

[0011] The sensitized particle of this embodiment is a sensitized particle having a ligand and a blocking protein immobilized by chemical bonds and a blocking protein immobilized by physical bonds on at least a part of the surface of a core particle, The surface irregularities of the sensitized particles, measured using an atomic force microscope according to the measurement procedure below, are configured so that the average height of the convex portions is 10.0 nm or less, and / or the proportion of particles having convex portions with a height of 10 nm or more is 40% or less.

[0012] According to the findings of the present inventors, it has been found that in sensitized particles in which a ligand such as an antibody is formed on the surface of a core particle such as a latex particle (organic particle) or an inorganic particle, the surface unevenness of the sensitized particle can be appropriately controlled by introducing a blocking protein that chemically bonds to the surface of the core particle. As a result of further investigation based on this finding, it was found that the uniformity (smoothness) of surface unevenness can be stably evaluated by using the "average height of convex portions" or the "proportion of particles having convex portions with a height of 10 nm or more" as an index of surface unevenness, and that, in such indexes, by setting the "average height of convex portions" to a predetermined value or less and / or the "proportion of particles having convex portions with a height of 10 nm or more" to a predetermined value or less, it is possible to improve dispersibility in the dispersion medium before the ligand and target substance bind to each other.

[0013] Although the detailed mechanism is unclear, it is thought that by introducing a blocking protein (BSA) onto the surface of the sensitized particles through chemical bonding in addition to physical adsorption, the blocking protein (BSA) penetrates into the gaps between the antibodies (ligands) and is stably fixed, making the surface irregularities of the sensitized particles more uniform (smooth surface). This stably suppresses self-aggregation of the sensitized particles in a dispersion medium that does not contain the target substance, thereby improving dispersibility as described above.

[0014] The upper limit of the average height of the convex portions is preferably 9 nm or less, and more preferably 8 nm or less. The lower limit of the average height of the convex portions is not particularly limited, but may be about the size difference between the antibody and BSA, for example, 1 nm or more.

[0015] The upper limit of the proportion of particles having protrusions with a height of 10 nm or more is preferably 30% or less, and more preferably 25% or less. The lower limit of the proportion of particles having convex portions with a height of 10 nm or more is not particularly limited, but may be 0% or more.

[0016] In addition, the upper limit of the diameter of the convex portions with a height of 10 nm or more in the surface irregularities is, for example, 35 nm or less, preferably 30 nm or less, and more preferably 25 nm or less, which makes it possible to increase the sensitivity. The lower limit of the diameter of the protrusions having a height of 10 nm or more is not particularly limited, but may be about the size of BSA, for example, 3 nm or more.

[0017] In the image, the upper limit of the average distance from the particle center to the bright area is, for example, 35 nm or less, preferably 30 nm or less, and more preferably 25 nm or less, which makes it possible to increase the sensitivity. The lower limit of the average value of the distance from the particle center to the bright portion is not particularly limited, but may be, for example, 0 nm or more.

[0018] In this embodiment, the procedure for measuring the surface irregularities of sensitized particles is as follows. Using an atomic force microscope under the following (observation conditions), four images are obtained for four particles contained in the sample obtained under the following (sample preparation). A one-dimensional profile is obtained in a direction parallel to the X-axis so as to pass through the brightest areas of each image, and the height of the bright areas is measured. The average value of the heights of the bright areas at four points is calculated as the average height of the convex portions. Furthermore, the number of particles having a convex portion with a height of 10 nm or more included in the one-dimensional profile is counted, and the percentage (%) of such particles in the total particles is calculated as the particle percentage. (Observation conditions) Cantilever (material: Si, spring constant: 0.1 N / m, tip curvature radius: 8 nm) Measurement atmosphere (23°C, HEPES aqueous solution (10 mM, pH 7.5)) ·Resolution: XY axis direction: 0.09nm, Z axis direction: 0.04nm (Sample preparation) 10 μL of a silane coupling agent having a primary amino group (for example, KBM903 manufactured by Shin-Etsu Silicone Co., Ltd.) and 490 μL of methanol are placed in a plastic container with a cap and stirred with a vortex to prepare a surface treatment agent. The surface treatment agent is dropped onto the surface of a silicon wafer and left to stand for 3 minutes, after which the droplets are blown away with a blower and the wafer is left to stand in an oven at 120°C to perform the surface treatment on the silicon wafer surface. The dispersion containing the sensitized particles is then dropped onto the surface of the surface-treated silicon wafer and allowed to stand for 10 minutes.The surface of the silicon wafer is then washed with pure water, and the sensitized particles on the surface are stored in a HEPES aqueous solution (10 mM, pH 7.5). However, the number of images (number of particles to be observed) is not limited to four, and may be any number between five and ten.

[0019] The image obtained by the atomic force microscope is color-coded so that bright areas represent relatively high values ​​and dark areas represent relatively low values. In a one-dimensional profile passing through a bright area in an image containing one particle, if the height at the apex of the convexity is H and the heights of the two ends on either side of the convexity (there is a left end and a right end on either side of the apex) are L1 and L2, respectively, the "height of the convexity" in one image can be calculated as [(H-L1)+(H-L2)] / 2. Then, the average value is calculated using the "height of the convex portion" calculated from the four images. The end portion may be determined as the position where a certain degree of flatness appears when moving from the apex of the convex portion toward the end, or the position of the bottom of a concave portion that appears along the way.

[0020] The diameter of the convex portion refers to the distance between the left and right ends of one particle contained in one image. The number of particles in the four images that satisfy a "convex portion height" of 10.0 nm or more is counted, and the proportion of particles present out of the total number of four is calculated.

[0021] In addition, the distance from the particle center to the bright area is given by √{(X1-X2) 2 +(Y1-Y2) 2 The average distance is calculated using the measurement results of the four images. It is presumed that the distance from the particle center to the bright area increases because the bright area, i.e., the aggregated portion formed on the surface of the particle (e.g., aggregates of blocking proteins), causes the particle's center of gravity to become unevenly distributed, causing the particle to roll on the substrate surface of the observation device.

[0022] FIG. 1 is a cross-sectional view schematically showing an example of the configuration of a sensitized particle 20 according to this embodiment. An example of a sensitized particle 20 has a core particle 1 and, on at least a portion of the surface of the core particle 1, a ligand 3 immobilized by a chemical bond, a blocking protein 5 immobilized by a chemical bond, and another blocking protein 5 immobilized by a physical bond.

[0023] The sensitized particles 20 of this embodiment can be used as particles for agglutination methods used to detect target substances by agglutination methods.

[0024] The agglutination method is a method in which a sample containing a target substance (e.g., an antigen) is contacted in a dispersion medium with sensitized particles 20 carrying a ligand 3 (e.g., an antibody) that specifically recognizes (binds to) the target substance, and the ligand 3 binds to the target substance, forming a complex between the target substance, the ligand 3, and the sensitized particles 20, thereby selectively agglutinating the sensitized particles 20. One of the agglutination methods, immunoagglutination (also called immunoturbidimetry), utilizes an antigen-antibody reaction to cause an antigen to react with an antibody present on the surface of the sensitized particles 20, thereby agglutinating the sensitized particles 20. In a method for detecting a target substance using the agglutination method, the degree of agglutination of the sensitized particles 20 can be characterized and quantified by optical measurement methods such as absorbance, scattered light intensity, and transmitted light intensity.

[0025] According to this embodiment, the dispersibility of the sensitized particles 20 in the dispersion medium can be increased, which prevents the sensitized particles 20 from aggregating with each other in the dispersion medium before reacting with the target substance, i.e., prevents a nonspecific reaction. Furthermore, when sensitized particles 20 with excellent dispersibility are used, the amount of sensitizer can be increased, and the sensitivity can be further improved.

[0026] Furthermore, according to this embodiment, the dispersibility of the sensitized particles 20 can be improved, thereby increasing the change in the degree of aggregation of the sensitized particles 20 before and after reacting with the target substance. In other words, when comparing the presence and absence of the target substance, the difference in optical measurement values ​​such as absorbance in a dispersion medium containing the sensitized particles 20 increases, making it possible to improve the sensitivity of the sensitized particles 20, i.e., the detection sensitivity of the target substance.

[0027] An example of the target substance detection reagent of this embodiment includes the particle group of the sensitized particles 20 and a dispersion medium. The concentration (wt %) of the sensitized particles 20 in the dispersion medium can be set appropriately. As the dispersion medium, for example, the above-mentioned aqueous solvents are preferably used. The aqueous solvent may contain, within the scope that does not impair the effects of the invention, a third substance such as a buffering agent, a preservative (such as sodium azide), a blocking agent such as a protein (albumin), a sensitizer such as a water-soluble polymer (such as a sugar, polyethylene glycol, or dextran), a salt (such as sodium chloride or an amino acid), or a surfactant. These may be used alone or in combination of two or more.

[0028] Examples of the sensitizer include polyethylene glycol, dextran, and a sensitizer having a phosphorylcholine group. Preferably, the sensitizer may include a sensitizer having a phosphorylcholine group. This can further improve the dispersibility of the sensitized particles 20.

[0029] Examples of sensitizers having a phosphorylcholine group include polymers having a phosphorylcholine group. These may be used alone or in combination of two or more. The polymer having a phosphorylcholine group has at least one or two or more structural units M having a phosphorylcholine group in the side chain of the polymer. The phosphorylcholine group may be partially modified as long as it can be used as a polar group of phospholipids, which are components of biomembranes. The polymer having a phosphorylcholine group may be a homopolymer having only the structural unit M, or may be a copolymer having the structural unit M and another structural unit N. The structural unit N may be a structural unit having, for example, any one of a hydrophobic group, an anionic group, a cationic group, and a hydrogen-bonding group on the side chain of the polymer. The main chain of the polymer having a phosphorylcholine group may have a structure in which polymerizable groups in the raw material monomers such as 2-methacryloyloxyethyl phosphorylcholine (MPC) are linked to each other.

[0030] The target substance detection reagent of this embodiment can also be in the form of a kit from the viewpoint of storage, transportation, distribution, etc. The form of the kit is not particularly limited as long as it is a form that enables the measurement method of the present invention. An example of the target substance detection kit of this embodiment includes a reagent container containing the above-described target substance detection reagent.

[0031] The kit can take various forms, such as the following: The kit of the first type comprises a first reagent container containing a particle group of sensitized particles 20 and a dispersion medium. The kit of the second embodiment includes a second reagent container A containing a particle group of sensitized particles 20, and a second reagent container B containing a dispersion medium. The first reagent container of the kit of the first form and the second reagent containers A and B of the kit of the second form may each contain at least one or more of the third substances exemplified above. Furthermore, each of the kits of the first and second forms may further include a third reagent container containing at least one of the third substances. Furthermore, each of the kits of the first and second forms may include, in addition to the above-mentioned reagent container, a fourth reagent container containing one or more of a positive control, a negative control, a diluent, a washing solution, instructions for use, etc. As the media for the positive and negative controls, serum, physiological saline, solvents, etc. that do not contain the target substance to be measured are used.

[0032] An example of the method for detecting a target substance according to this embodiment includes a contact step of mixing a target substance with a target substance detection reagent. In the contact step, a sample containing a target substance and sensitized particles 20 having ligands 3 are added to the same reaction vessel and allowed to coexist, allowing the ligands 3 bound to the sensitized particles 20 to come into contact with the target substance. The sample containing the standard substance and the target substance reagent containing the sensitized particles 20 may be added in any order. Furthermore, the target substance and the sensitized particles 20 may be mixed at a pH of about 3.0 or more and about 11.0 or less, and the temperature of the mixture may be about 20°C or more and about 50°C or less.

[0033] The target substance is brought into contact with the ligand 3 and reacted with each other, and the substance to be measured in the sample reacts with the antibody carried by the core particle, forming a complex between the target substance, the ligand 3, and the sensitized particle 20, causing the sensitized particle 20 to aggregate as a result of the complex formation.

[0034] The method for detecting a target substance may include, after the contact step, a detection step of optically measuring the agglutination of the sensitized particles 20. Examples of the method for optically measuring agglutination include a method of measuring absorbance, scattered light intensity, or transmitted light intensity with an optical device. The wavelength for measuring absorbance is usually 340 nm to 1000 nm, preferably 500 nm to 900 nm. The time for measuring an agglutination reaction can be measured by measuring the rate of change per unit time or the amount of change per unit time. For example, when measuring absorbance, the rate of change per unit time from 30 seconds to 5 minutes after the start of the agglutination reaction can be measured by measuring the amount of change in absorbance per unit time or the amount of change in absorbance per unit time.

[0035] The sample is not particularly limited as long as it has the potential to contain the target substance, and examples thereof include one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice. The sample is preferably whole blood, plasma, serum, or urine. The sample may be an aqueous solvent containing the target substance, for example, the target substance diluted with phosphate buffered saline or the like.

[0036] The aqueous solvent is not particularly limited as long as it allows the aggregation method, and examples thereof include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred. Examples of buffering agents contained in buffer solutions include phosphate-based, acetate-based, Tris-based, borate-based, carbonate-based, glycine-based, and Gut-based buffer solutions. Suitable Good's buffers include, for example, HEPES, PIPES, and MOPS. The buffering agent may be used alone or in combination of two or more kinds. Acids that can be used to adjust the pH of a buffer solution containing a buffer component include ordinary hydrochloric acid, sulfuric acid, nitric acid, and organic acids such as acetic acid. Alkalis that can be used include sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonium hydroxide.

[0037] The target substance is not particularly limited as long as it is a target substance that allows for the agglutination method, and examples thereof include CRP (C-reactive protein); prostate-specific antigen (PSA); ferritin; α2-macroglobulin; β2-microglobulin; myoglobin; fibrin; fibrinogen degradation products; D-dimer; thrombin-antithrombin III complex (TAT); soluble fibrin (SF); soluble interleukin-2 receptor (sIL-2R); and sodium-reducing peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Examples include urinary peptides; N-terminal pro-brain natriuretic peptide (NT-proBNP); antistreptolysin O; rheumatoid factor; transferrin; haptoglobin; α1-antitrypsin; α1-acidoglycoprotein; hemopexin; antithrombin-III; α-fetoprotein; CEA (carcinoembrionic antigen); HBs-Ag (hepatitis B envelope antigen); Anti-HBs (anti-hepatitis B envelope antibody); HBe-Ag (hepatitis B e antigen); Anti-HBe (anti-hepatitis B e antibody); Anti-HBc (anti-hepatitis B core antibody); IgG; IgA; IgM, etc. The target substance is not particularly limited as long as it is in a form that allows it to bind to an antibody, and may be, for example, in a free form as a simple substance, or in a complex form with another substance (for example, a protein, etc.).

[0038] Next, each component of the sensitized particle of this embodiment will be described.

[0039] An example of a sensitized particle 20 of this embodiment has a core particle 1 having multiple reactive functional groups (A1) on its surface, a ligand 3 chemically bonded to some of the reactive functional groups (A1), and a blocking protein 5 chemically bonded to other parts of the reactive functional groups (A1).

[0040] When the core particle 1 has a reactive functional group (A1) on its surface, the ligand 3 can be chemically bound to the surface of the core particle 1 via the reactive functional group (A1). Furthermore, when the core particle 1 has a reactive functional group (A1) on its surface, the blocking protein 5 can be chemically bound to the surface of the core particle 1 via the reactive functional group (A1).

[0041] The reactive functional group (A1) may contain one or more groups selected from the group consisting of nucleophilic groups such as carboxyl groups, amino groups, and thiol groups, and electrophilic groups such as epoxy groups, maleimide groups, acrylic groups, methacrylic groups, and acid anhydride groups. Examples of acid anhydride groups include maleic anhydride groups and succinic anhydride groups. Among these, the reactive functional group (A1) may contain a carboxyl group.

[0042] (core particle) The core particle 1 is a particle that serves as the core of the sensitized particle 20, and may be composed of either an organic particle or an inorganic particle.

[0043] The organic particles may be particles made of an organic polymer. Examples of organic polymers include synthetic resins such as polystyrene, polyvinyl chloride, polypropylene, (meth)acrylic resin, polymethyl methacrylate, and silicone rubber; cellulose derivatives such as nitrocellulose, cellulose, and methylcellulose; phenolic resins, polylactic acid, polyethylene, and melamine phenol; copolymers containing one of these as the main component (e.g., copolymers containing styrene as the main component); and copolymers containing two or more of these. These organic polymers may be used alone or in combination. The organic polymers constituting the organic particles are not particularly limited as long as they are composed of a polymer different from the surface-modifying polymer.

[0044] Known latex particles can be used as organic particles, and examples thereof include styrene-based latex particles such as polystyrene latex, acrylic acid-based latex particles, various modified latex particles (for example, carboxylic acid-modified latex particles in which a carboxyl group is introduced into the above-mentioned polystyrene), colored latex particles, and fluorescent latex particles. The latex particles can be produced by a known method, or commercially available particles may be used. Two or more types of the latex particles may be used in combination.

[0045] Examples of methods for producing organic particles (resin particles) include a method in which raw material monomers (styrene, methacrylic acid, etc.) are polymerized with a polymerization initiator (radical polymerization initiator, etc.) to form a granular organic polymer in a solvent (water, etc.). Methods for forming the granular organic polymer include radical polymerization methods such as emulsion polymerization, soap-free emulsion polymerization, and suspension polymerization, but are not limited to radical polymerization.

[0046] The inorganic particles may be particles made of an inorganic substance. Examples of inorganic substances include inorganic materials such as metals, ceramics, and glass. Specific examples of inorganic substances include silica. These may be used alone or in combination of two or more. Magnetic inorganic materials may also be used as inorganic substances. In this way, non-magnetic particles or magnetic particles may be used as the inorganic particles.

[0047] The shape of the core particle 1 is not particularly limited, but examples thereof include a spherical shape and an elliptical shape.

[0048] The average particle diameter (D50) of the particle group of core particles 1 may be, for example, 0.01 μm to 1.0 μm, 0.02 μm to 0.8 μm, or 0.03 μm to 0.6 μm. The particle size of the particle group of core particles 1 can be measured, for example, by a laser diffraction particle size distribution analyzer. The average particle size (D50) is defined as the particle size at which the cumulative value from the small particle side in the volume-based particle size distribution is 50%.

[0049] (ligand) Ligand 3 is a compound that specifically binds to a receptor possessed by the target substance described above. The site at which the ligand 3 binds to the target substance is fixed, and the ligand 3 has high affinity selectively or specifically. Examples of the ligand 3 include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, and nucleic acids. Examples of the ligand 3 include antibodies such as full-length antibodies and antigen-binding fragments (e.g., antibody fragments such as Fab, F(ab')2, F(ab'), Fv, and scFv); antigens; nucleic acids such as naturally occurring nucleic acids and artificial nucleic acids; proteins such as aptamers, peptide aptamers, oligopeptides, enzymes, and coenzymes; etc. Commercially available antibodies can be used as the antibodies that specifically recognize the target substance.

[0050] (Blocking protein) Examples of blocking proteins 5 include animal-derived albumins such as bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulin. Among these, naturally occurring blocking proteins are preferred from the viewpoint of dispersibility, and naturally occurring blocking proteins are preferred over synthetic polymers from the viewpoint of sensitivity. These may be used alone or in combination of two or more.

[0051] Here, one example of the method for producing sensitized particles of this embodiment includes a step of obtaining sensitized particles 20 in which ligands 3 and blocking proteins 5 are fixed to at least a portion of the surface of core particles 1 by chemical bonds. Specifically, the process of obtaining such sensitized particles 20 includes a reaction treatment in which some of the reactive functional groups (A1) present on the surface of the core particle 1 are chemically bonded to the blocking protein 5. An example of this reaction treatment is to add a condensing agent that chemically bonds the other part of the reactive functional group (A1) with the blocking protein 5 and the blocking protein 5. The addition of the blocking protein 5 and the condensing agent (reaction treatment) is not particularly limited, as long as it is performed during a series of treatments including the sensitization treatment, washing treatment, and blocking treatment described below, or before or after each treatment in the series of treatments, which are carried out during the process of obtaining the sensitized particles 20. Specifically, the addition of the blocking protein 5 and the condensing agent may be performed, for example, simultaneously with the blocking treatment, before the blocking treatment, simultaneously with any of a series of treatments other than the blocking treatment, or before or after any of the series of treatments. It is preferable that the addition of the blocking protein 5 and the condensing agent includes at least one of the following: adding the condensing agent after the blocking protein 5; adding the condensing agent and then adding the blocking protein 5; or adding a mixture of the blocking protein 5 and the condensing agent. Furthermore, the number of times that the blocking protein 5 and the condensing agent may each be added may be one or more than one. When adding them multiple times, one or more of the above-mentioned treatments may be performed between additions.

[0052] As the condensing agent, those used in the immobilization method utilizing a carboxy group or an amino group, which are known as methods for immobilizing antibodies, can be used. As the condensation agent, for example, a carbodiimide-based crosslinking agent or an amine-reactive crosslinking agent is used. Carboxy groups and amino groups can be reacted using a carbodiimide crosslinking agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as EDAC or WSC). Amine-reactive crosslinkers can also be used to react primary or secondary amino groups with synthetic chemical groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluoroesters.

[0053] In the above-mentioned sensitization treatment, the ligand 3 can be chemically reacted with at least a part of the surface of the core particle 1 . Sensitization treatment for binding (carrying) ligand 3 to core particle 1 can be performed, for example, by suspending ligand 3 (antibody) and core particle 1 in a solvent such as a buffer solution and reacting them at approximately 20 to 37°C for a predetermined period of time.

[0054] The buffer solution may have a pH of, for example, 5.0 to 10.0. The buffer solution may contain, for example, an aqueous solvent, a buffering agent, a preservative (such as sodium azide), a blocking agent such as a protein (albumin), a sensitizer such as a water-soluble polymer (such as a sugar, polyethylene glycol, or dextran), a salt (such as sodium chloride or an amino acid), a surfactant, etc. These may be used alone or in combination of two or more.

[0055] In the sensitization treatment, a conventionally known method can be used as a chemical reaction method for chemically immobilizing the reactive functional group (A1) present on the surface of the core particle 1 and the ligand 3. Examples of chemical reaction methods that can be used include, but are not limited to, a carbodiimide-mediated reaction and an NHS ester activation reaction. If necessary, the above-mentioned condensing agent may also be used.

[0056] The process for obtaining the sensitized particles 20 may include, in addition to the above-mentioned sensitization treatment, one or more treatments commonly used in the technical field of sensitized particles, such as a washing treatment and a blocking treatment. If necessary, other treatments such as centrifugation, ultrasonic treatment, heating (aging) treatment, etc. may be carried out at appropriate times. In the washing treatment, for example, the sensitized product obtained in the sensitization treatment can be washed by a known method. A washing solution or the like can be used for washing. The washing treatment may be carried out once or twice or more times. In the blocking treatment, another blocking protein that physically binds to the sensitized product obtained by the sensitization treatment, for example, by physical adsorption, can be added. At this time, a blocking solution containing the other blocking protein may be added.

[0057] In another embodiment, other blocking proteins may be present on at least a portion of the surface of the sensitized particle 20 and / or the surface of the blocking protein 5, by physical adsorption or the like. At least one or more of the washing solution that can be used in the washing process, the blocking solution that can be used in the blocking process, and the dispersion medium may contain other blocking proteins that physically bind by physical adsorption or the like, such as those exemplified for the above-mentioned blocking protein 5.

[0058] Furthermore, one example of the method for producing the sensitized particle dispersion of this embodiment includes a dispersing step of dispersing the obtained sensitized particles 20 in a dispersion medium. In this dispersing step, the sensitized particles 20 produced in the step of obtaining the sensitized particles 20 are dispersed in the above-mentioned dispersion medium, thereby producing a target substance detection reagent.

[0059] In another embodiment, the method may include an introduction step of introducing a surface-modifying polymer having the above-mentioned reactive functional group (A1) or a coupling agent having the above-mentioned reactive functional group (A1) onto at least a portion of the surface of the core particle. The introduction step can be performed at any timing during the process of obtaining the sensitized particles 20, but is preferably performed before the sensitization treatment or the binding treatment. As a result, the reactive functional group (A1) is formed on the surface of the core particle 1 via the surface-modifying polymer and / or the coupling agent. Sensitized particles 20 of this type may include a ligand 3 and / or a blocking protein 5 bound to at least some of the reactive functional groups (A1) of the surface-modifying polymer and / or coupling agent.

[0060] The surface-modifying polymer may be formed on at least a part of the surface of the core particle 1, and may form a polymer coating layer that covers a part or the entire surface of the core particle 1. The surface-modified polymer is physically bonded to the surface of the core particle 1, and / or a specific functional group contained in the surface-modified polymer is chemically bonded to a specific functional group present on the surface of the core particle 1.

[0061] The surface-modifying polymer may be composed of a (meth)acrylic polymer, a maleic acid polymer, a methylene malonate polymer, a phenolic resin, an epoxy resin, or the like. These may be used alone or in combination of two or more. However, the surface-modifying polymer may not include a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer. According to this embodiment, by using a surface-modified polymer in addition to the core particle 1, a wide variety of molecular designs become possible for the surface-modified polymer, and the degree of freedom in molecular design increases.

[0062] Examples of the (meth)acrylic polymer include methacrylic polymers and acrylic polymers. Examples of maleic acid-based polymers include maleic anhydride-norbornene copolymers, styrene-maleic anhydride copolymers, maleic anhydride homopolymers, methyl vinyl ether-maleic anhydride copolymers, and olefin-maleic anhydride copolymers.

[0063] The surface-modifying polymer may be any of a homopolymer, a random copolymer, an alternating copolymer, a block copolymer, a periodic copolymer, and the like.

[0064] The weight average molecular weight of the surface modifying polymer is, for example, 1,000 or more and 10,000,000 or less, preferably 2,000 or more and 5,000,000 or less, and more preferably 3,000 or more and 1,000,000 or less. The weight average molecular weight can be determined from a polystyrene equivalent value obtained from a calibration curve of standard polystyrene (PS) obtained by gel permeation chromatography measurement.

[0065] The surface modifying polymer may comprise a structural unit A1 that comprises a reactive functional group (A1) capable of binding to a ligand 3.

[0066] The method for forming the surface-modifying polymer on the surface of the core particle 1 uses, for example, a polymer introduction step in which a surface-modifying polymer is introduced onto the surface of the core particle 1 by a chemical reaction. For the chemical reaction, an immobilization method using a carboxy group or an immobilization method using an amino group, which are known as methods for immobilizing antibodies, can be used. These chemical reactions may involve the use of condensing agents, such as carbodiimide-based crosslinkers and amine-reactive crosslinkers, if desired. Carboxy groups and amino groups can be reacted using a carbodiimide crosslinking agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as EDAC or WSC). Amine-reactive crosslinkers can also be used to react primary or secondary amino groups with synthetic chemical groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluoroesters.

[0067] When the core particle 1 is an organic particle (resin particle), a method is used in which a polymer reactive group (such as a carboxy group or an amino group) contained in the raw material monomer of the organic particle reacts with a predetermined reactive group contained in the surface-modifying polymer. For example, in the manufacturing process of the organic particle, it is possible to incorporate a polymer reactive group into the organic polymer during the polymerization of the raw material monomer. Commercially available polystyrene particles containing a carboxy group may also be used as the core particle 1. As a specific example, when a surface-modifying polymer containing a carboxy group is immobilized on a core particle 1 containing a carboxy group, primary amino groups (-NH2) may be introduced by amino treatment to some of the carboxy groups in the core particle 1 (amino treatment). Then, the primary amino groups (polymer reactive groups) in the core particle 1 after the amino treatment and the predetermined reactive groups in the surface-modifying polymer are chemically reacted, if necessary, by adding the above-mentioned condensing agent. When the surface-modifying polymer contains an acrylic group / methacrylic group, it is possible to react the primary amino group (polymer reactive group) with the carboxy group (predetermined reactive group) contained in the acrylic group / methacrylic group via the above-mentioned condensing agent. In another embodiment, when the surface-modifying polymer contains an anhydride group, it is possible to react a primary amino group (polymer reactive group) with an anhydride group (predetermined reactive group) such as a maleic anhydride group without using the above-mentioned condensing agent. On the other hand, when the core particle 1 is an inorganic particle, a method is used in which a polymer reactive group is introduced onto the surface of the inorganic particle using a coupling agent or the like as needed, and then the polymer reactive group is reacted with a predetermined reactive group contained in the surface-modifying polymer.

[0068] Here, an example of a method for synthesizing a surface-modifying polymer will be described. The raw material monomers used in the polymer precursor production process can be one or more of raw material monomer A containing a diene compound and a diene parent compound, and raw material monomer B containing a functional group and an unsaturated group such as a double bond.

[0069] For the polymer of a diene compound and a diene parent compound, (i) if an anhydride group is contained, the reactive functional group (A1), the acidic functional group (A2), or other functional groups (functional functional groups) may be introduced into the side chain by ring-opening the anhydride group; (ii) if a carboxyl group is contained, various functional groups may be introduced into the side chain by a condensation reaction or the like; and (iii) if an unsaturated group such as a double bond is contained, the various functional groups described above may be introduced into the side chain by an addition reaction or the like. Examples of the other functional groups mentioned above include known functional groups such as various reactive groups, hydrophobic groups, hydrophilic groups (water-soluble groups), and acidic groups. Furthermore, (i) when an anhydride group is contained, the anhydride group may be ring-opened with water, an alcohol, etc., or may be ring-opened with an organic base. Furthermore, the functional group described above may be introduced into the side chain of the polymer precursor by a condensation reaction or the like with respect to a functional group such as a carboxyl group formed by ring-opening of the anhydride group, as in (ii) above.

[0070] For the synthesis of a polymer precursor of a maleic anhydride-based polymer, for example, a norbornene-based monomer such as norbornene can be used as the diene compound, and an unsaturated carboxylic acid anhydride having a cyclic structure in the molecule such as maleic anhydride can be used as the new diene compound.

[0071] On the other hand, the raw material monomer B may be a raw material monomer B1 having a reactive functional group (A1) and an acidic functional group (A2), or a combination of a raw material monomer B2 having a reactive functional group (A1) and a raw material monomer B2 having an acidic functional group (A2). If necessary, in addition to these, a raw material monomer B3 having a functional group other than the reactive functional group (A1) and the acidic functional group (A2) may be used.

[0072] To synthesize a polymer precursor of a (meth)acrylic polymer, for example, (meth)acrylic acid such as acrylic acid, methacrylic acid, (meth)acrylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, hexyl (meth)acrylate, and ethylhexyl (meth)acrylate can be used. One or more of these (meth)acrylic monomers can be used.

[0073] The polymerization may be, but is not limited to, addition polymerization and / or condensation polymerization. For example, addition polymerization may be carried out by radical polymerization. Alternatively, the reactive functional group (A1), the acidic functional group (A2), or other functional groups (functional functional groups) may be introduced into the side chains of the resulting polymer precursor by ring-opening reaction, condensation reaction, addition reaction, or the like. A specific example will be described. First, one or more raw material monomers and a polymerization initiator are dissolved in a solvent, and then the solution is heated for a predetermined period of time to carry out solution polymerization. The heating temperature can be, for example, 50°C to 80°C. The heating time can be, for example, 1 hour to 20 hours. It is more preferable to carry out solution polymerization after removing dissolved oxygen in the solvent by nitrogen bubbling.

[0074] As the polymerization initiator, for example, an azo compound, an organic peroxide, or the like can be used. Specific examples of the azo compound include azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobis(2-methylpropionate), and 1,1'-azobis(cyclohexanecarbonitrile) (ABCN). Examples of organic peroxides include hydrogen peroxide, ditertiary butyl peroxide (DTBP), benzoyl peroxide (benzoyl peroxide, BPO), and methyl ethyl ketone peroxide (MEKP). As for the polymerization initiator, one type may be used alone, or two or more types may be used in combination.

[0075] Furthermore, a molecular weight modifier or a chain transfer agent can be used as needed. Examples of the chain transfer agent include thiol compounds such as dodecyl mercaptan, mercaptoethanol, and 4,4-bis(trifluoromethyl)-4-hydroxy-1-mercaptobutane. These chain transfer agents can be used alone or in combination of two or more.

[0076] Examples of solvents used in the polymerization reaction include one or more esters such as methyl ethyl ketone (MEK), propylene glycol monomethyl ether, diethyl ether, tetrahydrofuran (THF), toluene, ethyl acetate, and butyl acetate. Examples of the polymerization initiator include one or more azo compounds and organic peroxides. Examples of azo compounds include azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobis(2-methylpropionate), and 1,1'-azobis(cyclohexanecarbonitrile) (ABCN). Examples of organic peroxides include hydrogen peroxide, ditertiarybutyl peroxide (DTBP), benzoyl peroxide (benzoyl peroxide (BPO)), and methyl ethyl ketone peroxide (MEKP).

[0077] The reaction solution containing the (co)polymer thus obtained is added to an alcohol or ether, such as hexane or methanol, to precipitate the polymer. The polymer is then filtered, washed with an alcohol, such as hexane or methanol, and then dried. In this manner, the polymer can be synthesized. This allows the removal of low-molecular-weight components, such as residual monomers, oligomers, and polymerization initiators.

[0078] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention. [Example]

[0079] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0080] <Production of dispersion containing sensitized particles> [Example 1] (sensitization treatment) 940 μL of latex solution A (0.4% w / vol) containing the following latex particles and to which the following condensation agent had been added was mixed with 940 μL of the following antibody A solution (0.07 mg / mL), and the mixture was shaken and stirred at 20°C for 180 minutes to obtain a mixed solution. Condensing agent: Carbodiimide crosslinking agent (EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as WSC) Latex solution A: Carboxyl group-modified polystyrene latex particles (manufactured by JSR, product name: IMMUNTEX, average particle diameter d: 400 × 10 -7 cm, solid content: 10%) Antibody A solution: Ferritin antibody (Mikuri Immuno Research Institute, Ferritin clone No. 14), 10 mM MES (pH 6.5)

[0081] (Cleaning process) The resulting mixture was centrifuged at 10,000 rpm for 15 minutes at 10° C. using a centrifuge (rotor: R15A (HITACHI)). The supernatant was decanted, and 5 mL of the following HEPES buffer solution was added to the resulting sediment, which was then dispersed by sonication. Subsequently, the mixture was centrifuged at 10,000 rpm for 15 minutes at 10° C. using the above centrifuge. The supernatant was decanted, and 5 mL of the following HEPES buffer solution was added to the resulting sediment, which was then dispersed by sonication to obtain a suspension. HEPES buffer: 0.1% BSA, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2), 0.1% Tween 20, 0.05% NaN

[0082] (Blocking process) To the resulting suspension, 5 mL of the following blocking solution A was added, followed by 30 μL of additional blocking solution B and 19 μL of the above condensing agent (5 mg / mL WSC solution), and after thorough dispersion, the mixture was stirred at 37°C for 1 hour (reaction treatment).Then, using the above centrifuge, the mixture was centrifuged at 10,000 rpm at 10°C for 15 minutes to obtain ligand-sensitized particles. Blocking solution A: Bovine serum albumin (BSA) solution (1% BSA, 20 mM MES buffer (pH 6.0), 0.05% NaN3) Blocking solution B: Bovine serum albumin (BSA) solution (the molecular weight M of BSA is approximately 66,500, the Stokes radius r of BSA is 3.5 nm, 0.2% BSA, 20 mM MES buffer (pH 6.0), 0.05% NaN3)

[0083] (reagent processing) The following components were mixed in a predetermined mixing ratio and uniformly dispersed by ultrasonic treatment (dispersion treatment) to prepare a reagent (sensitized particle dispersion) with a solid content of 0.1%. The ligand-sensitized particles obtained above HEPES buffer containing 0.5% bovine serum albumin (BSA) 100mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.0)

[0084] [Example 2] A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that the concentration of the antibody A solution during the sensitization treatment was changed to 0.21 mg / mL.

[0085] [Comparative Example 1] A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that in the blocking treatment, the amount of blocking solution A added was 5.03 mL, and blocking solution B and a condensing agent were not added.

[0086] Comparative Example 2 A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 2, except that in the blocking treatment, the amount of blocking solution A added was 5.03 mL, and blocking solution B and a condensing agent were not added.

[0087] <Surface roughness measurement procedure> In each of the examples and comparative examples, an atomic force microscope under the following (observation conditions) was used to obtain four images of four particles contained in the sample obtained in the following (sample preparation). As one of the four images, Example 1 is shown in FIG. 2, Example 2 in FIG. 4, Comparative Example 1 in FIG. 6, and Comparative Example 2 in FIG.

[0088] A one-dimensional profile was obtained in a direction parallel to the X-axis, passing through the brightest area of ​​each image, and the height of the bright area was measured. The average height of the bright area at four points was calculated as the average height of the convex portion. Furthermore, the number of particles having a protrusion with a height of 10 nm or more included in the one-dimensional profile was counted, and the percentage (%) of such particles in all particles was calculated as the above particle percentage. As one-dimensional profiles of the above images, Example 1 is shown in FIG. 3, Example 2 in FIG. 5, Comparative Example 1 in FIG. 7, and Comparative Example 2 in FIG.

[0089] In addition, in a one-dimensional profile passing through a bright area in one image containing one particle, the height at the apex of the convexity was defined as H, and the heights of the two ends on either side of the convexity (there is a left end and a right end on either side of the apex) were defined as L1 and L2, respectively. The "height of the convexity" in one image was calculated as [(H-L1)+(H-L2)] / 2, and the average value was calculated using the "heights of the convexity" calculated from the four images. The edge was determined as the position where a certain degree of flatness appeared when moving from the apex of the convex part toward the edge, or the bottom of a concave part that appeared along the way. In Figures 3, 7, and 9, circles and triangles correspond to the edge. However, Figure 5 was determined to have no edge because the overall slope is smooth.

[0090] The diameter of the convex portion was defined as the distance between the left and right ends of one particle contained in one image, and the number of particles in the four images that had a "convex portion height" of 10.0 nm or more was determined, and the proportion of particles present in the total number of four particles was calculated.

[0091] In addition, if the distance from the particle center to the bright area is the particle center coordinates (X1, Y1) and the bright area coordinates (X2, Y2), then √{(X1-X2) 2 +(Y1-Y2) 2} and the average distance was calculated using the measurement results of the four images.

[0092] (Observation conditions) Measurement device: NanoWizard4XP Measurement mode: QI Advanced mode Cantilever (AC40, material: Si (reflective surface: gold coated), spring constant: 0.1 N / m, tip curvature radius: 8 nm) Measurement atmosphere (23°C, HEPES aqueous solution (10 mM, pH 7.5)) Measurement range: 0.6 μm square, 0.2 μm square ·Resolution: XY axis direction: 0.09nm, Z axis direction: 0.04nm (Sample preparation) 10 μL of KBM903 (a silane coupling agent having a primary amino group) and 490 μL of methanol were placed in a plastic container with a cap and stirred with a vortex mixer to prepare a surface treatment agent. The surface treatment agent was dropped onto the surface of a silicon wafer and left to stand for 3 minutes. After that, the droplets were blown away with a blower, and the wafer was left to stand in an oven at 120°C to perform the surface treatment on the silicon wafer surface. The dispersion containing the sensitized particles obtained in each Example and Comparative Example was then dropped onto the surface of the surface-treated silicon wafer and allowed to stand for 10 minutes.The surface of the silicon wafer was then washed with pure water, and the sensitized particles on the surface were stored in a HEPES aqueous solution (10 mM, pH 7.5).

[0093] <Absorbance> Using an automatic analyzer TBA-120FR (manufactured by Canon Medical Systems Inc.), the absorbance at a wavelength of 700 nm was measured for each of the reagents of each example and comparative example, the measurement reagents listed below, and the target substance (ferritin antigen solution), all kept at 10°C. (measurement reagents) The following components were mixed in a predetermined ratio to prepare a measuring reagent. 0.1% Tween 20 Polyethylene glycol (PEG) (5000-50000) 1% bovine serum albumin (BSA) in HEPES buffer 150mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.6) Next, 20 μL of each reagent from each Example and Comparative Example was mixed with 80 μL of the following measurement reagent and 4 μL of the target substance (antigen amount 0 ng / mL). The mixture was stirred uniformly and maintained at 37°C, and the absorbance at a wavelength of 700 nm was measured immediately after adding the measurement reagent and after 5 minutes had elapsed. The absorbance of each reagent when the antigen amount was 0 ng / mL was calculated by subtracting the absorbance immediately after adding the measurement reagent from the absorbance after 5 minutes.

[0094] [Table 1]

[0095] The results in Table 1 above show that the sensitized particle groups of each Example have lower absorbance than the sensitized particle groups of each Comparative Example, and therefore exhibit superior dispersibility in the dispersion medium (before the addition of the antigen).

[0096] It was found that the dispersion containing the sensitized particle groups obtained in these examples can be suitably used as a sensitized particle dispersion (reagent) for use in the immunoagglutination method. [Explanation of symbols]

[0097] 1. Core particle 3. Ligand 5. Blocking Proteins 20 Sensitizing particles

Claims

1. A sensitized particle having a ligand and a blocking protein immobilized by chemical bonds and a blocking protein immobilized by physical bonds on at least a portion of the surface of a core particle, Sensitized particles, wherein the surface irregularities of the sensitized particles, as measured using an atomic force microscope according to the following measurement procedure, have an average height of convex portions of 10.0 nm or less, and / or the proportion of particles having convex portions with a height of 10 nm or more is 40% or less. <Surface roughness measurement procedure> Using an atomic force microscope under the following (observation conditions), four images are obtained for four particles contained in the sample obtained under the following (sample preparation). A one-dimensional profile is obtained in a direction parallel to the X axis so as to pass through the brightest part of each image, and the height of the bright part is measured. The average value of the heights of the bright parts at four points is calculated as the average height of the convex part. Furthermore, the number of particles having a protrusion with a height of 10 nm or more included in the one-dimensional profile is counted, and the ratio (%) of these particles to the total particles is calculated as the particle ratio. (Observation conditions) - Cantilever (material: Si, spring constant: 0.1 N / m, tip curvature radius: 8 nm) Measurement atmosphere (23°C, HEPES aqueous solution (10 mM, pH 7.5)) ・Resolution: XY axis direction: 0.09nm, Z axis direction: 0.04nm (Sample Preparation) 10 μL of a silane coupling agent having a primary amino group and 490 μL of methanol are placed in a plastic container with a cap and stirred with a vortex mixer to prepare a surface treatment agent. The surface treatment agent is dropped onto the surface of a silicon wafer and left to stand for 3 minutes, after which the droplets are blown away with a blower and the wafer is left to stand in an oven at 120° C. to perform the surface treatment on the surface of the silicon wafer. The dispersion containing the sensitized particles is then dropped onto the surface of the surface-treated silicon wafer and allowed to stand for 10 minutes.The surface of the silicon wafer is then washed with pure water, and the sensitized particles on the surface are stored in a HEPES aqueous solution (10 mM, pH 7.5).

2. 10. The sensitized particle of claim 1, The sensitized particle, wherein the diameter of the convex portions having a height of 10 nm or more in the surface irregularities is 35 nm or less.

3. The sensitized particle according to claim 1 or 2, Sensitized particles, wherein in the image, the average distance from the particle center to the bright area is 35 nm or less.

4. The sensitized particle according to claim 1 or 2, Sensitized particles, wherein the average particle size of the particle group of the sensitized particles is 0.01 μm or more and 1.0 μm or less.

5. The sensitized particle according to claim 1 or 2, The sensitized particle, wherein the core particle is an organic particle or an inorganic particle.

6. The sensitized particle according to claim 1 or 2, The sensitized particle, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid.

7. The sensitized particle according to claim 1 or 2, Sensitized particles used to detect target substances using agglutination methods.

8. A target substance detection reagent comprising the sensitized particles according to claim 1 or 2 and a dispersion medium.

9. A target substance detection kit comprising a reagent container containing the target substance detection reagent according to claim 8.

10. A method for detecting a target substance, comprising the step of mixing a target substance with the target substance detection reagent according to claim 8.

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