Particles and methods for manufacturing particles

Abstract

To provide a method for producing particles that can suppress non-specific adsorption without using BSA. [Solution] A method for producing particles, comprising: a first step of preparing an emulsion by mixing a radically polymerizable monomer, an organosilane compound having silicon atoms to which an alkoxy group is bonded and which is radically polymerizable, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium; and a second step of adding a specific reactive compound after the first step.

Description

[Technical Field]

[0001] This invention relates to particles and methods for producing particles. [Background technology]

[0002] Polymer particles are becoming increasingly important in the fields of basic biology and medicine. For example, there is a growing need for their application in in vitro diagnostic reagents using latex agglutination.

[0003] In principle, if a desired antigen or antibody can be adsorbed onto the particle surface, it can be used in a latex agglutination reaction. However, many impurities are present in samples such as serum, and these impurities also adsorb onto the particle surface (non-specific adsorption), inhibiting the adsorption of the desired antigen or antibody to the particle surface. Furthermore, if particles agglutinate with each other via impurities adsorbed onto them, the measurement accuracy decreases. Therefore, there is a method to suppress the adsorption of impurities (non-specific adsorption) onto the particle surface by coating the particle surface with bovine serum albumin (BSA) after adsorbing the desired antibody onto the particle (Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2004-151000 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] For example, in the method of coating the particle surface with BSA as described in Patent Document 1, the adsorption of impurities was sometimes sufficiently suppressed and sometimes not. Therefore, using BSA-coated particles in the latex agglutination method resulted in a problem of reduced reproducibility.

[0006] Therefore, the present invention aims to provide particles that can suppress nonspecific adsorption without using BSA, and a method for producing the same. [Means for solving the problem]

[0007] A particle manufacturing method according to one aspect of the present invention comprises a first step of preparing an emulsion by mixing a radically polymerizable monomer, an organosilane compound having silicon atoms to which an alkoxy group is bonded and which is radically polymerizable, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium, and a second step of adding a reactive compound after the first step, wherein the reactive compound is represented by the following formula (1). [ka]

[0008] In equation (1), R 1 and R 2 This represents a linear or branched alkyl group having 1 to 30 carbon atoms, which may contain -NH-CO-, -NH-, -O-, -S-, or -CO-, and is substituted with at least one carboxyl group or at least one amino group.

[0009] Furthermore, particles according to another aspect of the present invention are particles produced by a first step of preparing an emulsion by mixing a radical polymerizable monomer, an organosilane compound having silicon atoms to which an alkoxy group is bonded and which is radical polymerizable, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium, and a second step of adding a reactive compound after the first step, wherein the reactive compound is a particle represented by the above formula (1). [Effects of the Invention]

[0010] According to the present invention, it is possible to provide particles that can suppress nonspecific adsorption without using BSA, and a method for producing the same. [Modes for carrying out the invention]

[0011] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited thereto. (Particles and Method for Producing the Same) The method for producing particles according to this embodiment involves a first step of mixing a radically polymerizable monomer, an organosilane compound having a silicon atom bonded with an alkoxy group and having radical polymerizability, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium to prepare an emulsion, and a second step of adding a reactive compound after the first step. The reactive compound is represented by the following formula (1). [Chemical Formula]

[0012] In formula (1), R 1 and R 2 each represents a linear or branched alkyl group having 1 to 30 carbon atoms, which may contain -NH-CO-, -NH-, -O-, -S-, -CO- and is substituted with at least one carboxy group or at least one amino group.

[0013] Note that the particles produced by the production method according to this embodiment may be hereinafter referred to as polymer fine particles. Due to the presence of silica (silanol groups) derived from the organosilane compound, the particles produced by the production method according to this embodiment have high hydrophilicity on the particle surface, so non-specific adsorption can be suppressed without using BSA. Also, since BSA is a natural product, it is presumed that the degree of hydrophilicity and hydrophobicity varies for each lot. However, since the particles produced by the production method according to this embodiment do not use BSA, the difference in the ability to suppress non-specific adsorption for each particle is small. In other words, in producing particles, the reproducibility of the ability to suppress non-specific adsorption of the produced particles is high.

[0014] By adding a reactive compound as the second step after the first step, the following advantages are obtained. First, an effect of improving the uniformity of the core of the particles prepared in the first step can be expected. Also, an effect of improving the reactivity of the reactive compound on the surface of the particles obtained through the first step can be expected.

[0015] In the second step, one or more surfactants may be added. Adding surfactants can improve the dispersibility of polymer microparticles.

[0016] R in equation (1) above 1 and R 2 Preferably, at least one of these has at least one carboxyl group.

[0017] Furthermore, it is preferable that the particle manufacturing method according to this embodiment further includes a step of heating the emulsion after the first step.

[0018] In the first step, a monomer having two or more double bonds within a single molecule, such as divinylbenzene, may be used as a crosslinking agent.

[0019] R in equation (1) above 1 and R 2 Preferably, at least one of them has the structure shown in formula (2) or formula (3) below. [ka] [ka]

[0020] In formula (2), Z represents a linear or branched saturated or unsaturated alkyl group having 1 to 10 carbon atoms.

[0021] Furthermore, the particles according to this embodiment are produced by a first step of preparing an emulsion (mixture) by mixing a radically polymerizable monomer, an organosilane compound having silicon atoms to which an alkoxy group is bonded and which is radically polymerizable, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium, and a second step of adding a reactive compound after the first step, wherein the reactive compound is a particle represented by the above formula (1).

[0022] The particles according to this embodiment are particles produced by the method for producing particles according to the above-described embodiment of the present invention. That is, the particles according to this embodiment preferably have one or more structural units represented by the following formula (4).

Chemical formula

[0023] In formula (4), X represents a bond between Si of adjacent structures represented by formula (4) via O, a bond with the core of the particles prepared from the emulsion, or a hydroxy group, and at least one of the structural units represented by formula (4) has a bond with the core.

[0024] R 3 and R 4 At least one of them represents a linear or branched alkyl group having 1 to 30 carbon atoms which may contain -NH-CO-, -NH-, -O-, -S-, -CO- and is substituted with at least one carboxy group or at least one amino group.

[0025] Also, at least one of R 3 and R 4 in the formula (4) preferably has the structure represented by the above formula (2) or the structure represented by the above formula (3), and it is more preferable that at least one of R 3 and R 4 has the structure represented by the above formula (3).

[0026] (Radical polymerization monomer) As the radical polymerization monomer, a monomer containing no silicon atom can be used.

[0027] Furthermore, the radical polymerizable monomer can be at least one selected from the group consisting of styrene monomers, acrylate monomers, and methacrylate monomers. Specifically, as the radical polymerizable monomer, for example, at least one selected from the group consisting of styrene, butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, and methyl acrylate can be used. That is, these can be used individually or in combination.

[0028] Furthermore, the radical polymerizable monomer according to this embodiment can be used individually or in combination of multiple monomers that have high compatibility with each of the styrene monomers, acrylate monomers, and methacrylate monomers, and that have highly hydrophilic functional groups. For example, a styrene sulfonic acid monomer (such as sodium parastyrene sulfonate) that has a sulfonic acid group as a highly hydrophilic functional group and is highly compatible with styrene can be used.

[0029] In the first step of the particle manufacturing method according to this embodiment, further mixing in parastyrene sulfonate to prepare the emulsion can reduce the particle size and improve particle size uniformity. This is because, during the polymerization reaction of the radical polymerizable monomer and the organosilane compound, parastyrene sulfonic acid is also copolymerized, improving the hydrophilicity and dispersibility of the particles.

[0030] (Organosilane compounds) As the organosilane compound, at least one selected from the group consisting of vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane can be used. That is, these can be used individually or in combination.

[0031] (Radical polymerization initiator) As radical polymerization initiators, a wide range of azo compounds and organic peroxides can be used. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), and potassium persulfate (KPS).

[0032] (Water-soluble polymer) Water-soluble polymers act as protective colloids during the synthesis of polymer microparticles, contributing to the control of the particle size of the resulting polymer microparticles. Preferred water-soluble polymers include at least one selected from the group consisting of polyacrylamide, polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone (PVP), and polyvinylpyrrolidone-polyacrylic acid copolymers. In other words, one or more of these may be used in combination. Polyvinylpyrrolidone-polyacrylic acid copolymers are an example of polymers containing a pyrrolidone ring and functional groups capable of binding ligands, and copolymers containing a pyrrolidone ring and functional groups capable of binding ligands may also be used as water-soluble polymers.

[0033] The molecular weight of the water-soluble polymer is preferably between 10,000 and 1,000,000, and more preferably between 30,000 and 70,000. A molecular weight of 10,000 or more provides a high protective colloidal effect, while a molecular weight of 1,000,000 or less prevents an increase in the viscosity of the aqueous medium, making it difficult to handle. Furthermore, it is acceptable for some of these water-soluble polymers to adhere to the particle surface after synthesis through physical or chemical adsorption.

[0034] Hereinafter, copolymers containing a pyrrolidone ring and a functional group capable of binding ligands will be abbreviated as PVP copolymers. In PVP copolymers, the molar ratio of the pyrrolidone ring site to the ligand-binding functional group site is preferably around 2:8 to 9:1. If there are too few ligand-binding functional groups, the efficiency of the antigen-antibody reaction will decrease, and if there are too many, the performance in suppressing non-specific adsorption will decrease. The ligand-binding functional group in a PVP copolymer is not particularly limited as long as it is a functional group capable of binding antibodies, antigens, enzymes, etc., but carboxyl groups, amino groups, thiol groups, epoxy groups, maleimide groups, succinimidyl groups, and silicon alkoxide groups are commonly used. PVP copolymers may be either random polymers or block copolymers.

[0035] By incorporating the PVP copolymer from the particle synthesis stage, it becomes possible to simultaneously impart non-specific adsorption suppression ability and ligand binding ability to the particles. Furthermore, when particles are synthesized using a radical polymerizable monomer (or oligomer) containing an organosilane, silanol groups can be imparted to the particle surface. Hydrogen bonding between the silanol groups and PVP allows the PVP or PVP copolymer to be more strongly adsorbed onto the particle surface.

[0036] An example of a PVP polymer, specifically a polyvinylpyrrolidone-polyacrylic acid copolymer, is a random copolymer of N-vinyl-2-pyrrolidone and acrylic acid (manufactured by Polymer Source Co., Ltd.). Suitable molecular weights for this random copolymer can be selected from those of 60,000, 70,000, 79,000, etc. Similarly, the ratio of N-vinyl-2-pyrrolidone molecules to acrylic acid molecules can be selected from those of 4:6, 3.1:6.9, 2:8, etc.

[0037] (aqueous medium) The aqueous medium preferably contains 80% to 100% by mass of water. Examples of aqueous mediums include water and solutions of water mixed with water-soluble organic solvents, such as methanol, ethanol, isopropyl alcohol, and acetone. If the content of organic solvents other than water exceeds 20% by mass, dissolution of polymerizable monomers may occur during the production of polymer microparticles.

[0038] Furthermore, the above-mentioned aqueous medium preferably has a pH of 6 to 9. If the pH is between 6 and 9, the alkoxide and silanol groups in the organosilane compound will not undergo condensation polymerization or react with other functional groups before the formation of polymer fine particles, and there is no risk of the resulting particles agglomerating. In this embodiment, condensation polymerization of the alkoxide is not intentionally performed before the formation of polymer fine particles.

[0039] The pH of aqueous media is preferably adjusted using a pH buffer, but it can also be adjusted with acids or bases.

[0040] In aqueous media, surfactants, defoamers, salts, thickeners, etc., may be added in a proportion of 10% by mass or less relative to the aqueous media.

[0041] (Surfactants) As the surfactant added in the second step, nonionic surfactants, anionic surfactants, cationic surfactants, polymeric surfactants, or phospholipids can be used. These surfactants may be used individually or in combination of two or more types.

[0042] Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., compounds shown in formula (5)), Brij® 35, Brij® 58, Brij® 76, Brij® 98, Triton® X-100, Triton® X-114, Triton® X-305, Triton® N-101, Nonidet® P-40, IGEPAL® CO530, IGEPAL® CO630, IGEPAL® CO720, and IGEPAL® CO730. [ka]

[0043] In equation (5), R 21 ~R 24 Each of these is independently selected from -H and -OCR'. R' is a saturated or unsaturated alkyl group having 1 to 18 carbon atoms. In formula (5), w1, x1, y1, and z1 are integers such that the sum of w1, x1, y1, and z1 is between 10 and 30.

[0044] Examples of polyoxyethylene sorbitan fatty acid esters represented by formula (5) include Tween® 20, Tween® 40, Tween® 60, Tween® 80, and Tween® 85.

[0045] Examples of the above-mentioned anionic surfactants include sodium dodecyl sulfate, dodecylbenzenesulfonate, decylbenzenesulfonate, undecylbenzenesulfonate, tridecylbenzenesulfonate, nonylbenzenesulfonate, and their sodium, potassium, and ammonium salts.

[0046] Examples of the cationic surfactants mentioned above include cetyltrimethylammonium bromide, hexadecylpyridinium chloride, dodecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride.

[0047] Examples of the polymeric surfactants mentioned above include polyvinyl alcohol, polyoxyethylene polyoxypropylene glycol, and gelatin. Commercially available polyoxyethylene polyoxypropylene glycols include Pluronic F68 (manufactured by BASF) and Pluronic F127 (manufactured by BASF).

[0048] (Details of the particle manufacturing method according to this embodiment) In the particle manufacturing method according to this embodiment, the first step is to dissolve a water-soluble polymer in an aqueous medium whose pH is adjusted to 6 or higher and 9 or lower. The concentration of the water-soluble polymer is 0.01% by mass or higher and 20% by mass or lower, preferably 0.03% by mass or higher and 15% by mass or lower, relative to the aqueous medium. If the concentration is 0.01% by mass or higher, the effect of controlling the particle size can be sufficiently obtained. If the concentration is 20% by mass or lower, the viscosity of the aqueous medium does not increase, and stirring can be performed sufficiently.

[0049] Next, a radical polymerizable monomer (A) that does not contain silicon atoms and an organosilane compound (B) in which an alkoxy group is bonded to a silicon atom and which is further radical polymerizable are added to the aqueous medium to form an emulsion. The mass ratio of (A) to (B) is 4:6 to 9.7:0.3.

[0050] If the mass ratio of (A) to (B) is 4:6 or higher, the overall specific gravity of the particles will not increase too much, and there will be no risk of significant particle sedimentation. Also, if the mass ratio of (A) to (B) is 9.7:0.3 or lower, the silicon atom content will not decrease too much, and there will be no risk of a decrease in the ability to suppress nonspecific adsorption.

[0051] The mass ratio of the aqueous medium to the total amount of (A) and (B) is preferably 5:5 to 9.5:0.5. If the mass ratio of the aqueous medium to the total amount of (A) and (B) is 5:5 or higher, there is no risk of significant aggregation of the generated polymer fine particles. Also, if the mass ratio of the aqueous medium to the total amount of (A) and (B) is 9.5:0.5 or lower, there is no risk of a small amount being produced.

[0052] The reactive compound added in the second step can be used to immobilize the antibody. The reactive compound can be used in a proportion of 0.01% by mass or more and 60% by mass or less, based on the total mass of (A) and (B).

[0053] The radical polymerization initiator is used dissolved in water, a buffer, etc. The radical polymerization initiator can be used in a proportion of 0.1% to 10% by mass relative to the total mass of (A) and (B).

[0054] If the process further includes a step of heating the emulsion after the first step, any heating method may be used as long as the entire emulsion is heated uniformly. The heating temperature can be arbitrarily set between 50°C and 90°C, and the heating time between 2 hours and 24 hours.

[0055] In the second step, the polymer microparticles obtained after the first step are mixed with the reactive compound and the entire liquid is uniformly stirred. The stirring temperature can be set arbitrarily between 4°C and 90°C, and the stirring time can be set to 3 hours or more. The surfactant mentioned above may also be added in the second step.

[0056] In the first step, hybrid particles are obtained from a silicon atom-free radical polymerizable monomer, an organic silane compound in which an alkoxy group is bonded to a silicon atom and which is further radical polymerizable, and a water-soluble polymer. These hybrid particles have advantages compared to particles with a core-shell structure. Here, for example, the core particles of particles with a core-shell structure are polymers containing monomer units derived from a silicon atom-free radical polymerizable monomer. The shell of particles with a core-shell structure is a polymer containing monomer units derived from an organic silane compound in which an alkoxy group is bonded to a silicon atom and which is further radical polymerizable. The advantages of hybrid particles compared to the core-shell structure are as follows: Firstly, in the production of hybrid particles, the raw materials can be added all at once. In other words, in the case of the core-shell structure, the manufacturing process is complicated because it is necessary to form the shell after forming the core. Another advantage is that it is possible to change the reaction ratio (ratio of raw material input) between the silicon atom-free radical polymerizable monomer and the organic silane compound in which an alkoxy group is bonded to a silicon atom and which is further radical polymerizable. In other words, the composition ratio of the resulting polymer fine particles can be easily controlled. This makes it possible to control the properties (such as specific gravity) of the polymer nanoparticles obtained.

[0057] The particle manufacturing method according to this embodiment may include a step of adding a ligand after the second step.

[0058] The particle size of the polymer microparticles is preferably such that the average particle size is between 50 nm and 400 nm, and more preferably between 100 nm and 400 nm. Furthermore, the coefficient of variation (CV value) of the particle size of the polymer microparticles is preferably 5 or less.

[0059] While dynamic light scattering (DLS), laser diffraction (LD), and electron microscopy are the main methods for measuring average particle size, dynamic light scattering is preferably used in this embodiment. For example, the dispersion of polymer microparticles can be measured using dynamic light scattering, the resulting light intensity can be converted into a number distribution, and the average value can be used as the particle size. If the average particle size is 50 nm or more, there is no risk of requiring a long washing process during synthesis. Also, if it is 400 nm or less, there is no risk of significant aggregation due to sedimentation during storage.

[0060] The coefficient of variation (CV value) can be determined by taking images of polymer microparticles with an electron microscope, determining the diameter of, for example, 100 or more particles, and then performing statistical processing. If the coefficient of variation (CV value) is 5 or less (i.e., the particle size distribution is not too wide), there is no risk of instability in the reaction between the sample particles and the antigen.

[0061] The particle size of polymer microparticles can be controlled by the ratio of monomer to aqueous medium during synthesis, the amount of water-soluble polymer added, the reaction temperature, and the reaction time.

[0062] The dispersion of polymer microparticles produced in this manner is purified by classification and removal of unreacted substances, aggregates, etc., through methods such as filtration, decantation by centrifugation, and ultrafiltration. The method for producing a test reagent according to the present invention includes the step of dispersing polymer fine particles in a dispersion medium.

[0063] (Details of the particles according to this embodiment) The particles according to the embodiment of the present invention are a copolymer comprising repeating units represented by the following formula (I) and repeating units represented by the following formula (II), and particles having a structure represented by the following formula (III). [ka] [ka] [ka]

[0064] In equation (II), A 1 Or A 3 Each of these is independently one of the bonds that bond to Si in formula (II) via -H, -CH3, -CH2CH3, or -O-.

[0065] In equation (III), X 1 ~X 4 Each independently represents a bond with Si in adjacent structures represented by formula (III) via O, a bond with O bonded to Si in formula (II), or a hydroxyl group, and at least one of the structural units represented by formula (III) has a bond with the copolymer, R 3 and R 4 At least one of these represents a linear or branched alkyl group having 1 to 30 carbon atoms, which may contain -NH-CO-, -NH-, -O-, -S-, or -CO-, substituted with at least one carboxyl group or at least one amino group.

[0066] Furthermore, the R 3 ,R 4 Preferably, at least one of them has the structure shown in formula (2) or formula (3) below, R 3 and R 4 It is more preferable that at least one of each of them has the structure shown in formula (3) below. [ka] [ka]

[0067] Furthermore, a copolymer containing the repeating unit represented by formula (I) and the repeating unit represented by formula (II) may further contain the repeating unit represented by the following formula (IV). [ka]

[0068] In equation (IV), Y is one of H, Na, or K.

[0069] <Particles for specimen testing> Polymer microparticles are precursor particles for specimen testing. Antibodies can be immobilized onto these precursor particles, allowing them to be used as particles for specimen testing.

[0070] The immobilization method utilizes the functional groups of polymer nanoparticles and involves methods such as chemical bonding and physical adsorption. The method is not limited in this invention.

[0071] Antibodies immobilized on polymer microparticles are used for sample testing. When an antigen is present in the sample, the sample testing particles agglutinate via the antigen, and the antigen can be detected by observing this agglutination. If the target antigen is not present in the sample, agglutination is unlikely to occur.

[0072] (Affinity particles) In this embodiment, affinity particles can be provided, each having particles according to this embodiment and a ligand bound to a reactive compound. In this embodiment, a ligand is a compound that specifically binds to a receptor on a particular target substance. The site on which the ligand binds to the target substance is fixed, and it has selective or specific high affinity. Examples include antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, and nucleic acids, but the ligand in this embodiment is not limited to these. Examples of nucleic acids include deoxyribonucleic acid. The affinity particles in this embodiment have selective or specific high affinity for the target substance. It is preferable that the ligand in this embodiment is an antibody, an antigen, or a nucleic acid.

[0073] In this embodiment, the method for the chemical reaction to chemically bond the reactive functional group of the particles according to this embodiment with the ligand can be a conventionally known method, to the extent that the objective of the present invention can be achieved. Furthermore, when forming an amide bond with the ligand, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

[0074] In this embodiment, when affinity particles use an antibody (antigen) as a ligand and an antigen (antibody) as a target substance, they can be preferably applied to immunolatex agglutination assays, which are widely used in fields such as clinical testing and biochemical research.

[0075] (Diagnostic reagents for in vitro diagnostics) The in vitro diagnostic reagent in this embodiment, that is, the reagent used for detecting a target substance in a sample by in vitro diagnostics, comprises affinity particles according to this embodiment and a dispersion medium for dispersing the affinity particles. The amount of affinity particles according to this embodiment contained in the reagent in this embodiment is preferably 0.001% by mass or more and 20% by mass or less, and more preferably 0.01% by mass or more and 10% by mass or less.

[0076] The reagent according to this embodiment may contain, to the extent that the objectives of the present invention can be achieved, a third substance such as a solvent or a blocking agent in addition to the affinity particles according to this embodiment. Two or more types of third substances such as solvents or blocking agents may be included in combination.

[0077] Examples of solvents used in this embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents included in the reagents in this embodiment are not limited to these.

[0078] When used for the detection of antigens or antibodies in a sample by latex agglutination, the ligand can be either an antibody or an antigen.

[0079] (In vitro diagnostic test kit) The test kit used for detecting a target substance in a sample by in vitro diagnostics in this embodiment comprises the above-mentioned test reagent and a housing that encloses the above-mentioned test reagent.

[0080] The kit according to this embodiment may contain a sensitizer for latex agglutination measurement. Examples of sensitizers for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, and polyalginic acid, but the present invention is not limited to these.

[0081] Furthermore, the kit according to this embodiment may include a positive control, a negative control, a serum diluent, etc. As the medium for the positive control and negative control, in addition to serum and physiological saline that do not contain the target substance that can be measured, a solvent may be used.

[0082] The kit according to this embodiment can be used in the method for detecting a target substance according to this embodiment, in the same way as kits used for detecting a target substance in a sample by conventional in vitro diagnostics. Furthermore, the concentration of the target substance can also be measured by conventionally known methods, and it is preferable to use it for detecting a target substance in a sample by agglutination methods, particularly latex agglutination methods.

[0083] (Detection method) The method for detecting a target substance in a sample by in vitro diagnostics in this embodiment includes the step of mixing a sample that may contain the target substance with the test reagent according to this embodiment.

[0084] The mixing of the test reagent and the sample according to this embodiment is preferably carried out in the pH range of 3.0 to 11.0. The mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 1 minute to 20 minutes. Furthermore, it is preferable to use a solvent in this detection method. In addition, the concentration of affinity particles according to this embodiment in the detection method according to this embodiment is preferably 0.001% by mass or more and 5% by mass or less, more preferably 0.01% by mass or more and 1% by mass or less in the reaction system.

[0085] The detection method according to this embodiment preferably involves optically detecting the agglutination reaction that occurs as a result of mixing affinity particles according to this embodiment with a sample, that is, detecting the target substance in the sample by latex agglutination. Specifically, the method preferably includes the steps of: mixing the sample with the test reagent according to this embodiment to obtain a mixture; irradiating the mixture with light; and detecting at least one of the transmitted light or scattered light of the light irradiated onto the mixture.

[0086] By optically detecting the agglutination reaction that occurs in the mixture, the target substance in the sample can be detected, and its concentration can also be measured. The method for optically detecting the agglutination reaction involves using an optical instrument capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc., and measuring the changes in these values. [Examples]

[0087] The present invention will be described in more detail below using examples, comparative examples, and reference examples. However, the present invention is not limited to the following embodiments.

[0088] (Preparation of PSS-1) 90 g of phosphate buffer (Kishida Chemical Co., Ltd., pH 7.4) was placed in a 200 mL flask, and 0.9 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved in it. Next, 3.0 g of 3-methacrylateoxypropyltrimethoxysilane (product name: LS-3380, Shin-Etsu Chemical Co., Ltd.) and 9.0 g of styrene (Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature while blowing nitrogen into it for 10 minutes. After that, the emulsion in the flask was heated to 70°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 15 mL of phosphate buffer (Kishida Chemical Co., Ltd., pH 7.4) was added to the emulsion heated to 70°C. After stirring at 70°C for 7 hours, the mixture was returned to room temperature to obtain a dispersion of polystyrene silica hybrid microparticles. The obtained polystyrene silica hybrid microparticles were designated as PSS-1.

[0089] Observation using an electron microscope revealed that the average particle size of PSS-1 was 253 nm. The coefficient of variation for the particle size of PSS-1 was 1.5.

[0090] The obtained PSS-1 was dispersed in epoxy resin and cured. The resulting samples were then thinned using a microtome and subjected to transmission electron microscopy (TEM) observation and scanning transmission electron microscopy-energy dispersive X-ray analysis (STEM-EDS elemental analysis). Elemental analysis of the cross-sections of the thinned particles, both on the outer and central surfaces, revealed the presence of carbon, oxygen, and silicon. Since there was no significant difference in the amount of silicon detected relative to carbon, it was considered that there was no significant bias in the silicon distribution within the particles.

[0091] Furthermore, the PSS-1 dispersion was subjected to a centrifuge to recover the PSS-1, and the supernatant was discarded. The recovered PSS-1 was redispersed in deionized water and then subjected to another centrifuge. The recovery of PSS-1 by centrifugation and redispersion in deionized water was repeated four times.

[0092] The PSS-1 dispersion obtained in this manner was adjusted to have a particle concentration of 0.5% by mass and a particle dispersion volume of 200 mL.

[0093] (Preparation of PSS-2) 157 g of 50 mM 2-morpholinoethanesulfonic acid (MES, Tokyo Chemical Industries, Ltd.) buffer (pH 7.0) and 1.3 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) were mixed. Next, 4 g of 3-methacrylateoxypropyltrimethoxysilane (Shin-Etsu Chemical Co., Ltd., LS-3380) and 13 g of styrene (Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature while blowing in nitrogen for 10 minutes. After that, the emulsion in the flask was heated to 70°C in an oil bath. 0.5 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) was dissolved in deionized water, and the resulting solution was added to the emulsion heated to 70°C. After stirring at 70°C for 7 hours, heating was stopped and the mixture was stirred overnight to obtain a dispersion of polystyrene silica hybrid fine particles. The obtained polystyrene silica hybrid fine particles were designated as PSS-2.

[0094] Furthermore, the PSS-2 dispersion was subjected to a centrifuge to recover the PSS-2, and the supernatant was discarded. The recovered PSS-2 was then washed by repeatedly dispersing it in ion-exchanged water and then centrifugating it multiple times.

[0095] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the PSS-2 obtained in this manner was evaluated by dynamic light scattering and found to be 203 nm.

[0096] (Preparation of PSS-3) The following ingredients were mixed together. • 50 mM phosphate buffer (manufactured by Kishida Chemical Co., Ltd., pH 7.4): 157 g • Polyvinylpyrrolidone K-30 (manufactured by Kishida Chemical Co., Ltd., molecular weight 40000): 0.43g Random copolymer of N-vinyl-2-pyrrolidone and acrylic acid (manufactured by Polymer Source, molecular weight 60,000, ratio of N-vinyl-2-pyrrolidone molecules to acrylic acid molecules = 4:6, hereinafter abbreviated as PVP-PAA): 0.87g

[0097] Next, 4 g of 3-methacrylateoxypropyltrimethoxysilane (trade name: LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) and 13 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature for 10 minutes while blowing in nitrogen. After that, the emulsion in the flask was heated to 70°C in an oil bath. 0.5 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in deionized water, and the resulting solution was added to the emulsion heated to 70°C. After stirring at 70°C for 7 hours, the heating was stopped and the mixture was stirred overnight to obtain a dispersion of polystyrene silica hybrid fine particles. The obtained polystyrene silica hybrid fine particles were designated as PSS-3.

[0098] Furthermore, the PSS-3 dispersion was subjected to a centrifuge to recover the PSS-3, and the supernatant was discarded. The recovered PSS-3 was then washed by repeatedly dispersing it in ion-exchanged water and then centrifugating it multiple times.

[0099] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the PSS-3 obtained in this manner was evaluated by dynamic light scattering and found to be 301 nm.

[0100] (Preparation of PSS-4) The following ingredients were mixed together. 25 mM 2-morpholinoethanesulfonic acid (MES, manufactured by Tokyo Chemical Industry Co., Ltd.) buffer (pH 7.0): 157 g • Polyvinylpyrrolidone K-30 (manufactured by Kishida Chemical Co., Ltd., molecular weight 40000): 1.3g • Sodium parastyrene sulfonate (NaPSS): 0.05g

[0101] Next, 4 g of 3-methacrylateoxypropyltrimethoxysilane (trade name: LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) and 13 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and the mixture was stirred for 10 minutes at room temperature while blowing in nitrogen. After that, the emulsion in the flask was heated to 70°C in an oil bath. 0.5 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in deionized water, and the resulting solution was added to the emulsion heated to 70°C. After stirring at 70°C for 7 hours, the heating was stopped and the mixture was stirred overnight to obtain a dispersion of polystyrene silica hybrid fine particles. The obtained polystyrene silica hybrid fine particles were designated as PSS-4.

[0102] Furthermore, the PSS-4 dispersion was subjected to a centrifuge to recover the PSS-4, and the supernatant was discarded. The recovered PSS-4 was then washed by repeatedly dispersing it in ion-exchanged water and then centrifugating it multiple times.

[0103] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the PSS-4 obtained in this manner was evaluated by dynamic light scattering and found to be 153 nm.

[0104] (Preparation of PSS-5) In the preparation of PSS-4, the amount of NaPSS used was changed to 0.3g. Otherwise, PSS-5 was prepared in the same manner as PSS-4.

[0105] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the obtained PSS-5 was evaluated by dynamic light scattering and found to be 93 nm.

[0106] (Example 1) 2 g of Tween20 (manufactured by Tokyo Chemical Industry Co., Ltd.) was added as a surfactant to the PSS-1 dispersion, and then 0.1 mL of X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent containing a carboxyl group, was added and the mixture was stirred at room temperature for 14 hours. The resulting particles were designated as PSSX-1.

[0107] Observation using an electron microscope revealed that the average particle size of PSSX-1 was 250 nm. The coefficient of variation of the particle size of PSSX-1 was 1.4.

[0108] Furthermore, the PSSX-1 dispersion was subjected to centrifugation to recover the PSSX-1, and the supernatant was discarded. The PSSX-1 was dispersed in ion-exchanged water and adjusted to a particulate solid content concentration of 1.0 mass%.

[0109] (Example 2) The amount of X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) used in Example 1 was changed to 0.01 mL. Otherwise, the procedure was carried out in the same manner as in Example 1, and the resulting particles were designated as PSSX-2. PSSX-2 was dispersed in ion-exchanged water to obtain a dispersion with a particle solid content concentration of 1.0% by mass.

[0110] Observation using an electron microscope revealed that the average particle size of PSSX-2 was 254 nm. The coefficient of variation of the particle size of PSSX-2 was 1.5.

[0111] (Example 3) In Example 1, X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was replaced with KBP-90 (manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent containing an amino group, and the amount used was 0.01 mL. Otherwise, the procedure was carried out in the same manner as in Example 1. The obtained polymer fine particles were dispersed in ion-exchanged water to obtain a dispersion with a particle solid content concentration of 1.0% by mass.

[0112] Next, succinic anhydride dissolved in dimethyl sulfoxide was added to the obtained polymer microparticle dispersion at a ratio of 0.1% by mass relative to the dispersion, and the mixture was stirred at room temperature for 14 hours to modify the surface of the polymer microparticles with carboxyl groups. The resulting particles were designated PSSX-3.

[0113] Observation using an electron microscope revealed that the average particle size of PSSX-3 was 255 nm. The coefficient of variation of the particle size of PSSX-3 was 1.4.

[0114] (Example 4) In Example 1, X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was replaced with KBP-64 (manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent containing ethylenediamine, and the amount used was 0.01 mL. The procedure was otherwise carried out in the same manner as in Example 1, and the resulting polymer fine particles were dispersed in ion-exchanged water to obtain a dispersion with a particle solid content concentration of 1.0% by mass.

[0115] Next, the surface of PSSX-4 was modified with carboxyl groups in the same manner as the modification of polymer microparticles with carboxyl groups in Example 3. The resulting particles were designated as PSSX-4.

[0116] Observation using an electron microscope revealed that the average particle size of PSSX-4 was 255 nm. The coefficient of variation for the particle size of PSSX-4 was 1.4.

[0117] (Examples 5-16) PSS-2 to PSS-5 were used as the original particles and mixed with 0.5 M 2-morpholinoethanesulfonic acid (MES, manufactured by Tokyo Chemical Industry Co., Ltd.) buffer (pH 7.0), Tween® 20 (manufactured by Tokyo Chemical Industry Co., Ltd.) as a surfactant, and deionized water. After stirring this mixture at room temperature for 15 minutes, X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent containing a carboxyl group, was added and the mixture was stirred at room temperature overnight. Each material was used in the amounts shown in Table 1, or in amounts that resulted in the final concentrations shown in Table 1.

[0118] Next, this dispersion was subjected to a centrifuge, and the supernatant was discarded. The recovered particles were redispersed in ion-exchanged water and washed by repeating the centrifugation process several times. The particles obtained from Examples 5 to 16 were designated PSSX-5 to PSSX-16, respectively.

[0119] [Table 1]

[0120] The average particle size was evaluated by dynamic light scattering using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical). As a result, the average particle size of PSSX-11 was 165 nm, the average particle size of PSSX-12 was 171 nm, and the average particle size of PSSX-13 was 176 nm.

[0121] (Comparative Example 1) IMMUTEX polymer particles P0113 for immunodiagnosis (manufactured by JSR, polymer particles in which carboxyl groups are bonded to polystyrene particles with a particle size of 0.187 μm, hereinafter simply referred to as P0113) were used as the particles for Comparative Example 1. The concentration of P0113 was adjusted to 1.0% by mass using deionized water.

[0122] Observation using an electron microscope revealed that the average particle size of P0113 was 190 nm. The coefficient of variation for the particle size of P0113 was 2.1.

[0123] (Comparative Example 2) IMMUTEX polymer particles P0307 for immunodiagnosis (manufactured by JSR, polymer particles in which carboxyl groups are bonded to polystyrene particles with a particle size of 0.351 μm, hereinafter simply referred to as P0307) were used as the particles for Comparative Example 2. The concentration of P0307 was adjusted to 1.0% by mass using deionized water.

[0124] Observation using an electron microscope revealed that the average particle size of P0307 was 355 nm. The coefficient of variation for the particle size of P0307 was 2.8.

[0125] (Reference example 1) 90 g of phosphate buffer (Kishida Chemical Co., Ltd., pH 6.4) was added to a 200 mL flask, and 0.9 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved in it. Next, 0.4 g of 3-methacrylateoxypropyltrimethoxysilane (Shin-Etsu Chemical Co., Ltd., LS-3380) and 11.6 g of styrene (Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature while blowing in nitrogen for 10 minutes. After that, the emulsion in the flask was heated to 75°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 15 mL of water was added to the emulsion heated to 75°C. After stirring at 75°C for 6 hours, the mixture was returned to room temperature to obtain a dispersion of polystyrene silica hybrid microparticles. The obtained polystyrene silica hybrid microparticles were designated as PSS-7.

[0126] Furthermore, the PSS-7 dispersion was subjected to a centrifuge to recover the PSS-7, and the supernatant was discarded. The recovered PSS-7 was then redispersed in deionized water and subjected to another centrifuge. The recovery of PSS-7 by centrifuge and redispersion in deionized water was repeated four times. The PSS-7 dispersion obtained in this manner was adjusted to a concentration of 1.0% by mass.

[0127] Observation using an electron microscope revealed that the average particle size of PSS-7 was 140 nm. The coefficient of variation for the particle size of PSS-7 was 2.0.

[0128] (Reference example 2) 90 g of phosphate buffer (Kishida Chemical Co., Ltd., pH 9.0) was added to a 200 mL flask, and 0.4 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved in it. Next, 7.2 g of 3-methacrylateoxypropyltrimethoxysilane (Shin-Etsu Chemical Co., Ltd., LS-3380) and 4.8 g of styrene (Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature while blowing nitrogen into it for 10 minutes. After that, the emulsion in the flask was heated to 80°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 15 mL of water was added to the emulsion heated to 80°C. After stirring at 80°C for 4 hours, the mixture was returned to room temperature to obtain a dispersion of polystyrene silica hybrid microparticles. The obtained polystyrene silica hybrid microparticles were designated as PSS-8.

[0129] Furthermore, the PSS-8 dispersion was subjected to a centrifuge to recover the PSS-8, and the supernatant was discarded. The recovered PSS-8 was then redispersed in deionized water and subjected to another centrifuge. The recovery of PSS-8 by centrifuge and redispersion in deionized water was repeated four times.

[0130] The PSS-8 dispersion obtained in this manner was adjusted to a concentration of 1.0% by mass.

[0131] Observation using an electron microscope revealed that the average particle size of PSS-8 was 295 nm. The coefficient of variation for the particle size of PSS-8 was 3.0.

[0132] (Reference example 3) The PSS-1 dispersion was diluted to 0.1% by mass, and aminopropyltrimethoxysilane was added at a ratio of 0.05% by mass relative to the dispersion. The mixture was stirred at room temperature for 14 hours to modify the surface of PSS-1 with amino groups. The resulting particles were designated as PSSZ-1.

[0133] Observation using an electron microscope revealed that the average particle size of PSSZ-1 was 255 nm. The coefficient of variation for the particle size of PSSZ-1 was 1.5.

[0134] (Reference example 4) The PSS-1 dispersion was diluted to 0.1% by mass, and 2% by mass of 28% by mass aqueous ammonia and 0.05% by mass of mercaptopropyltrimethoxysilane were added to the dispersion. The mixture was then stirred at room temperature for 14 hours to modify the surface of PSS-1 with thiol groups. The resulting particles were designated as PSSZ-2.

[0135] Observation using an electron microscope revealed that the average particle size of PSSZ-2 was 262 nm. The coefficient of variation of the particle size of PSSZ-2 was 1.7.

[0136] (Reference example 5) The PSS-1 dispersion was diluted to 0.1% by mass, and aminopropyltrimethoxysilane was added at a ratio of 0.05% by mass relative to the dispersion. The mixture was stirred at room temperature for 14 hours to modify the surface of PSS-1 with amino groups. Furthermore, the surface of the particles was modified with carboxyl groups in the same manner as the modification of polymer microparticles with carboxyl groups in Example 3. The resulting particles were designated as PSSZ-3.

[0137] Observation using an electron microscope revealed that the average particle size of PSSZ-3 was 255 nm. The coefficient of variation for the particle size of PSSZ-3 was 1.5.

[0138] (Reference example 6) The PSS-1 dispersion was diluted to 0.1% by mass, and Si-tag fusion protein A (manufactured by Silicon Bio, Inc., a fusion protein of an antibody-binding protein (protein A) and a peptide that binds to a silanol group) was added. The Si-tag fusion protein A was then immobilized on the surface of the PSS-1. The resulting particles were designated as PSSZ-4.

[0139] Observation using an electron microscope revealed that the average particle size of PSSZ-4 was 253 nm. The coefficient of variation for the particle size of PSSZ-4 was 1.5.

[0140] (Reference example 7) To the PSS-1 dispersion, 0.5% by mass of 3-glycidyloxypropyltrimethoxysilane, 0.5% by mass of glycine, and 2% by mass of 28% by mass of aqueous ammonia were added, and the mixture was stirred at room temperature for 14 hours. The surface of the polymer microparticles was then modified with glycidyl groups. The resulting particles were designated as PSSZ-5.

[0141] Observation using an electron microscope revealed that the average particle size of PSSZ-5 was 260 nm. The coefficient of variation for the particle size of PSSZ-5 was 2.1.

[0142] (Reference example 8) A dimethyl sulfoxide solution of trimethoxypropyl succinic anhydride was added to a PSS-1 dispersion at a ratio of 0.4% by mass relative to the dispersion, and the mixture was stirred at room temperature for 14 hours to modify the surface of PSS-1 with dicarboxyl groups. The resulting particles were designated PSSZ-6.

[0143] Observation using an electron microscope revealed that the average particle size of PSSZ-3 was 265 nm. The coefficient of variation for the particle size of PSSZ-3 was 1.8.

[0144] (Reference example 9) In Example 1, X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was replaced with X-12-967C (manufactured by Shin-Etsu Chemical Co., Ltd.), a silane coupling agent containing succinic anhydride, and the amount used was set to 0.01 mL. The procedure was otherwise carried out in the same manner as in Example 1, and the resulting particles were designated as PSSX-17. PSSX-17 was dispersed in ion-exchanged water to obtain a dispersion with a particle solid content concentration of 1.0% by mass.

[0145] Observation using an electron microscope revealed that the average particle size of PSSX-17 was 252 nm. The coefficient of variation for the particle size of PSSX-17 was 1.5.

[0146] (Reference example 10) The concentration of the MES buffer and the amount of each material used in the preparation of PSS-2 were changed as shown in Table 1. Otherwise, polystyrene-silica hybrid microparticles were obtained in the same manner as the preparation of PSS-2. The obtained polystyrene-silica hybrid microparticles were designated as PSS-9 to PSS-17, respectively.

[0147] Furthermore, using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical), the average particle size of the obtained PSS-9 to PSS-17 was evaluated by dynamic light scattering, and the results are shown in Table 2.

[0148] [Table 2]

[0149] (Reference example 11) The amounts of each material used in the preparation of PSS-3 were changed as follows: • 50mM phosphate buffer (Kishida Chemical Co., Ltd., pH 7.4): 39g • Polyvinylpyrrolidone K-30 (manufactured by Kishida Chemical Co., Ltd., molecular weight 40000): 0g (not used) Random copolymer of N-vinyl-2-pyrrolidone and acrylic acid (manufactured by Polymer Source, molecular weight 60,000, ratio of N-vinyl-2-pyrrolidone molecules to acrylic acid molecules = 4:6, hereinafter abbreviated as PVP-PAA): 0.33g • 3-Methacryloxypropyltrimethoxysilane (product name: LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.): 1g • Styrene (manufactured by Kishida Chemical Co., Ltd.): 3g • Potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.): 0.125g

[0150] Otherwise, polystyrene-silica hybrid fine particles were obtained in the same manner as the preparation of PSS-3. The obtained polystyrene-silica hybrid fine particles were designated as PSS-18.

[0151] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the PSS-18 obtained in this manner was evaluated by dynamic light scattering and found to be 203 nm.

[0152] (Reference example 12) In the preparation of PSS-4, the concentration of the MES buffer used was changed to 50 mM. Otherwise, PSS-19 was prepared in the same manner as PSS-4.

[0153] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the obtained PSS-19 was evaluated by dynamic light scattering and found to be 138 nm.

[0154] (Reference example 13) In the preparation of PSS-4, the amount of NaPSS used was changed to 0.1g. Otherwise, PSS-20 was prepared in the same manner as PSS-4.

[0155] Using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical Corporation), the average particle size of the obtained PSS-20 was evaluated by dynamic light scattering and found to be 126 nm.

[0156] Compared to PSS-4 particles (particle size 153 nm), PSSX-11 (average particle size 165 nm), PSSX-12 (average particle size 171 nm), and PSSX-13 (average particle size 176 nm) all had larger particle sizes. Furthermore, a tendency for particle size to increase was observed as the amount of silane coupling agent added increased. In other words, it is possible to adjust the thickness of the layer coating the particles by adjusting the amount of silane coupling agent.

[0157] By performing acid-base titration while measuring the electrical conductivity of the particle solution, the amount of carboxyl groups in the particles can be calculated from the titration curve. This method was used to evaluate PSSX-5, PSSX-6, PSSX-7, PSSX-11, PSSX-12, and PSSX-13 particles. The results are shown in Table 3.

[0158] [Table 3]

[0159] The results shown in Table 3 indicate that the amount of carboxyl groups in the particles can be adjusted by adjusting the amount of silane coupling agent.

[0160] <Antibody binding example 1> Anti-C-reactive protein (hereinafter abbreviated as CRP) antibodies were conjugated to PSSX-1, PSSX-2, PSSZ-3, PSSZ-5, PSSZ-6, PSSX-17, and PSS-2 as follows.

[0161] First, each particle dispersion was centrifuged at 15,000 rpm (20,400 g) for 15 minutes to precipitate the particles. After removing the supernatant, the particle pellet was redispersed in MES buffer, and water-soluble carbodiimide (WSC) was added. N-hydroxysuccinimide was then added. The particle dispersion was stirred at room temperature for 30 minutes, and the particles were recovered by centrifugation. The recovered particles were washed with MES buffer, redispersed in MES buffer, and anti-CRP antibody was added to a final concentration of 100 μg / mL. After stirring at room temperature for 180 minutes, the particles were recovered by centrifugation. The recovered particles were thoroughly washed with tris(hydroxymethyl)aminomethane (Tris) buffer to obtain particles bound to the anti-CRP antibody. The particles to which the obtained CRP antibody was bound were designated PSSX-1-Ab, PSSX-2-Ab, PSSZ-3-Ab, PSSZ-5-Ab, PSSZ-6-Ab, PSSX-17-Ab, and PSS-2-Ab, respectively.

[0162] The binding of antibodies to each obtained particle was confirmed by measuring the decrease in antibody concentration in the antibody-added buffer using a BSA assay. It was found that approximately 100 to 500 antibodies were bound to each particle. All obtained particles were stably dispersed in the buffer, and post-coating with BSA was not necessary.

[0163] <Antibody binding example 2> 0.1 mL (1 mg particle) of each dispersion (1.0% by mass solution, 10 mg / mL) of PSSX-6, PSSX-7, PSSX-11, PSSX-12, and PSSX-13 was transferred to a microcentrifuge tube (1.5 mL capacity). To each, 0.12 mL of activation buffer (25 mM MES buffer, pH 6.0) was added, and the mixture was centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes. After centrifugation, the supernatant was discarded using a pipette. Next, 0.12 mL of activation buffer was added, and the mixture was dispersed ultrasonically using an ultrasonic cleaner (product name: MODEL VS-100III AS ONE 3-frequency ultrasonic cleaner, manufactured by AS ONE, 28 kHz). Then, the mixture was centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes. The supernatant was discarded using a pipette, and 0.12 mL of activation buffer was added, and the mixture was dispersed ultrasonically. Next, the mixture was centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes, and the supernatant was discarded using a pipette. Subsequently, 60 μL each of WSC solution (50 mg of WSC dissolved in 1 mL of activation buffer) and N-hydroxysulfosuccinimide (Sulfo NHS) solution (50 mg of Sulfo NHS dissolved in 1 mL of activation buffer) were added. After addition, the mixture was dispersed using ultrasound. Further stirring at room temperature for 30 minutes converted the carboxyl groups of the particles to the activated ester.

[0164] Next, the dispersion was centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes, and the supernatant was discarded using a pipette. 0.2 mL of immobilization buffer (25 mM MES buffer, pH 5.0) was added, and the mixture was dispersed by sonication. The mixture was centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes, and the supernatant was discarded using a pipette. 50 μL of immobilization buffer was added per 1 mg of particles, and the particles with activated carboxyl groups were dispersed by sonication.

[0165] Anti-human CRP antibody (polyclonal antibody) was diluted with immobilization buffer to concentrations of 25 μg / 50 μL, 50 μg / 50 μL, 100 μg / 50 μL, and 200 μg / 50 μL (referred to as antibody solution). 50 μL of antibody solution of each concentration was added to 50 μL of a solution of carboxyl-activated particles (containing 1 mg of particles), and the particles were dispersed by sonication. The amount of antibody used for binding was 25, 50, 100, and 200 μg per 1 mg of particle, as shown in Table 4. The tubes were agitated at room temperature for 60 minutes to immobilize the antibody onto the carboxyl groups of the particles. Then, the tubes were centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes, and the supernatant was discarded using a pipette.

[0166] 0.24 mL of an active ester inactivation buffer containing tris(hydroxymethyl)aminomethane (Tris) (1 M Tris buffer, pH 8.0 with 0.1% Tween® 20) was added and dispersed by sonication. The mixture was stirred at room temperature for 1 hour to allow Tris to bind to the remaining active ester, and then allowed to stand overnight at 4°C.

[0167] Next, the mixture was centrifuged at 4°C and 15000 rpm (20400 g) for 20 minutes, and the supernatant was discarded using a pipette. 0.2 mL of washing and storage buffer (10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.9) was added, and the mixture was dispersed by sonication. After repeating the washing procedure with 0.2 mL of washing and storage buffer twice, 1.0 mL of washing and storage buffer was added, and the mixture was dispersed by sonication. Since almost no particle loss was observed in the above steps, the final antibody-sensitized particle concentration was 0.1% by mass (1 mg / mL). The mixture was stored in a refrigerator and redispersed by sonication before use.

[0168] The average particle size of the obtained antibody-sensitized particles was evaluated by dynamic light scattering using a particle size analyzer (product name: Zetasizer, manufactured by Malvern Panalytical). The types of antibody-sensitized particles obtained and their average particle sizes are shown in Table 4.

[0169] (Measurement of antibody sensitization efficiency) In antibody binding example 2, antibody-sensitized particles were prepared, and their sensitization (immobilization) was confirmed by protein quantification.

[0170] Specifically, antibody-sensitized particles were reacted with BCA reagent. First, 25 μL (particle volume 25 μg) of the dispersion (0.1% by mass solution) of each antibody-sensitized particle was taken. 7 mL of solution A and 140 μL of solution B from the Protein Assay BCA Kit (Wako Pure Chemical Industries, Ltd.) were mixed to make solution AB. 200 μL of solution AB was added to 25 μL of the dispersion and allowed to stand at 60°C for 30 minutes. After standing, the solution was centrifuged at 4°C and 15000 rpm (20400 g) for 5 minutes, and 200 μL of the supernatant was collected with a pipette. The absorbance of the collected solution to light at a wavelength of 562 nm was measured using a multimode microplate reader (product name: SynergyMX, BioTek). Separately, several standard samples were prepared by dissolving antibodies in 10 mM HEPES to concentrations ranging from 0 to 200 μg / mL. Similarly, standard curves were created by measuring the absorbance of solutions prepared by reacting the standard samples with BCA reagents to light at a wavelength of 562 nm. Using the obtained standard curves, the amount of antibody bound to the antibody-sensitized particles was determined.

[0171] The amount of antibody sensitized to the antibody-sensitized particles (amount of antibody immobilized per unit mass of antibody-sensitized particles (μg / mg)) was determined by dividing the amount of antibody calculated above by the mass of the antibody-sensitized particles used for measurement. Furthermore, the antibody sensitization efficiency was determined from the amount of antibody used for binding and the amount of antibody sensitization.

[0172] The results are shown in Table 4. Measurements of antibody sensitization efficiency revealed that the antibody sensitization efficiency increased as the amount of silane coupling agent used increased. One reason for this is that as the amount of silane coupling agent used increases, the amount of carboxyl groups on the particle surface increases, which increases the number of points on which antibodies can be immobilized.

[0173] [Table 4]

[0174] <Antibody binding example 3> Antibodies were conjugated to PSSX-8, PSSX-9, PSSX-10, PSS-3 prepared in Examples 8-10, and PSS-18 prepared in Reference Example 11.

[0175] In antibody binding example 2, the anti-human CRP antibody (polyclonal antibody) was replaced with two types of anti-human prostate-specific antigen (hereinafter referred to as PSA) antibodies (monoclonal antibodies). Otherwise, the procedure was carried out in the same manner as in antibody binding example 2, and two types of antibody-sensitized particles, each containing a different antibody, were obtained.

[0176] The antibody sensitization efficiency of the two types of antibody-sensitized particles obtained was measured according to the method described above. Equal amounts of the two antibody-sensitized particles containing each antibody were also mixed. Table 5 shows the obtained antibody-sensitized particles and the results of the antibody sensitization efficiency measurements. All of the particles were capable of sensitizing antibodies with high antibody sensitization efficiency.

[0177] [Table 5]

[0178] <Antibody binding example 4> Anti-CRP antibodies were conjugated to particles according to Comparative Example 1 and Comparative Example 2. The dispersions prepared in Comparative Examples 1 and 2 were centrifuged at 15,000 rpm (20,400 g) for 20 minutes to precipitate the particles. After removing the supernatant, the particle pellet was redispersed in MES buffer, and WSC solution was added. Anti-CRP antibody was then added to a final concentration of 100 μg / mL. After stirring at room temperature for 180 minutes, BSA solution was added. Subsequently, the BSA-coated particles were recovered by centrifugation. The recovered particles were washed with phosphate buffer to obtain particles coated with BSA and conjugated with anti-CRP antibody.

[0179] The binding of antibodies to the obtained particles was confirmed by measuring the decrease in antibody concentration in the antibody-added buffer using a BCA assay. It was found that approximately 500 antibodies were bound to each particle.

[0180] Immediately after the antibody was bound to the particles, the particles were visually observed to aggregate and precipitate. Because the antibody-bound particles aggregated and precipitated, they could not be used to evaluate the latex agglutination reaction.

[0181] <Antibody binding example 5> An anti-CRP antibody was conjugated to PSSZ-2 prepared in Reference Example 4. To obtain a thiol-reactive antibody, EMCS (6-maleimidohexanoate N-succinimidyl) was added to the anti-CRP antibody solution to introduce a maleimide group into the antibody. The PSSZ-2 dispersion was centrifuged at 15000 rpm (20400 g) for 15 minutes to precipitate the PSSZ-2. After removing the supernatant, the PSSZ-2 pellet was redispersed in phosphate buffer, and the pre-maleimidized anti-CRP antibody was added. After stirring at room temperature for 180 minutes, the particles were collected by centrifugation. The collected particles were thoroughly washed with phosphate buffer to obtain microparticles conjugated with the anti-CRP antibody. The obtained CRP antibody-conjugated particles were designated PSSZ-2-Ab.

[0182] The binding of antibodies to PSSZ-2-Ab was confirmed by measuring the decrease in antibody concentration in the antibody-added buffer using a BSA assay. It was found that approximately 100 to 500 antibodies were bound to each particle. PSSZ-2-Ab was stably dispersed in the buffer, and post-coating with BSA was not necessary.

[0183] <Antibody binding example 6> Anti-CRP antibody was conjugated to PSSZ-4 prepared in Reference Example 6. The PSSZ-4 dispersion was centrifuged at 15000 rpm (20400 g) for 20 minutes to precipitate the PSSZ-4. After removing the supernatant, the PSSZ-4 pellet was redispersed in Tris buffer, and anti-CRP antibody was added to a final concentration of 200 μg / mL. After stirring at 4°C for 16 hours, the particles were collected by centrifugation. The collected particles were thoroughly washed with Tris buffer to obtain particles conjugated with anti-CRP antibody. The obtained particles conjugated with CRP antibody were designated as PSSZ-4-Ab.

[0184] The binding of antibodies to PSSZ-4-Ab was confirmed by measuring the decrease in antibody concentration in the antibody-added buffer using a BSA assay. It was found that approximately 500 antibodies were bound to each particle. PSSZ-4-Ab was stably dispersed in the buffer, and post-coating with BSA was not necessary.

[0185] <Evaluation of non-specific aggregation suppression> 30 μL of PSS-1 particle dispersion, adjusted to a concentration of 1.0% by mass, and 30 μL of each particle dispersion prepared in Examples 1-4, Comparative Examples 1 and 2, and Reference Examples 1-9 were each added to 60 μL of buffer solution and 4 μL of human serum solution, and incubated at 37°C for 5 minutes. Absorbance to light at a wavelength of 572 nm was measured before and after incubation, and the change in absorbance between the two states was measured three times, with the average value calculated. A change in absorbance of less than 0.1 was considered to indicate suppression of nonspecific aggregation, while a change of 0.1 or more was considered to indicate the occurrence of nonspecific aggregation. The results are shown in Table 6.

[0186] Furthermore, as can be seen from Table 6, the particles produced in Examples 1-4 and Reference Examples 1-9 were found to disperse stably in human serum without agglutination. On the other hand, the particles in Comparative Examples 1 and 2 exhibited agglutination (non-specific agglutination) in reaction with human serum. This is thought to be due to the non-specific adsorption of proteins to the surface of the microparticles, resulting in crosslinking between particles via the adsorbed proteins. When the particles in Comparative Examples 1 and 2 were post-coated with BSA beforehand, they dispersed stably in human serum without agglutination. This is thought to be because albumin adsorbed to the surface of the polystyrene particles, preventing the adsorption of proteins derived from human serum.

[0187] Furthermore, similar evaluations of non-specific aggregation suppression were performed on the dispersions of PSS-2, 3, and 9-18. As a result, the change in absorbance was less than 0.1 in all polymer microparticle dispersions, indicating that no non-specific aggregation occurred.

[0188] [Table 6]

[0189] <Evaluation of Latex Agglutination Reaction 1: Evaluation of Antigen Recognition Ability of Antibody-Sensitized Particles against CRP> A 0.1% by mass dispersion of antibody-sensitized particles PSSX-1-Ab, PSSX-2-Ab, PSSZ-2-Ab~PSSZ-6-Ab, and PSSX-17-Ab, prepared using antibody binding examples 1, 5, and 6, was used to perform a latex agglutination reaction.

[0190] Human serum-derived CRP (Sigma-Aldrich) was mixed with various antibody-sensitized particle dispersions, and the absorbance to light at a wavelength of 572 nm was measured before and after mixing. A UV-Vis spectrophotometer (product name: GeneQuant 1300, GE Healthcare) was used to measure the absorbance, and the samples were injected into plastic cells with a path length of 10 mm. If the anti-CRP antibody on the antibody-sensitized particles can capture the antigen CRP, an aggregation reaction between the microparticles occurs via CRP. This results in a decrease in transmittance, leading to an observed increase in absorbance.

[0191] First, a CRP solution was prepared by mixing 1 μL of 32 mg / L CRP with 50 μL of phosphate buffer. Subsequently, a reaction solution prepared by adding 51 μL of CRP solution to 50 μL of antibody-sensitized particle dispersion, and a reaction solution prepared by adding 51 μL of buffer instead of CRP solution to 50 μL of antibody-sensitized particle dispersion, were treated at 37°C for 5 minutes. For each reaction solution, the percentage change in absorbance before and after treatment was measured three times, and the average value was calculated. The results are shown in Table 7.

[0192] As shown in Table 7, an agglutination reaction occurred due to the antigen-antibody reaction when CRP was added, and a clear difference was observed when comparing the rate of change with that when buffer solution was added instead of CRP solution (without CRP).

[0193] As can be seen from Table 7, the antibody-sensitized particles used in this evaluation recognized CRP and aggregated with each other, as confirmed by the evaluation of the latex agglutination reaction. In other words, the particles prepared in Examples 1 and 2 and Reference Examples 4-9 were found to be able to bind antibodies without using BSA, and the bound antibodies were found to have antigen recognition ability.

[0194] The results shown in Tables 6 and 7 indicate that particles were obtained that had a scaffold capable of binding ligands such as antibodies, while suppressing nonspecific adsorption of serum proteins without the use of BSA.

[0195] [Table 7]

[0196] <Evaluation of Latex Agglutination Reaction 2: Sensitivity Evaluation of Antibody-Sensitized Particles to CRP> The sensitivity of antibody-sensitized particles prepared in antibody binding example 2 was evaluated using latex immunoaggregation. Specifically, antibody-sensitized particles were reacted with an antigen to form immunocomplex aggregates, and these aggregates were irradiated with light. The attenuation of the irradiated light due to scattering (absorbance) was measured using a spectrophotometer. The proportion of aggregates and the absorbance increased depending on the amount of antigen contained in the sample. For sensitivity evaluation, it is desirable that the increase in absorbance (expressed as ΔOD × 10000) when using a solution containing a predetermined concentration of CRP is large. A UV-Vis spectrophotometer (product name: GeneQuant 1300, GE Healthcare) was used to measure absorbance, and the sample was injected into a plastic cell and measured with a path length of 10 mm. The measurement method is described below in detail.

[0197] 1 μL of CRP standard solution (CRP concentration 5-160 μg / mL) and 50 μL of R1 buffer (phosphate-based buffer) were mixed in a plastic cell and heated at 37°C for 5 minutes. 50 μL of antibody-sensitized particle dispersion solution (particle concentration 0.1% by mass, 10 mM HEPES, pH 7.9, 0.01% by mass Tween® 20) was added to 51 μL of R1 buffer containing the CRP standard solution. Then, the mixture was quickly pipetted, taking care not to introduce air bubbles, to prepare the sample for measurement. The absorbance of the sample to light at a wavelength of 572 nm was read and designated as Abs1. After heating the sample at 37°C for 5 minutes, the absorbance of the sample to light at a wavelength of 572 nm was read and designated as Abs2. The value obtained by subtracting Abs1 from Abs2 and multiplying by 10000 was taken as the ΔOD × 10000 value. To evaluate non-specific reactions, the ΔOD × 10000 value was similarly calculated for samples prepared using a solution obtained by mixing 1 μL of serum solution without CRP (CRP concentration 0 μg / mL) with 50 μL of R1 buffer, and a dispersion solution of antibody-sensitized particles.

[0198] The results are shown in Table 8. For all antibody-sensitized particles used in this evaluation, an increase in ΔOD×10000 was observed with increasing CRP concentration. This indicates that the antibody-sensitized particles bound to the antigen CRP and formed particle aggregates, demonstrating their function as particles for use in latex immunoaggregation. Furthermore, the antibody-sensitized particles used in this evaluation had a ΔOD×10000 of 100 or less at a CRP concentration of 0 μg / mL, indicating that non-specific agglutination did not occur. In addition, it was shown that increasing the amount of antibody used to sensitize the particles increased the value of ΔOD×10000.

[0199] [Table 8]

[0200] <Evaluation of Latex Agglutination Reaction 3: Sensitivity Evaluation of Antibody-Sensitized Particles to Human PSA> The antibody-sensitized particles prepared in Antibody Binding Example 3 were evaluated for PSA sensitivity following the method described in Evaluation of Latex Agglutination Reaction 2.

[0201] Specifically, 16 μL of PSA standard solution (PSA concentration 91.7 ng / mL) and 60 μL of R1 buffer were mixed in a plastic cell and heated at 37°C for 5 minutes. 30 μL of antibody-sensitized particle dispersion solution (particle concentration 0.2 mass%) was added to R1 buffer (76 μL) containing the PSA standard solution, and the mixture was quickly pipetted, taking care to avoid air bubbles, to prepare the sample for measurement. The absorbance of the sample to light at a wavelength of 572 nm was read and designated as Abs3. After heating the sample at 37°C for 5 minutes, the absorbance of the sample to light at a wavelength of 572 nm was read and designated as Abs4. The value obtained by subtracting Abs3 from Abs4 and multiplying by 10000 was taken as the ΔOD × 10000 value. To evaluate non-specific reactions, the ΔOD × 10000 value was similarly calculated for samples prepared using a solution obtained by mixing 1 μL of PSA-free serum solution (PSA concentration 0 ng / mL) with 50 μL of R1 buffer, and a dispersion solution of antibody-sensitized particles.

[0202] The results are shown in Table 9. For all antibody-sensitized particles used in this evaluation, an increase in ΔOD × 10000 was observed in the presence of PSA. This indicates that the antibody-sensitized particles bound to the antigen, PSA, and formed particle aggregates, demonstrating their function as particles for use in latex immunoaggregation. Furthermore, the antibody-sensitized particles used in this evaluation had a ΔOD × 10000 of 100 or less at a PSA concentration of 0 ng / mL, indicating that non-specific agglutination did not occur.

[0203] [Table 9]

[0204] (Example 17) Evaluation of PSSX-1's performance in suppressing nonspecific aggregation of albumin To evaluate the non-specific aggregation suppression performance of PSSX-1 obtained in Example 1, non-specific aggregation of albumin was confirmed. First, 50 μL of a 0.1% by mass aqueous dispersion of PSSX-1 and 50 μL of a 0.05% or 0.5% bovine serum albumin solution (bovine serum albumin dissolved in phosphate-buffered saline) were mixed and incubated at 37°C for 5 minutes. The absorbance at 572 nm was measured before and after incubation, and the change in absorbance between the two was measured. If non-specific aggregation of albumin occurs, the value of the change in absorbance × 10000 will be 1000 or more. The value obtained by multiplying the change in absorbance by 10000 is shown below. 0.05% albumin solution: 20 0.5% albumin solution: 80 These results clearly show that PSSX-1 does not aggregate with albumin.

[0205] (Example 18) Evaluation of PSSX-1's performance in inhibiting nonspecific aggregation of human serum globulin fractions. To evaluate the non-specific aggregation inhibitory performance of PSSX-1 obtained in Example 1, non-specific aggregation of human serum globulin fractions was confirmed. First, 16 μL of globulin solution with a globulin concentration of 0.48 mg / mL or 48 mg / mL was added to 60 μL of phosphate buffer and incubated at 37°C for 5 minutes. Then, 30 μL of a 0.1% by mass aqueous dispersion of PSSX-1 was added and incubated at 37°C for 5 minutes. The absorbance at 572 nm was measured before and after incubation, and the change in absorbance between the two was measured. If non-specific aggregation of human serum globulin fractions occurs, the value of the change in absorbance × 10000 will be 1000 or more. The value of the change in absorbance multiplied by 10000 is shown below. Globulin concentration 0.48 mg / mL: 0 Globulin concentration 48 mg / mL: 60 These results clearly show that PSSX-1 does not agglutinate with human serum globulin fractions.

[0206] (Example 19) Evaluation of PSSX-1's inhibitory performance in suppressing nonspecific aggregation of human serum fibrin To evaluate the non-specific aggregation inhibitory performance of PSSX-1 obtained in Example 1, non-specific aggregation of human serum fibrin was confirmed. First, 50 μL of a 0.1% by mass aqueous dispersion of PSSX-1 and 50 μL of a human serum fibrin solution (prepared on-site) were mixed and incubated at 37°C for 5 minutes. After incubation, particle aggregation was observed visually, and no particle aggregation was observed. These results clearly show that PSSX-1 does not agglutinate with human serum fibrin.

[0207] (Example 20) Synthesis and evaluation of particles with different particle sizes Polystyrene-silica hybrid nanoparticles were obtained by changing the amount of each material used in the preparation of PSS-2 and PSS-4 as shown in Table 10, except that the preparation was otherwise the same as for PSS-2 and PSS-4. The obtained particles were designated PSSX-18 to PSSX-33. Table 10 shows the particle size and dispersibility. By changing the formulation, the particle size could be controlled within the range of 93.1 nm to 423.2 nm. When the particle size exceeded 340 nm, the dispersibility of the particles in water tended to deteriorate.

[0208] [Table 10]

[0209] (Example 21) Measurement of COOH content of particles and evaluation of nonspecific reaction suppression The amount of COOH on the particle surface and the ability to suppress nonspecific reactions of the particles were evaluated for PSSX-23, PSSX-24, and PSSX-26 to PSSX-33 obtained in Example 8. The results are shown in Table 11. By changing the formulation, the amount of COOH could be controlled from 18 nmol / mg to 334 nmol / mg. When human serum was added to these particle solutions, the change in absorbance, which is an indicator of the absence of nonspecific reactions, was less than 1000, indicating that no nonspecific reactions to human serum were observed.

[0210] [Table 11]

[0211] The present invention is not limited to the embodiments described above, and various modifications and variations are possible without departing from the spirit and scope of the invention. Accordingly, the following claims are attached to make the scope of the invention public.

Claims

1. A first step involves mixing a radically polymerizable monomer, an organosilane compound having silicon atoms bonded to an alkoxy group and possessing radical polymerizability, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium to prepare an emulsion. A second step is to add a reactive compound after the first step, It has, A method for producing particles represented by the following formula (1) in which the reactive compound is described above. 【Chemistry 1】 (In equation (1), R １ and R ２ Each of these represents a linear or branched alkyl group having 1 to 30 carbon atoms, which may contain -NH-CO-, -NH-, -O-, -S-, or -CO-, substituted with at least one carboxyl group or at least one amino group.

2. R in formula (1) １ and R ２ The method for producing particles according to claim 1, wherein at least one of the members has at least one carboxyl group.

3. The method for producing particles according to claim 1 or 2, further comprising the step of heating the emulsion after the first step.

4. The method for producing particles according to any one of claims 1 to 3, wherein the radical polymerizable monomer is a monomer that does not contain silicon atoms.

5. The method for producing particles according to any one of claims 1 to 4, wherein the radical polymerizable monomer is at least one selected from the group consisting of styrene monomers, acrylate monomers, and methacrylate monomers.

6. The method for producing particles according to any one of claims 1 to 5, wherein the radical polymerizable monomer is at least one selected from the group consisting of styrene, butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, and methyl acrylate.

7. A method for producing particles according to any one of claims 1 to 6, wherein in the first step, parastyrene sulfonate is further mixed to prepare the emulsion.

8. The method for producing particles according to any one of claims 1 to 7, wherein the water-soluble polymer is at least one selected from the group consisting of polyacrylamide, polyvinyl alcohol, polyethylene oxide, and polyvinylpyrrolidone and polyvinylpyrrolidone-polyacrylic acid copolymer.

9. The method for producing particles according to any one of claims 1 to 8, wherein the organosilane compound is at least one selected from the group consisting of vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane.

10. The method for producing particles according to any one of claims 1 to 9, wherein the aqueous medium has a pH of 6 or more and 9 or less.

11. R in formula (1) １ and R ２ A method for producing particles according to any one of claims 1 to 10, wherein at least one of the particles has a structure represented by the following formula (2). 【Chemistry 2】 (In formula (2) above, Z represents a linear or branched saturated or unsaturated alkyl group having 1 to 10 carbon atoms.)

12. R in formula (1) １ and R ２ A method for producing particles according to any one of claims 1 to 10, wherein at least one of the particles is represented by the following formula (3). 【Transformation 3】

13. A method for producing particles according to any one of claims 1 to 12, further comprising the step of adding a ligand after the second step.

14. A method for producing particles according to any one of claims 1 to 12, wherein the average particle size of the particles produced by the method according to any one of claims 1 to 13 is 100 nm or more and 400 nm or less, and the coefficient of variation of the particle size is 5 or less.

15. A method for producing a test reagent, comprising the step of dispersing particles produced by the manufacturing method described in any one of claims 1 to 14 in a dispersion medium.

16. A copolymer comprising repeating units represented by the following formula (I) and repeating units represented by the following formula (II), and particles having a structure represented by the following formula (III). 【Chemistry 4】 【Transformation 5】 【Transformation 6】 (In formula (II), A １ to A ３ are each independently any of the bonds that are bonded to Si in formula (II) via -H, -CH ３ , -CH ２ CH ３ ; In formula (III), X １ to X ４ each independently represents a bond with Si in the adjacent structure represented by formula (III via O), a bond with O bonded to Si in formula (II), or a hydroxy group, and at least one of the structural units represented by formula (III) has a bond with the copolymer, and R ３ and R ４ at least one of them represents a linear or branched alkyl group having 1 to 30 carbon atoms which may contain -NH-CO-, -NH-, -O-, -S-, -CO- and is substituted with at least one carboxy group or at least one amino group.)

17. The particle according to claim 16, wherein the copolymer further comprises repeating units represented by the following formula (IV). 【Transformation 7】 (In formula (IV), Y is either H, Na, or K.)

18. R in formula (III) above ３ and R ４ At least one of these is represented by the following formula (3), the particle according to any one of claims 15 to 17. 【Transformation 8】

19. A first step involves mixing a radically polymerizable monomer, an organosilane compound having silicon atoms bonded to an alkoxy group and possessing radical polymerizability, a radical polymerization initiator, a water-soluble polymer, and an aqueous medium to prepare an emulsion. Particles produced by a second step of adding a reactive compound after the first step, The reactive compound is a particle represented by the following formula (1). 【Chemistry 9】 (In formula (1) above, R １ and R ２ Each of these represents a linear or branched alkyl group having 1 to 30 carbon atoms, which may contain -NH-CO-, -NH-, -O-, -S-, or -CO-, substituted with at least one carboxyl group or at least one amino group.

20. R in formula (1) １ and R ２ The particle according to claim 19, wherein at least one of the particles has at least one carboxyl group.

21. R in formula (1) １ and R ２ The particle according to claim 19 or 20, wherein at least one of the particles has a structure represented by the following formula (2). 【Chemistry 10】 (In formula (2) above, Z represents a linear or branched saturated or unsaturated alkyl group having 1 to 10 carbon atoms.)

22. R in formula (4) ３ and R ４ The particle according to claim 19 or 20, wherein at least one of each of the particles has a structure represented by the following formula (3). 【Chemistry 11】

23. The particle according to any one of claims 19 to 22, wherein the radical polymerizable monomer is a monomer that does not contain silicon atoms.

24. The particle according to any one of claims 19 to 23, wherein the radical polymerizable monomer is at least one selected from the group consisting of styrene monomers, acrylate monomers, and methacrylate monomers.

25. The particle according to any one of claims 19 to 24, wherein the radical polymerizable monomer is at least one selected from the group consisting of styrene, butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, and methyl acrylate.

26. The particles according to any one of claims 19 to 25, wherein in the first step, parastyrene sulfonate is further mixed to prepare the emulsion.

27. The particles according to any one of claims 19 to 26, wherein the water-soluble polymer is at least one selected from the group consisting of polyacrylamide, polyvinyl alcohol, polyethylene oxide, and polyvinylpyrrolidone and polyvinylpyrrolidone-polyacrylic acid copolymer.

28. A particle according to any one of claims 19 to 27, having one or more structural units represented by the following formula (4). 【Chemistry 12】 (In formula (4), X independently represents a bond with Si in adjacent structures represented by formula (3) via O, a bond with the core of the particles prepared from the emulsion, or a hydroxyl group, and at least one of the structural units represented by formula (4) has a bond with the core.) R ３ and R ４ At least one of these represents a linear or branched alkyl group having 1 to 30 carbon atoms, which may contain -NH-CO-, -NH-, -O-, -S-, or -CO-, substituted with at least one carboxyl group or at least one amino group.

29. R in formula (4) ３ and R ４ The particle according to claim 28, wherein at least one of each of the particles has a structure represented by the following formula (2). 【Chemistry 13】 (In formula (2), Z represents a linear or branched saturated or unsaturated alkyl group having 1 to 10 carbon atoms.)

30. R in formula (4) ３ and R ４ The particle according to claim 28, wherein at least one of each of the particles has a structure represented by the following formula (3). 【Chemistry 14】

31. Affinity particles comprising particles according to any one of claims 19 to 30 and a ligand bound to the reactive compound.

32. The affinity particle according to claim 31, wherein the ligand is any of an antibody, an antigen, and a nucleic acid.

33. An in vitro diagnostic test reagent comprising affinity particles according to claim 31 or 32, and a dispersion medium for dispersing the affinity particles.

34. The diagnostic reagent according to claim 33, wherein the ligand is an antibody or antigen, and is used for detecting an antigen or antibody in a sample by agglutination.

35. An in vitro diagnostic test kit comprising a test reagent according to claim 33 or 34 and a housing containing the test reagent.

36. A method for detecting a target substance in a sample, A detection method comprising the step of mixing a sample with the test reagent according to claim 33 or 34.

37. A method for detecting a target substance in a sample by agglutination, A step of mixing a sample with the test reagent according to claim 33 or 34 to obtain a mixed solution, The aforementioned mixture is irradiated with light, A detection method comprising the step of detecting at least one of transmitted light or scattered light of light irradiated onto the mixture.

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