PARTICLES, AFFINITY PARTICLES HAVING LIGAND FOR TARGET SUBSTANCE, AND IN VITRO DIAGNOSTIC REAGENTS AND KIT COMPRISING SAME, AND METHOD FOR DETECTING TARGET SUBSTANCE - Patent application

Abstract

Provided are: particles and affinity particles suitable for latex agglutination, the particles and affinity particles having little nonspecific adsorption and having a reactive functional group for chemically binding a ligand; an in vitro diagnostic reagent and a kit that include these particles as particles for latex agglutination; and a method for detecting a target substance.　Particles in which the particle surface layer includes a copolymer having repeating units A that each have a side chain A having a carboxy group for binding with a ligand at the side chain end, and repeating units B that each have a side chain B having a hydroxyl group at the side chain end, the particles being characterized in that the particle surface layer hydrates and forms a swollen layer when the particles are dispersed in ion-exchanged water, the carboxy group density contained in the swollen layer satisfies 0.04-0.15 / nm3, and the ratio of the dry grain size measured when the particles are dried and the grain size in water measured when the particles are dispersed in ion-exchanged water satisfies 1.10-1.40.

Description

[Technical field]

[0001] The present invention relates to particles, affinity particles having a ligand for a target substance, and in vitro diagnostic reagents and kits containing the same, as well as a method for detecting a target substance. [Background technology]

[0002] Latex agglutination is a simple and rapid immunological test method. In this method, a dispersion of affinity particles, which are composed of particles bound to ligands that have affinity for the target substance, is mixed with a specimen that may contain the target substance. If the specimen contains the target substance, the affinity particles will cause an agglutination reaction, and this agglutination reaction can be optically detected as changes in scattered light intensity, transmitted light intensity, absorbance, etc. to identify the presence or absence of a disease. In general, the combination of a ligand and a target substance is an antibody and an antigen, or an antigen and an antibody. For the particles used in the latex agglutination method and affinity particles characterized by having a ligand for a target substance on the surface of the particles, it is desirable that the particles have a property of adsorbing substances other than the target substance, that is, a small nonspecific adsorption, in order to prevent nonspecific agglutination reactions not derived from the target substance. In addition, depending on the testing institution, a dispersion of affinity particles may be stored stationary for several weeks, so that devising a way to maintain the dispersion state of affinity particles in the dispersion is an extremely important technical issue.

[0003] One method for reducing nonspecific adsorption of particles or affinity particles is to coat the particle surface with biological substances such as albumin, casein, gelatin, etc. However, the physical properties of these biological substances may differ depending on the production lot, and there are also concerns about future biological contamination due to the use of large amounts.

[0004] Coating the surface of particles or affinity particles with an amphiphilic polymer compound is also an effective method for reducing nonspecific adsorption. However, since the adsorption of the polymer compound to particles or affinity particles is due to physical adsorption, it may be possible for the polymer compound to be released by dilution, and nonspecific adsorption may not be sufficiently suppressed.

[0005] Particles with polyglycidyl methacrylate on the surface are known as particles with low nonspecific adsorption. It is believed that the nonspecific adsorption is suppressed by the polyglycidyl methacrylate on the surface of the particles, which has some of the glycidyl groups ring-opened to form glycols.

[0006] Patent Document 1 discloses an example in which particles having a surface on which polyglycidyl methacrylate is arranged and a ligand is chemically bonded via a polyethylene glycol chain are used for bioseparation.

[0007] Patent Document 2 discloses an example in which particles having polyglycidyl methacrylate disposed on the surface thereof and having a ligand chemically bonded via an amino acid are applied to a latex agglutination method. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2000-351814 A [Patent Document 2] International Publication No. 2007 / 063616 Summary of the Invention [Problem to be solved by the invention]

[0009] As a result of extensive investigations, the inventors have found that the particles of Patent Documents 1 and 2 may undergo non-specific adsorption in high concentration samples, may not have sufficient sensitivity when applied to latex agglutination methods, and there is a concern that electrostatic hetero-agglutination may occur under stationary conditions.

[0010] The present invention has been made in consideration of these background art and technical problems. An object of the present invention is to provide particles that have low non-specific adsorption, have reactive functional groups for chemically binding ligands, and are suitable for latex agglutination, and affinity particles that have excellent dispersion stability. Another object of the present invention is to provide in vitro diagnostic reagents and kits that contain these particles or affinity particles as particles for latex agglutination, and a method for detecting a target substance.

[0011] The present invention relates to particles, affinity particles having a ligand for a target substance, and in vitro diagnostic reagents and kits containing the same, as well as a method for detecting a target substance. [Means for solving the problem]

[0012] That is, the present invention relates to a particle having a surface layer comprising a copolymer having a repeating unit A and a repeating unit B, wherein the repeating unit A has a side chain A, the side chain A has a carboxy group at a side chain end for binding to a ligand, the repeating unit B has a side chain B, the side chain B has a hydroxyl group at a side chain end, the surface layer of the particle is configured to absorb water and form a swollen layer when the particle is dispersed in ion-exchanged water, the density of the carboxy groups contained in the swollen layer satisfies formula (1-1), and the dry particle size measured when the particle is dried and the underwater particle size measured when the particle is dispersed in ion-exchanged water satisfy formula (1-2). 0.040≦[Carboxy group density (pieces / nm 3 )]≦0.150...Equation (1-1) 1.10≦[Particle size in water / Dry particle size]≦1.40...Equation (1-2)

[0013] The present invention also relates to an affinity particle comprising the above particle bound to a ligand.

[0014] The present invention also relates to a reagent for use in detecting a target substance in a specimen by in vitro testing and diagnosis, characterized in that it contains the affinity particles, and further relates to the reagent for use in detecting a target substance in a specimen by an agglutination method (latex agglutination method). Another aspect of the present invention relates to an affinity particle having a ligand on the particle surface, characterized in that the area ratio of the ligand to the particle surface satisfies the relationship of formula (2-1), the zeta potential of the particle and the ligand satisfies the relationship of formula (2-2), and the particle has a repeating unit A represented by formula (2-3). 10≦[occupied area ratio (%)]≦40...Equation (2-1) 0≦[||Zeta potential of particle|-|Zeta potential of ligand||]≦20 Equation (2-2)

[0015] [ka]

[0016] (In formula (2-3), R1 represents a methyl group or a hydrogen atom. R2 represents a carboxy group or a hydrogen atom. L1 represents an alkylene group having 1 to 15 carbon atoms which may have a substituent or an oxyalkylene group having 1 to 15 carbon atoms which may have a substituent.)

[0017] Another aspect of the present invention relates to a reagent for use in detecting a target substance in a specimen by in vitro diagnosis, characterized in that it contains the affinity particles, and further, the reagent is for use in detecting a target substance in a specimen by an agglutination method (latex agglutination method).

[0018] That is, a further first aspect of the present invention is a particle having a copolymer containing a repeating unit A represented by the following formula (3-1), in which the repeating unit A has a structure containing a sulfide group, thereby enabling ligands to be chemically bonded to the particle surface with high yield.

[0019] [ka]

[0020] A second aspect of the present invention relates to particles for use in latex agglutination methods, characterized in that a ligand is chemically bonded via the repeating unit A.

[0021] Furthermore, a third aspect of the present invention relates to a reagent for use in the detection of a target substance in a specimen by in vitro diagnosis, characterized by containing the particles for latex agglutination, a kit for use in the detection of the target substance in a specimen by in vitro diagnosis, characterized by comprising at least the reagent, and a detection method, characterized by mixing the particles for latex agglutination with a specimen that may contain the target substance. Effect of the Invention

[0022] According to the present invention, it is possible to provide particles which have low non-specific adsorption, have reactive functional groups for binding to ligands, and are suitable for agglutination methods (latex agglutination methods).

[0023] Furthermore, in the present invention, affinity particles to which ligands are bound are used as particles for agglutination methods (latex agglutination methods), and it is possible to provide in vitro diagnostic reagents and kits containing these, as well as a method for detecting a target substance. According to another aspect of the present invention, there is provided an affinity particle having low non-specific adsorption and excellent dispersion stability. According to still another aspect of the present invention, there is provided a highly sensitive in vitro diagnostic reagent and kit containing the affinity particle for use in an agglutination method (latex agglutination method), and a method for detecting a target substance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] [Embodiment 1] Hereinafter, the first embodiment of the present invention will be described in detail, but the technical scope of the present invention is not limited to these embodiments.

[0025] The particle according to the first embodiment of the present invention is a particle whose surface layer contains a copolymer having a repeating unit A having a side chain A with a carboxy group at an end of the side chain for binding to a ligand, and a repeating unit B having a side chain B with a hydroxyl group at an end of the side chain.

[0026] The particles used in the aggregation method (for example, latex aggregation method) preferably have excellent dispersion stability in an aqueous dispersion liquid, and therefore, it is preferable that the carboxyl group forms a carboxylate. Examples of the carboxyl salt include metal salts such as sodium salts and potassium salts, and organic salts such as ammonium salts. Considering the reactivity when bonding the carboxyl group and the ligand by chemical reaction, it is more preferable to use an organic salt. Examples of organic bases for forming an organic salt with a carboxyl group include ammonia, diethylamine, triethylamine, ethanolamine, diethylaminoethanol, etc., but the present invention is not limited to these. Considering experimental operability such as boiling point and solubility in various solvents, triethylamine is easy to use. The organic bases for forming a carboxyl salt may be used alone or in combination of two or more types within a range that can achieve the object of embodiment 1. Similarly, a metal base and an organic base may be used in combination.

[0027] When the particles of embodiment 1 are dispersed in water or an aqueous solution, the particle surface becomes hydrated to form a hydrogel-like swollen layer. The characteristic of this swollen layer is that the carboxyl group density contained in the swollen layer satisfies the following formula (1-1), and the dry particle size measured when the particles are dried and the particle size in water measured when the particles are dispersed in ion-exchanged water satisfy the following formula (1-2). 0.04≦[Carboxy group density (pieces / nm 3 )]≦0.15...Equation (1-1) 1.10≦[Particle size in water / Dry particle size]≦1.40...Equation (1-2)

[0028] Carboxy group density is 0.04 / nm 3 If the density of carboxy groups is less than 0.15 / nm, the reactivity between the particles and the ligand (hereinafter referred to as the sensitization rate) is insufficient. 3 If the carboxyl group density is larger than 0.15 / nm, the water binding force of the swollen layer is high, and when the particles of embodiment 1 are applied to a latex agglutination method, osmotic pressure aggregation occurs, which is observed as a pseudo non-specific adsorption phenomenon, and is not preferable. 3 If it is larger, the zeta potential of the particle becomes large, and when the particle is bound to a ligand to form an affinity particle, electrostatic interaction occurs between the ligand and the particle surface, impairing the reactivity of the ligand with the target substance, and decreasing the detection sensitivity of the latex agglutination method. Furthermore, as the carboxy group density increases, the zeta potential difference with the ligand also increases. This means that the affinity particle surface with the ligand bound to the particle exhibits charges of opposite signs, which promotes electrostatic aggregation of the affinity particle. For this reason, it is preferable that the zeta potential of the particle of embodiment 1 satisfies formula (1-3). -30≦[Zeta potential (mV)]≦-10 Equation (1-3)

[0029] As described above, there is a preferred range for the carboxy group density in terms of the sensitization rate, osmotic agglutination, detection sensitivity when affinity particles are applied to the latex agglutination method, and electrostatic agglutination of affinity particles. For this reason, the carboxy group density is preferably 0.04 / nm 3 More than 0.15 pieces / nm 3 Less than or equal to 0.10 particles / nm 3 More than 0.13 pieces / nm 3 The following is the result.

[0030] Formula (1-2) correlates with the thickness of the swollen layer. The smaller the value of formula (1-2), the thinner the swollen layer, and the larger the value of formula (1-2), the thicker the swollen layer. When the [particle size in water / particle size in dry] is less than 1.10, it may be difficult to expect the effect of suppressing non-specific adsorption due to the water content of the swollen layer. In addition, since the water content of the swollen layer is insufficient, the mobilities of the carboxyl groups derived from the repeating unit A of the swollen layer in the aqueous dispersion are not large, and it may be difficult to achieve a sufficient sensitization rate. Furthermore, when the particles are bound to a ligand to form affinity particles, the mobilities of the ligand are also inhibited, impairing the reactivity of the ligand with the target substance, which may be a factor in reducing the detection sensitivity of the latex agglutination method. The [particle size in water / particle size in dry] being 1.10 or more also contributes greatly to the dispersion stability of the particles. It is preferable that the particles used in the latex agglutination method have excellent dispersion stability in normal samples and buffer solutions such as physiological saline. In this regard, when the [particle size in water / particle size in dry] is 1.10 or more, the swollen layer is sufficiently hydrated, and the particles are provided with dispersion stability based on the excluded volume effect. On the other hand, when the [particle size in water / particle size in dry] is more than 1.40, the water binding force of the swollen layer is too large, and therefore, when the particles of embodiment 1 are applied to the latex aggregation method, osmotic aggregation occurs, which is observed as a pseudo non-specific adsorption phenomenon, and is not preferable.

[0031] As described above, there is a preferred range for the thickness of the swollen layer in terms of the sensitization rate, the detection sensitivity when the affinity particles are applied to the latex agglutination method, the dispersion stability of the particles, and the osmotic pressure agglutination. For this reason, the ratio of [particle size in water / particle size in dry state] is preferably 1.10 or more and 1.40 or less, more preferably 1.15 or more and 1.30 or less.

[0032] It is preferable that the repeating unit A and the repeating unit B of the copolymer constituting the particle surface layer of the particle of embodiment 1 each have side chain A having a carboxy group at the side chain terminal for bonding to a ligand, and side chain B having a hydroxyl group at the side chain terminal, and that the number of moles of repeating unit A and the number of moles of repeating unit B satisfy formula (1-4). 0.05≦[number of moles of repeating unit A] / [number of moles of repeating unit B]≦1.00 Formula (1-4)

[0033] If the mole number of repeating unit A / molar number of repeating unit B is less than 0.05, there is a concern that the sensitization rate may decrease, although this is not fatal. In addition, a decrease in the ratio of repeating unit A to the particle surface layer is synonymous with a decrease in the amount of carboxyl groups, which are the source of negative charges, and there is a potential concern that it may become difficult to impart dispersion stability derived from electrostatic repulsion to the particles. If the mole number of repeating unit A / molar number of repeating unit B is 1.00 or more, the water binding force of the swollen layer formed by the water absorption of the particle surface layer becomes large, and when mixed with a high concentration sample, osmotic aggregation may occur.

[0034] A specific chemical structure of the repeating unit A of the copolymer constituting the particle surface layer of the particle of embodiment 1 will be described. The repeating unit A is characterized in that it has a carboxy group for binding to a ligand at the end of its side chain, and while its chemical structure is not limited within the scope that can achieve the object of embodiment 1, it is more preferable that the side chain structure be represented by formula (1-5).

[0035] [ka] R1: Methyl group or hydrogen atom R2: Carboxy group or hydrogen atom L1: an optionally substituted alkylene or oxyalkylene group having 1 to 15 carbon atoms

[0036] The hydroxyl group adjacent to the ester bond in formula (1-5) plays a role in reducing nonspecific adsorption of the particles. The sulfide bond is a chemical structure that exhibits a weak hydrophobic tendency, and the repeating unit A having the side chain structure shown in formula (1-5) is expected to moderately weaken the water binding force of the swollen layer formed by the water-containing surface of the particles, thereby suppressing osmotic aggregation that may occur when the particle is mixed with a high concentration sample.

[0037] In formula (1-5), L1 is preferably an alkylene group having a carbon number of 1. The carbon number of L1 correlates with the hydrophilicity or hydrophobicity of the repeating unit A, and if the carbon number is too large, it may promote nonspecific adsorption to particles.

[0038] From the viewpoint of the sensitization rate of the particles of embodiment 1, R2 in formula (1-5) is preferably a carboxy group.

[0039] A description will now be given of a specific chemical structure of the repeating unit B of the copolymer constituting the particle surface layer of the particle of embodiment 1. The repeating unit B of embodiment 1 is characterized by having a side chain B having a hydroxyl group at the end of the side chain, and although the chemical structure is not limited within the scope in which the object of embodiment 1 can be achieved, it is more preferable that the side chain structure be represented by formula (1-6).

[0040] [ka] R1: Methyl group or hydrogen atom L2: an alkylene group or oxyalkylene group having 2 to 15 carbon atoms which may be substituted, and which satisfies the following relationship: [number of carbon atoms in L1] + 2 ≧ [number of carbon atoms in L2] X: Sulfur atom or optionally substituted nitrogen atom

[0041] The hydroxyl group adjacent to the ester bond in formula (1-6) and the hydroxyl group at the end of the side chain play a role in reducing nonspecific adsorption of the particles. X in formula (1-6) may be either a sulfur atom or a nitrogen atom, but is preferably a sulfur atom. A sulfide bond is a structure that exhibits a weak hydrophobic tendency, and the repeating unit A having the side chain structure shown in formula (1-6) is expected to moderately weaken the water binding force of the swollen layer formed by the water-containing surface layer of the particle, thereby suppressing osmotic aggregation that may occur when mixed with a high concentration sample.

[0042] When comparing formula (1-5) and formula (1-6), the relationship between the number of carbon atoms constituting L1 in formula (1-5) and the number of carbon atoms constituting L2 in formula (1-6) is preferably the relationship of formula (1-7). If the relationship of formula (1-7) is not satisfied, side chain B will be relatively longer than side chain A, which may inhibit the reactivity of the carboxy group that side chain A has at the side chain end. For this reason, when L1 in formula (1-5) is an alkylene group having one carbon atom, L2 in formula (1-6) is preferably an alkylene group having two or three carbon atoms. [Number of carbon atoms in L1] + 2 ≧ [Number of carbon atoms in L2] Formula (1-7)

[0043] When L2 in formula (1-6) is an alkylene group having 3 carbon atoms, from the viewpoint of reducing non-specific adsorption of the particles, it is more preferable that the chemical structure has one of the alkylene groups substituted with a hydroxyl group.

[0044] The particles of embodiment 1 preferably have at least one repeating unit C selected from the group consisting of styrenes and (meth)acrylates, and more preferably contain repeating units derived from styrenes and glycidyl (meth)acrylate. As described in the background art of this specification, the repeating unit derived from glycidyl (meth)acrylate has the effect of reducing non-specific adsorption of the particles. On the other hand, the reason why it is preferable to contain a repeating unit derived from a styrene is as follows. When the particles of embodiment 1 are purified by a method such as centrifugation or ultrafiltration, the presence of a repeating unit of a styrene, which has a high glass transition temperature and excellent mechanical strength, contributes to suppressing damage such as cracking and chipping of the particles. Examples of styrenes include styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene, but are not limited thereto as long as the object of the first embodiment can be achieved. Two or more types of styrenes may be used in combination. The content of styrenes is preferably 10 parts by mass or more and 70 parts by mass or less, assuming that the mass of the particles is 100 parts by mass, because this can reduce nonspecific adsorption while imparting sufficient strength to the particles.

[0045] The particles of embodiment 1 can further contain repeating units derived from radically polymerizable monomers having crosslinking properties. Examples of radically polymerizable monomers having crosslinking properties include diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene glycol diacrylate, 2,2'-bis(4-(acryloxydiethoxy)phenyl)propane, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and triethylene glycol dimethacrylate. Examples of the monomer include, but are not limited to, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, 1,6-hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis(4-(methacryloxydiethoxy)phenyl)propane, 2,2'-bis(4-(methacryloxypolyethoxy)phenyl)propane, trimethylolpropane trimethacrylate, tetramethylolmethane tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether, as long as the object of the first embodiment can be achieved. In addition, two or more types of radical polymerizable monomers having crosslinking properties may be used in combination.

[0046] The particle size of the particles in embodiment 1 is preferably 0.05 μm to 1.00 μm, more preferably 0.05 μm to 0.50 μm, and even more preferably 0.05 μm to 0.30 μm. When the particle size is 0.05 μm to 0.30 μm, the particles are easy to handle and are less likely to settle when the particles are stored for a long period of time as a dispersion.

[0047] A description will be given of a typical method for producing the particles of embodiment 1. However, the method for producing the particles of embodiment 1 is not limited as long as the object of embodiment 1 can be achieved.

[0048] The method includes a step 1 of mixing glycidyl (meth)acrylate with styrene, divinylbenzene with water, and a radical polymerization initiator to form a granular copolymer, and obtaining an aqueous dispersion of the granular copolymer. The method also includes a step 2 of mixing the aqueous dispersion with 3-mercapto-1,2-propanediol and mercaptosuccinic acid to prepare a mixed solution, and reacting the epoxy groups derived from the glycidyl (meth)acrylate of the granular copolymer with the thiol groups derived from the 3-mercapto-1,2-propanediol and mercaptosuccinic acid to form the particles of the first embodiment. The radical polymerization initiator is at least any one of 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]tetrahydrate, 2,2'-azobis(2-methylpropionamidine)dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, and 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate. The method includes a step of adjusting the pH of the mixture of the step 2 to an alkaline region using an organic base that does not have a primary amine. In the step 2, the epoxy group derived from glycidyl (meth)acrylate of the granular copolymer is reacted with the thiol group derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid in the depth direction from the surface of the granular copolymer. In order to form a surface layer of sufficient thickness on the particles of embodiment 1, it is preferable to select triethylamine, which has permeability to the granular copolymer, as the organic base.

[0049] The method for forming the granular polymer is not limited to radical polymerization within the scope of the object of the first embodiment. When radical polymerization is applied, it is preferable to use emulsion polymerization, soap-free emulsion polymerization, or suspension polymerization, and it is more preferable to use emulsion polymerization or soap-free emulsion polymerization. It is more preferable to use soap-free emulsion polymerization. In general, emulsion polymerization and soap-free emulsion polymerization can obtain granular polymers with a sharp particle size distribution compared to suspension polymerization. In addition, when particles are bound to a ligand to form affinity particles, there is a concern that the ligand will be denatured if an anionic surfactant or cationic surfactant generally used in emulsion polymerization exists as a residue. For this reason, when a granular polymer is formed by emulsion polymerization, it is preferable to use a nonionic surfactant.

[0050] A ligand is a compound that specifically binds to a receptor of a specific target substance. The site where the ligand binds to the target substance is fixed, and the ligand has a selective or specific high affinity. Examples of the ligand include antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, and the like, but are not limited to these as long as the object of the first embodiment can be achieved. Specific examples of the ligand include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, and the like.

[0051] In the first embodiment, it is intended that the particles of the first embodiment are used as particles for latex agglutination, which can be applied to the latex agglutination method in immunoassays.

[0052] In the first embodiment, the method of chemical reaction for binding the carboxy group or carboxylate derived from the repeating unit A to a ligand can be a conventionally known method within the scope of achieving the object of the first embodiment. For example, carbodiimide-mediated reaction and NHS ester activation reaction are suitable examples of chemical reactions. Avidin may be bound to the carboxy group, and a biotin-modified ligand may be bound to the carboxy group. However, within the scope of achieving the object of the first embodiment, the method of chemical reaction for binding the carboxy group or carboxylate derived from the repeating unit A to a ligand is not limited to these.

[0053] In the first embodiment, when an antibody (antigen) is used as the ligand and an antigen (antibody) is used as the target substance, the method can be very preferably applied to the latex agglutination method in immunoassays, which is widely used in the fields of clinical testing, biochemical research, etc. as a method for detecting a target substance in a specimen in in vitro diagnosis. When general particles are used as particles for the latex agglutination method, the target substance, the antigen (antibody) or foreign matter in serum or plasma, etc., nonspecifically adsorbs to the particle surface, which may cause unintended inter-particle agglutination and impair the accuracy of the immunoassay.

[0054] The reagent for use in detecting a target substance in a specimen by in vitro diagnosis according to the first embodiment is characterized in that it contains latex agglutination particles. The amount of latex agglutination particles contained in the reagent according to the first embodiment is preferably 0.001% by mass to 20% by mass, more preferably 0.01% by mass to 10% by mass. The reagent according to the first embodiment may contain a third substance such as a solvent or a blocking agent in addition to the latex agglutination particles, within a range that can achieve the object of the first embodiment. Examples of the solvent used in the first embodiment include various aqueous buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, Hepes buffer, Meth buffer, and ammonia buffer, but the solvent contained in the reagent according to the first embodiment is not limited to these.

[0055] The kit for use in detecting a target substance in a specimen by in vitro diagnosis according to the first embodiment is characterized by comprising at least the reagent according to the first embodiment. The kit according to the first embodiment preferably further comprises a reaction buffer solution containing albumin (hereinafter, reagent 2) in addition to the reagent according to the first embodiment (hereinafter, reagent 1). The albumin may be serum albumin or the like, which may be protease-treated. The amount of albumin contained in the reagent 2 is approximately 0.001% by mass to 5% by mass, but the kit according to the first embodiment is not limited thereto. A sensitizer for latex agglutination measurement may be contained in both or either of the reagents 1 and 2. Examples of sensitizers for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, alginic acid, and the like, but the kit according to the first embodiment is not limited thereto. The kit according to the first embodiment may also comprise a positive control, a negative control, a serum dilution solution, and the like in addition to the reagents 1 and 2. As a medium for the positive control and the negative control, a solvent may be used in addition to serum and physiological saline that do not contain a measurable target substance. The kit of embodiment 1 can be used in the method for detecting a target substance of embodiment 1 in the same manner as a kit for use in detecting a target substance in a specimen by normal in vitro diagnosis. In addition, the concentration of the target substance can also be measured by a conventionally known method, and is particularly suitable for detecting a target substance in a specimen by a latex agglutination method.

[0056] The method for detecting a target substance in a specimen by in vitro diagnosis according to the first embodiment is characterized by mixing the affinity particles according to the first embodiment with a specimen that may contain the target substance. The affinity particles according to the first embodiment are preferably mixed with the specimen at a 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 to 20 minutes. In addition, it is preferable to use a solvent in this detection method. The concentration of the affinity particles according to the first embodiment in the detection method according to the first embodiment is preferably 0.001% by mass to 5% by mass, preferably 0.01% by mass to 1% by mass, in the reaction system. The detection method according to the first embodiment is characterized by optically detecting interparticle aggregation that occurs as a result of mixing the affinity particles according to the first embodiment with the specimen, and the target substance in the specimen can be detected by optically detecting the interparticle aggregation, and the concentration of the target substance can also be measured. The agglutination reaction can be optically detected by measuring the change in scattered light intensity, transmitted light intensity, absorbance, etc. using an optical device capable of detecting these values.

[0057] A method for measuring the dry particle size of particles in the first embodiment will be described. The dry particle size in the first embodiment is a number average particle size, which is measured when the particles are sufficiently dried. Specifically, the particles are dispersed in ion-exchanged water at a concentration of 5% by mass, the dispersion is dropped onto aluminum foil, and the particles are dried at 25°C for 48 hours, and then further dried for 24 hours using a vacuum dryer, after which the particle size is measured. A scanning electron microscope and an image processing analyzer are used to measure the dry particle size. Specifically, 100 individual particles are randomly extracted from a particle image in a dry state obtained using a scanning electron microscope (S-4800: Hitachi High-Technologies Corporation), and analyzed using an image processing analyzer "Luzex AP" (Nireco Corporation), to calculate the number average particle size.

[0058] A method for measuring the particle diameter in water of a particle in the first embodiment will be described. The particle diameter in water in the first embodiment is a number average particle diameter, and is measured in a state where the particles are dispersed in ion-exchanged water to a concentration of 0.001% by mass. Ion-exchanged water with an electrical conductivity of 10 μS / cm or less is used. A dynamic light scattering method is applied to measure the particle diameter in water. Specifically, a Zetasizer (Nano-ZS: Spectris Inc.) is used and the measurement is performed at 25° C. The analysis parameters are latex (n≒1.59) as the refractive index of the particles and pure water as the solvent. The measurement is performed 10 times, and the average value of the 10 measured values ​​is used as the particle diameter in water.

[0059] A method for measuring the zeta potential of particles in the first embodiment will be described. The zeta potential in the first embodiment is measured in a state where the particles are dispersed in a 0.01N potassium aqueous solution of pH 7.8 to a concentration of 0.001% by mass. The 0.01N potassium aqueous solution of pH 7.8 is obtained by appropriately mixing a 0.01N potassium chloride aqueous solution, which is adjusted using ion-exchanged water with an electrical conductivity of 10 μS / cm or less, a 0.01N potassium hydroxide aqueous solution, and a 0.01N hydrochloric acid aqueous solution. The measurement device is a Zetasizer (Nano-ZS: Spectris Inc.), and the measurement is performed at 25° C. The analysis parameters are latex (n≒1.59) as the refractive index of the particles and pure water as the solvent. The measurement is performed 10 times, and the average value of the 10 measured values ​​is used as the zeta potential.

[0060] The carboxy group density in the first embodiment is calculated from the relationship between the amount of carboxy groups in the particles and the volume of a swollen layer formed on the surface of the particles when the particles are dispersed in ion-exchanged water.

[0061] First, a method for measuring the amount of carboxy groups on the particles will be described. Using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC / HCl) as a catalyst, the carboxy groups on the particles are activated esterified with N-hydroxysuccinimide (NHS). After that, this activated ester is reacted with aminoethanol to release NHS, and the amount of free NHS is quantified by a high-performance liquid chromatograph to calculate the amount of carboxy groups per unit particle mass. Note that the unit of the amount of carboxy groups per unit particle amount is nmol / mg. The specific method will be described below.

[0062] <Activated esterification> Add an aqueous dispersion of particles containing an amount equivalent to 2.5 mg of particles to a 1.5 mL microtube, separate the particles from the dispersion using a centrifuge, and perform the operation of redispersing the particles in dimethylformamide (DMF) three times. Further, with the particles sedimented in the microtube by a centrifuge, remove the DMF from the microtube. Next, add 400 μL of DMF, 19.2 mg of EDC·HCl, and 100 μL of a 1 mol / L DMF solution of N-hydroxysuccinimide to this microtube, and shake at 25°C for 2 hours to activate esterify the carboxy groups on the particles.

[0063] <Release of NHS> To remove excess NHS from the DMF dispersion of the activated esterified particles, separate the activated esterified particles from the dispersion using a centrifuge, and perform the operation of redispersing the particles in DMF three times. Further, with the activated esterified particles sedimented in the microtube by a centrifuge, remove the DMF from the microtube. Next, add 500 μL of a 1 mol / L DMF solution of aminoethanol to this microtube, and shake at 25°C for 2 hours to release NHS from the particles.

[0064] <Quantification of NHS> The DMF dispersion of the particles containing the free NHS is centrifuged, the particles are allowed to settle in a microtube, and the DMF solution containing the free NHS is collected. The amount of the free NHS contained in the DMF solution is quantified using a high performance liquid chromatograph. The carboxyl group mass per unit particle weight is calculated from the quantified value.

[0065] The instruments and reagents used for quantifying the amount of carboxyl groups are as follows.

[0066] <Equipment> High-performance liquid chromatograph: LC20A-FL (Shimadzu Corporation) Column: Kinetex 5μm C18 100A LC Column 150×4.6mm (phenomenex) Eluent: 4mmol / L ammonium acetate aqueous solution Flow rate: 1.0mL / min Oven temperature: 40℃ Sample injection volume: 1 μL To quantify the amount of free NHS contained in the DMF solution, a calibration curve of NHS-derived peak area and NHS amount is prepared in the range of 0.1 mmol / L to 10 mmol / L, and the amount of free NHS is quantified from the NHS peak area.

[0067] <Reagents> EDC / HCl (Dojindo Chemical Research Institute Co., Ltd.) NHS (Kishida Chemical Co., Ltd.) Aminoethanol (Tokyo Chemical Industry Co., Ltd.) DMF (FUJIFILM Wako Pure Chemical Corporation) Ammonium acetate (Tokyo Chemical Industry Co., Ltd.)

[0068] Next, a method for calculating the carboxy group density of particles will be described. When calculating the amount of carboxy groups per unit volume contained in the swollen layer formed on the surface of a particle when the particle is dispersed in water or an aqueous solution, i.e., the carboxy group density, the particle size in water when the particle is dispersed in ion-exchanged water and the dry particle size of the particle are used. The particle density is 1 (g / cm) for both the dry particle size and the particle size in water. 3) is calculated by applying formula (1-8). [Carboxylic group density (pcs / nm 3 )]=Dd 3 / (Dw 3 -Dd 3 )×A×N A ×10 -27 ...Formula (1-8) In formula (1-8), Dw is the particle diameter in water (nm), Dd is the particle diameter in dry state (nm), A is the amount of carboxyl group per unit particle mass (nml / mg), N A is the Avogadro constant.

[0069] [Embodiment 2] Hereinafter, the second embodiment of the present invention will be described in detail, but the technical scope of the present invention is not limited to this embodiment.

[0070] Embodiment 2 relates to an affinity particle having a ligand on the particle surface, characterized in that the area ratio of the ligand to the particle surface satisfies the relationship of formula (2-1), the zeta potential of the particle and the ligand satisfies the relationship of formula (2-2), and the particle has a repeating unit A represented by formula (2-3). 10≦[occupied area ratio (%)]≦40...Equation (2-1) 0≦[||Zeta potential of particle|-|Zeta potential of ligand||]≦20 Equation (2-2)

[0071] [ka]

[0072] (In formula (2-3), R1 represents a methyl group or a hydrogen atom. R2 represents a carboxy group or a hydrogen atom. L1 represents an alkylene group having 1 to 15 carbon atoms which may have a substituent or an oxyalkylene group having 1 to 15 carbon atoms which may have a substituent.)

[0073] First, the particles will be described. In the second embodiment, the particles to which the ligand is bound to obtain affinity particles are simply called particles. The particles have a repeating unit A represented by the above formula (2-3).

[0074] The hydroxyl group adjacent to the ester bond in formula (2-3) plays a role in reducing non-specific adsorption of the affinity particles according to the second embodiment.

[0075] In formula (2-3), L1 is preferably a methylene group having one carbon atom. The number of carbon atoms in L1 correlates with the hydrophilicity or hydrophobicity of the repeating unit A, and if the number of carbon atoms in L1 is too large, it may promote nonspecific adsorption to affinity particles.

[0076] It is preferable that the surface layer of the particle contains a copolymer having a repeating unit A, and when the particle is dispersed in water or an aqueous solution, the surface layer absorbs water to form a swollen layer. The thickness of the swollen layer correlates with the ratio of the dry particle size of the particle measured when the particle is dried to the particle size in water measured when the particle is dispersed in ion-exchanged water, and it is preferable that the dry particle size and the particle size in water satisfy formula (2-5). 1.10≦[Particle size in water / Dry particle size]≦1.40...Equation (2-5)

[0077] The surface layer of the particle forms a swollen layer containing water in water or an aqueous solution, which contributes to the following: reducing non-specific adsorption to affinity particles, improving sensitivity when the affinity particles are used as particles for agglutination methods (e.g., latex agglutination methods), and improving the dispersion stability of affinity particles.

[0078] In particular, a [particle size in water / particle size on a dry basis] of 1.10 or more means that the swollen layer is sufficiently hydrated and the particle surface is hydrophilic, so that non-specific adsorption to affinity particles is significantly suppressed. Also, a [particle size in water / particle size on a dry basis] of 1.10 or more means that the swollen layer is sufficiently hydrated and the mobility of the ligands on the affinity particles is improved, improving the reactivity of the ligands with the target substance. Also, the swollen layer is sufficiently hydrated, so that the affinity particles are endowed with dispersion stability based on the excluded volume effect.

[0079] On the other hand, when the ratio of particle diameter in water to particle diameter in dry water is greater than 1.40, the water binding force of the swollen layer is large, and therefore, when the affinity particles of embodiment 2 are applied to the latex agglutination method, osmotic aggregation may occur, which is difficult for a testing institution to distinguish from a pseudo nonspecific agglutination phenomenon.

[0080] Thus, there is a preferred range for the thickness of the swollen layer from the viewpoints of non-specific adsorption, sensitivity when affinity particles are applied to the latex agglutination method, dispersion stability of affinity particles, and osmotic aggregation. For this reason, the ratio of [particle size in water / particle size in dry state] is preferably 1.10 or more and 1.40 or less, more preferably 1.15 or more and 1.30 or less.

[0081] When the particle surface layer forms a swollen layer containing water in water or an aqueous solution, the sulfide bond in the repeating unit A represented by formula (2-3) has a chemical structure that exhibits a weak hydrophobic tendency, and therefore moderately weakens the water binding force of the swollen layer, which is expected to suppress osmotic aggregation that may occur when the affinity particle according to embodiment 2 is mixed with a sample.

[0082] In formula (2-3), R2 is preferably a carboxy group. When R2 is a carboxy group, the particle surface layer is more likely to absorb water or an aqueous solution, which is advantageous for forming a swelling layer.

[0083] Next, the ligand will be described. A ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is fixed, and the ligand has a selective or specific high affinity. Examples of the ligand include antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, and the like, but are not limited to these as long as the object of the second embodiment can be achieved. Specific examples of the ligand include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, and the like.

[0084] The method for binding the particles to the ligand is not limited as long as the object of embodiment 2 can be achieved, but it is preferable to bind the ligand to the particle surface using a chemical reaction via a carboxy group derived from the repeating unit A represented by formula (2-3).

[0085] In the second embodiment, the chemical reaction method for binding the carboxy group derived from the repeating unit A to the ligand can be a conventionally known method within the scope of achieving the object of the second embodiment. For example, a carbodiimide-mediated reaction or an NHS ester activation reaction is a suitable example. Alternatively, avidin may be bound to the carboxy group, and then a biotin-modified ligand may be bound to the carboxy group.

[0086] When binding the carboxy group derived from the repeating unit A to a ligand, a part of the carboxy group may be converted to another chemical structure by a chemical reaction. In the second embodiment, such a conversion reaction is referred to as a masking treatment, and a reagent used in the masking treatment is referred to as a masking agent. Amines are often used as masking agents, and trishydroxymethylaminomethane is generally used among them. In response to this, the present inventors have confirmed that the detection sensitivity is greatly improved when the affinity particles according to the second embodiment are applied to the latex agglutination method by doing the following. That is, ethanolamine is used as a masking agent, and the carboxy group of a part of the repeating unit A is converted to a chemical structure represented by formula (2-6).

[0087] [ka]

[0088] (In formula (2-6), R1 represents a methyl group or a hydrogen atom. R2 represents a carboxy group or a hydrogen atom. L1 represents an alkylene group having 1 to 15 carbon atoms which may have a substituent or an oxyalkylene group having 1 to 15 carbon atoms which may have a substituent.)

[0089] In the second embodiment, when an antibody (antigen) is used as the ligand and an antigen (antibody) is used as the target substance, the present invention can be very preferably applied to the latex agglutination method in immunoassays. Immunoassays are widely used in the fields of clinical testing, biochemistry research, and the like as a method for detecting target substances in specimens in in vitro diagnosis. When general affinity particles are used as particles for latex agglutination, the target substances, such as antigens (antibodies) and foreign substances in serum or plasma, are nonspecifically adsorbed to the particle surface. This may cause unintended agglutination between affinity particles, which may impair the accuracy of the immunoassay.

[0090] A description will now be given of the relationship between the zeta potential of the particle and the zeta potential of the ligand in embodiment 2. The affinity particle according to embodiment 2 is characterized in that the zeta potential of the particle and the zeta potential of the ligand satisfy the relationship of formula (2-2). 0≦[||Particle zeta potential (mV)|-|Ligand zeta potential (mV)||]≦20 ···Equation (2-2)

[0091] When [||Particle Zeta Potential (mV)|-|Ligand Zeta Potential (mV)||] is greater than 20 mV, it means that the affinity particle surface formed by binding the ligand to the particle exhibits charges of opposite signs. In this case, when the affinity particle is stored stationary, there is a high possibility that electrostatic heteroaggregation will occur, and care must be taken when handling the affinity particle in the testing laboratory. In the second embodiment, when an antibody or antigen is used as the ligand, since the zeta potential of a general antigen or antibody is around -10 mV, it is preferable that the zeta potential of the particle is -10 mV or more and -30 mV or less.

[0092] The affinity particles according to the second embodiment are characterized in that the area ratio of the ligand to the particle surface satisfies the relationship of formula (2-1). 10≦[occupied area ratio (%)]≦40...Equation (2-1)

[0093] The occupied area ratio will be explained. The occupied area of ​​one ligand is calculated by approximating the size of the ligand to a perfect sphere and using a known formula to calculate the area of ​​a circle. Meanwhile, the surface area of ​​one particle is calculated by approximating the particle to a perfect sphere and using a known formula to calculate the surface area of ​​a perfect sphere. The particle size is assumed to be the particle size in water. Meanwhile, the amount of ligand sensitized to the particle (μg / mg) is quantified through an experiment, and the number of ligands per affinity particle is calculated from this quantified value. The occupied area ratio is calculated by applying a monolayer adsorption model that assumes that the affinity particle of embodiment 2 is formed by binding the ligand to the particle surface in a single layer. That is, the occupied area ratio is defined as a value calculated using formula (2-7). [Occupancy rate (%)] = [Occupancy area of ​​one ligand (nm 2)] × [number of ligands per affinity particle (units)] / [surface area per particle (nm 2 )]×100...Equation (2-7)

[0094] When the ligand is an antibody, a typical antibody can be considered as a perfect sphere with a diameter of 8 nm, so the surface area occupied by one antibody is approximately 50.2 nm 2 An occupied area rate of 50% means that 50% of the affinity particle surface is occupied by the antibody, and the remaining 50% is the underlying particle surface exposed.

[0095] If the occupied area ratio is less than 10%, when the affinity particles are used as latex agglutination particles, the affinity particles may not be agglutinated with each other via the target substance at a frequency low, and sufficient test sensitivity may not be obtained. In addition, if the occupied area ratio of the ligand is too small, there is a concern that the chemical composition of the affinity particle surface may become non-uniform, which may impair the dispersion stability of the affinity particles. On the other hand, if the occupied area ratio is more than 40%, interactions may occur between the ligands, which may inhibit the reactivity of the ligand with the target substance that it originally has. In the second embodiment, the more preferable range of the occupied area ratio is 15% or more and less than 30%.

[0096] A more preferable form of the particles will now be described. In addition to having the repeating unit A represented by formula (2-3), the particle preferably has a repeating unit B characterized by having a hydroxyl group at the side chain end. Affinity particles obtained by binding a ligand to a particle having such characteristics significantly suppress non-specific adsorption. For the purpose of reducing non-specific adsorption, it is more preferable that the surface layer of the particle contains a copolymer having the repeating unit A and the repeating unit B, and that when the particle is dispersed in water or an aqueous solution, the surface layer absorbs water and forms a swollen layer.

[0097] A specific chemical structure of the repeating unit B will be described. The repeating unit B is characterized by having a hydroxyl group at the end of the side chain, and although the chemical structure is not limited within the scope of the object of the second embodiment, it is more preferable that the repeating unit B is a repeating unit having a side chain structure represented by formula (2-4).

[0098] [ka]

[0099] (In formula (2-4), R1 represents a methyl group or a hydrogen atom. L2 represents an alkylene group having 2 to 15 carbon atoms which may have a substituent or an oxyalkylene group having 2 to 15 carbon atoms which may have a substituent. X represents a sulfur atom or a nitrogen atom which may have a substituent. L1 in formula (2-3) and L2 in formula (2-4) satisfy the relationship [number of carbon atoms in L1]+2≧[number of carbon atoms in L2].)

[0100] The hydroxyl group adjacent to the ester bond in formula (2-4) and the hydroxyl group at the end of the side chain play a role in reducing nonspecific adsorption of the particles. X in formula (2-4) may be either a sulfur atom or a nitrogen atom, but is preferably a sulfur atom. Since the sulfide bond is a structure that exhibits a weak hydrophobic tendency, when a swollen layer containing water is formed on the surface of the particle, it is expected to moderately weaken the water binding force of the swollen layer and suppress osmotic aggregation that may occur when mixed with a high concentration sample.

[0101] When comparing formula (2-3) and formula (2-4), it is preferable that the relationship between the number of carbon atoms constituting L1 in formula (2-3) and the number of carbon atoms constituting L2 in formula (2-4) satisfies the relationship of formula (2-8). If the relationship of formula (2-8) is not satisfied, the side chain derived from repeating unit B will be relatively longer than the side chain derived from repeating unit A, and the carboxy group at the end of the side chain of repeating unit A may be blocked. For this reason, when L1 in formula (2-3) is an alkylene group having one carbon atom, it is preferable that L2 in formula (2-4) is an alkylene group having two or three carbon atoms. [Number of carbon atoms in L1] + 2 ≧ [Number of carbon atoms in L2] Equation (2-8)

[0102] When L2 in formula (2-4) is an alkylene group having 3 carbon atoms, from the viewpoint of reducing non-specific adsorption of the particles, it is more preferable that the chemical structure has one of the alkylene groups substituted with a hydroxyl group.

[0103] The particles preferably have at least one repeating unit C selected from the group consisting of styrenes and (meth)acrylates, and more preferably contain repeating units derived from styrenes and glycidyl (meth)acrylate. In this specification, "(meth)acrylate" means "acrylate or methacrylate". As described in the background art of this specification, the repeating unit derived from glycidyl (meth)acrylate has the effect of reducing non-specific adsorption of particles. On the other hand, the reason why it is preferable to contain a repeating unit derived from a styrene is as follows. When the particles are purified by a method such as centrifugation or ultrafiltration, having a repeating unit of a styrene, which has a high glass transition temperature and excellent mechanical strength, contributes to suppressing damage such as cracking and chipping of the particles. Examples of styrenes include styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene, but are not limited thereto as long as the object of embodiment 2 can be achieved. Two or more types of styrenes may be used in combination.

[0104] The content of styrenes is preferably 10 parts by mass or more and 70 parts by mass or less, assuming that the mass of the particles is 100 parts by mass, because this can impart sufficient strength to the particles while reducing nonspecific adsorption.

[0105] The particles may further contain repeating units derived from radically polymerizable monomers having crosslinking properties. Examples of radically polymerizable monomers having crosslinking properties include diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene glycol diacrylate, 2,2'-bis(4-(acryloxydiethoxy)phenyl)propane, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and triethylene glycol dimethacrylate. , tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, 1,6-hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis(4-(methacryloxydiethoxy)phenyl)propane, 2,2'-bis(4-(methacryloxypolyethoxy)phenyl)propane, trimethylolpropane trimethacrylate, tetramethylolmethane tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether, but are not limited to these as long as the object of embodiment 2 can be achieved. In addition, two or more types of radical polymerizable monomers having crosslinking properties may be used in combination.

[0106] The particle size of the particles is preferably 0.05 μm to 1.00 μm, more preferably 0.05 μm to 0.50 μm, and even more preferably 0.05 μm to 0.30 μm. When the particle size is 0.05 μm to 0.30 μm, the particles are easy to handle and are less likely to settle when the particles are stored for a long period of time as a dispersion.

[0107] A typical method for producing the particles will be described below. However, the method for producing the particles is not limited as long as the object of the second embodiment can be achieved.

[0108] The method for producing the particles includes a step 1 of mixing glycidyl (meth)acrylate, styrene, divinylbenzene, water, and a radical polymerization initiator to form a granular copolymer, and obtaining an aqueous dispersion of the granular copolymer.

[0109] The method for producing particles also includes step 2 of forming particles by the following steps. That is, first, the aqueous dispersion is mixed with 3-mercapto-1,2-propanediol and mercaptosuccinic acid to prepare a mixed solution. Then, the epoxy groups derived from the glycidyl (meth)acrylate of the granular copolymer are reacted with the thiol groups derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid.

[0110] The radical polymerization initiator is preferably at least any one of 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] tetrahydrate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate.

[0111] The method for producing particles also includes a step of adjusting the pH of the mixed solution of step 2 to an alkaline region using an organic base that does not have a primary amine. In step 2, the epoxy group derived from glycidyl (meth)acrylate contained in the granular copolymer is reacted in the depth direction from the surface of the granular copolymer using thiol groups derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid. In order to form a surface layer of sufficient thickness on the particles, it is preferable to select triethylamine, which has permeability to the granular copolymer, as the organic base.

[0112] The method for forming the granular polymer is not limited to radical polymerization, so long as the object of the second embodiment can be achieved.

[0113] When radical polymerization is applied, it is preferable to use emulsion polymerization, soap-free emulsion polymerization, or suspension polymerization, and it is more preferable to use emulsion polymerization or soap-free emulsion polymerization. It is further preferable to use soap-free emulsion polymerization.

[0114] In general, emulsion polymerization and soap-free emulsion polymerization can produce granular polymers with a sharper particle size distribution than suspension polymerization. In addition, when a ligand is bound to a particle to produce an affinity particle, if an anionic surfactant or cationic surfactant generally used in emulsion polymerization exists as a residue, it is feared that the ligand may be denatured. For this reason, when a granular polymer is formed by emulsion polymerization, it is preferable to use a nonionic surfactant.

[0115] A reagent for use in detecting a target substance in a specimen by in vitro diagnosis according to the second embodiment is characterized in that it contains the affinity particles according to the second embodiment as particles for latex agglutination.

[0116] The amount of particles for latex agglutination contained in the reagent is preferably from 0.001% by mass to 20% by mass, and more preferably from 0.01% by mass to 10% by mass.

[0117] The reagent according to the second embodiment may contain a third substance, such as a solvent or a blocking agent, in addition to the affinity particles according to the second embodiment, as long as the object of the second embodiment can be achieved.

[0118] Examples of the solvent used in embodiment 2 include various aqueous buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, Hepes buffer, Meth buffer, and ammonia buffer, but the solvent contained in the reagent according to embodiment 2 is not limited to these.

[0119] A kit for use in in vitro diagnosis for detecting a target substance in a specimen according to the second embodiment is characterized by comprising at least the reagent according to the second embodiment.

[0120] The kit according to the second embodiment preferably further includes a reaction buffer solution containing albumin (hereinafter referred to as Reagent 2) in addition to the reagent according to the second embodiment (hereinafter referred to as Reagent 1). The albumin may be serum albumin or the like, and may be albumin treated with a protease. The amount of albumin contained in Reagent 2 is approximately 0.001% by mass or more and 5% by mass or less, but the kit according to the second embodiment is not limited thereto.

[0121] A sensitizer for latex agglutination assay may be contained in both or either one of Reagent 1 and Reagent 2. Examples of sensitizers for latex agglutination assay include polyvinyl alcohol, polyvinylpyrrolidone, alginic acid, etc., but the kit according to the second embodiment is not limited to these.

[0122] Furthermore, the kit according to the second embodiment may include a positive control, a negative control, a serum dilution solution, and the like, in addition to the reagent 1 and the reagent 2. As a medium for the positive control and the negative control, a solvent may be used in addition to serum or physiological saline that does not contain a measurable target substance.

[0123] The kit according to embodiment 2 can be used in the method for detecting a target substance according to embodiment 2 in the same manner as a kit for use in detecting a target substance in a specimen by normal in vitro diagnosis. In addition, the concentration of the target substance can also be measured by a conventionally known method, and is particularly suitable for detecting a target substance in a specimen by a latex agglutination method.

[0124] The method for detecting a target substance in a specimen by in vitro diagnosis according to embodiment 2 is characterized by mixing the affinity particles according to embodiment 2 with a specimen that may contain the target substance.

[0125] The affinity particles according to the second embodiment are preferably mixed with the specimen at a 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 to 20 minutes. In the present detection method, a solvent is preferably used. The concentration of the affinity particles according to the second embodiment in the detection method according to the second embodiment is preferably 0.001% by mass to 5% by mass, preferably 0.01% by mass to 1% by mass, in the reaction system. The detection method according to the second embodiment is characterized in that it optically detects interparticle aggregation that occurs as a result of mixing the affinity particles according to the second embodiment with the specimen. By optically detecting the interparticle aggregation, the target substance in the specimen can be detected, and the concentration of the target substance can also be measured. As a method for optically detecting the agglutination reaction, it is sufficient to measure the amount of change in these values ​​using an optical instrument capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc.

[0126] A method for measuring the dry particle diameter of particles in the second embodiment will be described. The dry particle diameter in the second embodiment is a number average particle diameter, which is measured when the particles are sufficiently dried. Specifically, the particles are dispersed in ion-exchanged water at a concentration of 5% by mass, the dispersion is dropped onto aluminum foil, and the particles are dried at 25°C for 48 hours, and then further dried for 24 hours using a vacuum dryer before measurement. A scanning electron microscope and an image processing analyzer are used to measure the dry particle diameter. Specifically, for example, 100 individual particles are randomly extracted from a particle image in a dry state obtained using a scanning electron microscope (product name: S-4800, manufactured by Hitachi High-Technologies Corporation), and analyzed using an image processing analyzer (product name: LuzexAP, manufactured by Nireco Corporation) to calculate the number average particle diameter.

[0127] A method for measuring the particle diameter in water of a particle in the second embodiment will be described. The particle diameter in water in the second embodiment is a number average particle diameter, and is measured in a state where the particles are dispersed in ion-exchanged water to a concentration of 0.001% by mass. Ion-exchanged water with an electrical conductivity of 10 μS / cm or less is used. A dynamic light scattering method is applied to measure the particle diameter in water. Specifically, a Zetasizer (product name: Nano-ZS, manufactured by Spectris) is used and the measurement is performed at 25° C. The analysis parameters are latex (n≒1.59) as the refractive index of the particles and pure water as the solvent. The measurement is performed 10 times, and the average value of the 10 measured values ​​is used as the particle diameter in water.

[0128] A method for measuring the particle size in water of affinity particles in embodiment 2 will be described. The particle size in water in embodiment 2 is the number average particle size, and is measured in a state where the particles are dispersed in a 0.01N potassium aqueous solution of pH 7.8 to a concentration of 0.001% by mass. The 0.01N potassium aqueous solution of pH 7.8 is prepared by appropriately mixing a 0.01N potassium chloride aqueous solution, a 0.01N potassium hydroxide aqueous solution, and a 0.01N hydrochloric acid aqueous solution, which are prepared using ion-exchanged water with an electrical conductivity of 10 μS / cm or less. A dynamic light scattering method is applied to measure the particle size in water. Specifically, a Zetasizer (product name: Nano-ZS, manufactured by Spectris) is used to measure at 25° C. The analysis parameters are latex (n≒1.59) as the refractive index of the particles and pure water as the solvent. The measurement is performed 10 times, and the average value of the 10 measured values ​​is used as the particle size in water.

[0129] A method for measuring the zeta potential of particles or affinity particles in the second embodiment will be described. The zeta potential in the second embodiment is measured in a state where the particles are dispersed in a 0.01N potassium aqueous solution of pH 7.8 to a concentration of 0.001% by mass. The 0.01N potassium aqueous solution of pH 7.8 is obtained by appropriately mixing a 0.01N potassium chloride aqueous solution, which is prepared using ion-exchanged water with an electric conductivity of 10 μS / cm or less, a 0.01N potassium hydroxide aqueous solution, and a 0.01N hydrochloric acid aqueous solution. The measurement device is a Zetasizer (product name: Nano-ZS, manufactured by Spectris), and the measurement is performed at 25° C. The analysis parameters are latex (n≒1.59) as the refractive index of the particles and pure water as the solvent. The measurement is performed 10 times, and the average value of the 10 measured values ​​is used as the zeta potential.

[0130] A method for measuring the zeta potential of an antibody in the second embodiment will be described. The antibody zeta potential in the second embodiment is measured in a state where the antibody solution is diluted with at least 10 times the amount of a 0.01N potassium aqueous solution of pH 7.8 to a protein concentration of 0.5 to 2.0 mg / mL. The 0.01N potassium aqueous solution of pH 7.8 is obtained by appropriately mixing a 0.01N potassium chloride aqueous solution, which is prepared using ion-exchanged water with an electric conductivity of 10 μS / cm or less, a 0.01N potassium hydroxide aqueous solution, and a 0.01N hydrochloric acid aqueous solution. The measurement device is a Zetasizer (product name: Nano-ZS, manufactured by Spectris), and the measurement is performed at 25°C. For the analysis parameters, protein (n≈1.4) is selected as the refractive index of the particles, and pure water is selected as the solvent. The measurement is performed 10 times, and the average value of the 10 measured values ​​is used as the zeta potential.

[0131] [Embodiment 3] Hereinafter, the third embodiment of the present invention will be described in detail, but the technical scope of the present invention is not limited to these embodiments. First, the background of the third embodiment and the problem to be solved will be described.

[0132] (Background Art and Problems to be Solved by the Third Embodiment) In recent years, research has been widely conducted on purifying or quantifying target substances using affinity particles formed by chemically bonding a ligand having affinity for the target substance to the particle. It is desirable for particles used for such purposes to have a property of adsorbing substances other than the target substance, that is, a small non-specific adsorption. For example, Bioseparation Using Affinity Latex (1995) pp. 11-30 discloses resin particles (hereinafter referred to as SG particles) whose surfaces are coated with polyglycidyl methacrylate by emulsion polymerization using both monomers of styrene and glycidyl methacrylate. In addition, JP 2014-193972 A discloses a method for controlling the particle size of SG particles.

[0133] In SG particles, the epoxy groups derived from polyglycidyl methacrylate are partially opened to form glycols, and nonspecific adsorption is suppressed due to the hydrophilicity of these glycols. On the other hand, when chemically bonding a ligand to the surface of SG particles, it is also possible to use the epoxy groups derived from polyglycidyl methacrylate as they are. However, it is common to undergo a process of converting the epoxy groups to other reactive functional groups such as carboxyl groups, amino groups, and thiol groups, and then chemically react these reactive functional groups with the ligand. Among these, particles in which the epoxy groups of SG particles are converted to carboxyl groups are the most versatile and preferred form for chemically bonding a ligand to the particle surface.

[0134] In recent years, immuno-latex agglutination assay has been attracting attention as a simple and rapid immunological testing method. In this method, a dispersion of particles formed by chemically binding antibodies or antigens as ligands is mixed with a specimen that may contain a target substance (antigen or antibody). If the specimen contains a target substance (antibody or antigen), the particles will cause an agglutination reaction, and the presence or absence of a disease can be identified by optically detecting this agglutination reaction as a change in scattered light intensity, transmitted light intensity, absorbance, etc. In order to reduce false positives, it is preferable that the particles used in the immuno-latex agglutination assay have small non-specific adsorption and reactive functional groups for immobilizing ligands, such as SG particles. The present inventors synthesized SG particles according to Bioseparation using Affinity Latex (1995) pp. 11-30, and further chemically reacted the epoxy groups derived from glycidyl methacrylate in the SG particles with amino acids to obtain particles in which the epoxy groups were converted to carboxy groups. However, the particles obtained in this way had a worse ability to suppress nonspecific adsorption than the SG particles.

[0135] On the other hand, a method of introducing carboxyl groups into SG particles is known. This is obtained by treating SG particles with ammonia water, chemically reacting them with ethylene glycol diglycidyl ether, and then chemically reacting the epoxy groups derived from the ethylene glycol diglycidyl ether with amino acids. The particles obtained in this way miraculously suppress non-specific adsorption, but because the dispersion stability is so excellent, the particles are unlikely to aggregate with each other, and when used in the immuno-latex agglutination method, sufficient sensitivity may not be obtained. Another issue is that the chemical reaction process is multi-step and complicated, making it unsuitable for industrialization.

[0136] The third embodiment has been made in consideration of these background techniques and problems. The object of the third embodiment is to provide particles that have low non-specific adsorption, have reactive functional groups for chemically binding a ligand, and are suitable for latex agglutination. Another object of the third embodiment is to provide particles for latex agglutination to which a ligand is chemically bound, and in vitro diagnostic reagents and kits containing the particles, and a method for detecting a target substance.

[0137] Specifically, the object of the third embodiment is to provide particles having a reactive functional group capable of suppressing non-specific adsorption as excellent as or better than SG particles and capable of chemically bonding a ligand to the particle surface with high yield. The object is to provide a novel method for producing the particles simply and with high yield. Furthermore, the object of the third embodiment is to provide affinity particles formed by chemically bonding an antigen or an antibody as a ligand, and in vitro diagnostic reagents and kits containing the same, and a method for detecting a target substance.

[0138] (Effects of the invention according to embodiment 3) According to the third embodiment, it is possible to provide particles that have a superior ability to suppress non-specific adsorption to that of SG particles or more and have reactive functional groups for chemically bonding a ligand to the particle surface with high yield, and a novel method for easily and highly-yieldingly producing the particles.Furthermore, the third embodiment can provide affinity particles formed by chemically bonding an antigen or an antibody as a ligand, in vitro diagnostic reagents and kits including the same, and a method for detecting a target substance. The particles of the third embodiment will be described in detail below.

[0139] The particle according to the third embodiment of the present invention has a copolymer. The repeating unit A contained in the copolymer has a reactive functional group for chemically bonding a ligand to a side chain. The repeating unit A has a carboxy group as a reactive functional group for chemically bonding a ligand to a side chain. Specifically, the particle according to this embodiment is characterized in that it contains a copolymer containing "repeating unit A", and the repeating unit A is represented by formula (3-1).

[0140] [ka] In formula (3-1), R1 represents a methyl group or a hydrogen atom, R2 represents a carboxy group or a hydrogen atom, and L1 represents an optionally substituted alkylene group having 1 to 15 carbon atoms or an oxyalkylene group having 1 to 15 carbon atoms.

[0141] The "repeating unit A" in the third embodiment has a carboxy group via a sulfide group. The carboxy group is for chemically bonding with a ligand, and by having the structure of the repeating unit A, the ligand can be chemically bonded to the particle surface with high yield, and when used in an immuno-latex agglutination assay, the target substance can be detected with high sensitivity. Although the details are unclear, it is presumed that this is because the side chain is less likely to be protonated compared to when the carboxy group is present via an amino group, and the particles are electrostatically stabilized. In addition, although the details of the mechanism are unclear, it is possible to detect with high sensitivity by appropriately controlling the distance between the carboxylic acid and the sulfide group.

[0142] Furthermore, the particles of embodiment 3 preferably have a carboxylic acid amount per unit mass of 5 [nmol / mg] or more, more preferably 100 [nmol / mg] or more. This allows many ligands to be chemically bound to the particle surface with high yield, and when used in an immuno-latex agglutination assay, the target substance can be detected with high sensitivity. Furthermore, the particles of embodiment 3 preferably have a carboxylic acid amount per unit mass of 300 [nmol / mg] or less. By controlling the amount within this range, non-specific adsorption of substances other than the target substance can be suppressed.

[0143] The number-average particle size of the particles of embodiment 3 is preferably 0.05 μm or more and 1 μm or less. By controlling the particle size within this range, when used in an immunolatex agglutination assay, it is possible to achieve both dispersion stability of the particles in the non-agglutination state and a turbidity change in the agglutination state, i.e., excellent detection sensitivity.

[0144] The particles of the third embodiment can also be expressed in terms of the amount of carboxylic acid per unit area. In this case, the amount of carboxylic acid per unit area is 0.1 to 20 [pieces / nm 2 This makes it possible to suppress non-specific adsorption of substances other than the target substance, to achieve both dispersion stability of the particles in the non-agglutinated state and a change in turbidity in the agglutinated state, i.e., excellent detection sensitivity.

[0145] In addition, the particles of embodiment 3 may contain a "repeating unit B" having a hydrophilic structure to suppress non-specific adsorption. The "repeating unit B" is preferably a glycol structure in which an epoxy group derived from polyglycidyl methacrylate is partially ring-opened, a structure having a sulfide group or a secondary amine in a side chain, and two hydroxyl groups. By having these side chains, it is possible to suppress non-specific adsorption of substances other than the target substance.

[0146] In addition, the particles of the third embodiment may contain a "repeating unit C" having a hydrophobic structure from the viewpoint of particle strength and solvent resistance. The "repeating unit C" is not limited in its chemical structure within the scope of the achievement of the object of the third embodiment, but is preferably at least one selected from the group derived from styrenes and (meth)acrylates. In particular, when the repeating unit C is derived from styrene, methyl methacrylate, or both, the glass transition temperature of the repeating unit C is high, and the particles have sufficient strength, which is preferable. Below, styrenes and (meth)acrylates that can be used for the "repeating unit C" are exemplified, but the "repeating unit C" is not limited to these. In addition, the hydrophobic "repeating unit C" may be two or more oil-based radical polymerizable monomers.

[0147] Styrenes: styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene, etc.

[0148] (Meth)acrylates: methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, n-amyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate, cyclohexyl acrylate, benzyl acrylate, dimethyl phosphate ethyl acrylate, diethyl phosphate ethyl acrylate, dibutyl phosphate ethyl acrylate methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, iso-propyl methacrylate, n-butyl methacrylate, iso-butyl methacrylate, tert-butyl methacrylate, n-amyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, n-nonyl methacrylate, diethyl phosphate ethyl methacrylate, and dibutyl phosphate ethyl methacrylate.

[0149] In addition, in the third embodiment, a crosslinkable radical polymerizable monomer can be included to further improve particle strength. The crosslinkable radical polymerizable monomer is a compound having two or more radical polymerizable groups in the molecule, and examples thereof include aromatic divinyl compounds such as divinylbenzene (DVB), divinyltoluene, divinylxylene, divinanthracene, divinylnaphthalene, and divinyldurene, aromatic trivinyl compounds such as trivinylbenzene, aromatic tetravinyl compounds such as tetravinylbenzene, and other radical polymerizable aromatic compounds having two or more radical polymerizable groups and aromatic rings, and radical polymerizable aliphatic compounds having two or more radical polymerizable groups and aliphatic groups such as pentaerythritol tetraacrylate.

[0150] When the hydrophobic repeating unit C is used, the composition ratio between the repeating units A and B is not limited, so long as the object of the third embodiment can be achieved.

[0151] The particle size of the particles in embodiment 3 is 0.05 μm to 1 μm, preferably 0.1 μm to 0.5 μm, more preferably 0.15 μm to 0.3 μm, in terms of number average particle size in water. When it is 0.15 μm to 0.3 μm, the particles have excellent handleability in centrifugation and a large specific surface area, which is a characteristic of the particles. The number average particle size of the particles in embodiment 3 is evaluated by dynamic light scattering.

[0152] Moreover, the third embodiment relates to an affinity particle in which a carboxy group derived from the "repeating unit A" of the particle of the third embodiment is chemically bonded to a ligand.

[0153] A ligand is a compound that specifically binds to a receptor of a specific target substance. The site where the ligand binds to the target substance is fixed, and the ligand has a selective or specific high affinity. Examples of the ligand include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc. Specific examples of the ligand include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, etc. The affinity particles in the third embodiment refer to particles that have a selective or specific high affinity for a target substance.

[0154] In the third embodiment, the method of chemical reaction for chemically bonding the carboxy group derived from the "repeating unit A" of the particle of the third embodiment with the ligand can be a conventionally known method within the scope of achieving the object of the third embodiment. For example, carbodiimide-mediated reaction and NHS ester activation reaction are commonly used chemical reaction methods. There is also a method of binding avidin to the carboxy group of the particle and binding a biotin-modified ligand to the avidin. However, the chemical reaction method for chemically bonding the carboxy group derived from the "repeating unit A" of the particle of the third embodiment with the ligand is not limited to these.

[0155] When the affinity particles of embodiment 3 use an antibody (antigen) as the ligand and an antigen (antibody) as the target substance, they can be preferably applied to an immunolatex agglutination assay that is widely used in the fields of clinical testing, biochemical research, etc. When general particles are applied to an immunolatex agglutination assay, there is a problem that the target substance, the antigen (antibody), foreign matter in serum, etc., are nonspecifically adsorbed to the particle surface, which causes unintended particle agglutination to be detected, hindering accurate measurement.

[0156] Therefore, in order to reduce noise such as false positives, particles are usually coated with a biological substance such as albumin as a blocking agent to suppress non-specific adsorption to the particle surface. However, since the properties of such biological substances vary slightly depending on the lot, particles coated with them have different abilities to suppress non-specific adsorption for each coating process. Therefore, there is a problem in stably supplying particles with the same level of ability to suppress non-specific adsorption. In addition, biological substances coated on the particle surface may become hydrophobic due to denaturation, and are not necessarily excellent in the ability to suppress non-specific adsorption. Biological contamination is also an issue.

[0157] Patent Document 2 discloses affinity particles for in vitro diagnosis, characterized in that the particles are coated with a polymer having a repeating unit with a sulfenyl group in the side chain as a blocking agent. However, since the polymer having a repeating unit with a sulfenyl group in the side chain is water-soluble and coats the particle surface by physical adsorption, there is a concern that it may be essentially released by dilution. In addition, the inventors evaluated the ability to inhibit nonspecific adsorption in chyme of SG particles obtained by the method described in Bioseparation Using Affinity Latex (1995) pp. 11-30 and particles adsorbed on polystyrene particles obtained by the method described in JP-A-2014-153140.

[0158] The particles obtained by the method described in JP 2014-153140 A were obtained by soap-free emulsion polymerization of a polymer having a repeating unit having a sulfenyl group on the side chain.

[0159] As a result, although it is possible that the results of bioseparation using affinity latex (1995) p11-p30 were not completely reproduced, SG particles were superior in suppressing nonspecific adsorption. Here, SG particles are particles in which the epoxy groups derived from glycidyl methacrylate are converted to glycols by heating in an acidic aqueous solution.

[0160] The reagent for use in detecting a target substance in a specimen by in vitro diagnosis according to embodiment 3 is characterized in that it contains the affinity particles according to embodiment 3. The amount of the affinity particles according to embodiment 3 contained in the reagent according to embodiment 3 is preferably 0.001% by mass to 20% by mass, more preferably 0.01% by mass to 10% by mass. The reagent according to embodiment 3 may contain a third substance such as a solvent or a blocking agent in addition to the affinity particles according to embodiment 3, within a range in which the object of embodiment 3 can be achieved. The third substance such as a solvent or a blocking agent may be contained in combination of two or more types. Examples of the solvent used in the third embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Tris buffer, ammonia buffer, MES (2-morpholinoethanesulfonic acid), ADA (N-(2-acetamido)iminodiacetic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), cholamine hydrochloride, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), acetamidoglycine, tricine, glycineamide, and Good's buffer of bicine, but the solvent contained in the reagent of the third embodiment is not limited to these.

[0161] The kit for use in detecting a target substance in a specimen by in vitro diagnosis according to the third embodiment is characterized by comprising at least the reagent according to the third embodiment. The kit according to the third embodiment preferably further comprises a reaction buffer solution containing albumin (hereinafter, reagent 2) in addition to the reagent according to the third embodiment (hereinafter, reagent 1). The albumin may be serum albumin or the like, which may be protease-treated. The amount of albumin contained in the reagent 2 is approximately 0.001% by mass to 5% by mass, but the kit according to the third embodiment is not limited thereto. A sensitizer for latex agglutination measurement may be contained in both or either of the reagents 1 and 2. Examples of sensitizers for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, alginic acid, and the like, but the kit according to the third embodiment is not limited thereto. The kit according to the third embodiment may also comprise a positive control, a negative control, a serum dilution solution, and the like in addition to the reagents 1 and 2. As a medium for the positive control and the negative control, a solvent may be used in addition to serum not containing a measurable target substance, physiological saline, and the like. The kit of embodiment 3 can be used in the method for detecting a target substance of embodiment 3 in the same manner as a kit for use in detecting a target substance in a specimen by ordinary in vitro diagnosis. In addition, the concentration of the target substance can also be measured by a conventionally known method, and it is suitable for use in detecting a target substance in a specimen by an agglutination method, particularly a latex agglutination method.

[0162] The method for detecting a target substance in a specimen by in vitro diagnosis according to the third embodiment is characterized by mixing the affinity particles according to the third embodiment with a specimen that may contain the target substance, and can be used for an agglutination method. The affinity particles according to the third embodiment are preferably mixed with the specimen at a 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 to 20 minutes. The present detection method preferably uses a solvent. The concentration of the affinity particles according to the third embodiment in the detection method according to the third embodiment is preferably 0·001% by mass to 5% by mass, more preferably 0.01% by mass to 1% by mass, in the reaction system. The detection method according to the third embodiment is characterized by optically detecting an agglutination reaction that occurs as a result of mixing the affinity particles according to the third embodiment with the specimen, and the target substance in the specimen can be detected by optically detecting the agglutination reaction, and the concentration of the target substance can also be measured. The above-mentioned agglutination reaction can be optically detected by measuring the change in scattered light intensity, transmitted light intensity, absorbance, etc. using an optical device capable of detecting these values.

[0163] Next, a preferred method for producing the particles of embodiment 3 will be described. Embodiment 3 is a method for producing particles, characterized by including step 1 of mixing glycidyl (meth)acrylate, styrene or methyl (meth)acrylate, water, and a radical polymerization initiator to form a granular copolymer and obtain an aqueous dispersion of the granular copolymer, and step 2 of mixing the aqueous dispersion with mercaptosuccinic acid or mercaptopropionic acid to prepare a mixed liquid and reacting the epoxy groups derived from the glycidyl (meth)acrylate of the granular copolymer with the thiol groups derived from mercaptosuccinic acid or mercaptopropionic acid.

[0164] First, step 1 will be described. Step 1 is a step for forming a granular polymer, but is not limited to radical polymerization within the scope of the object of embodiment 3. Among radical polymerizations, it is preferable to use emulsion polymerization, soap-free emulsion polymerization, and suspension polymerization, and it is more preferable to use emulsion polymerization or soap-free emulsion polymerization. It is more preferable to use soap-free emulsion polymerization. In general, emulsion polymerization and soap-free emulsion polymerization can obtain a granular polymer with a sharp particle size distribution compared to suspension polymerization. In addition, when particles are chemically bonded to a ligand, there is a concern that the ligand will be denatured if an anionic surfactant such as that generally used in emulsion polymerization is present. Therefore, soap-free emulsion polymerization is the most preferable method for forming a granular polymer.

[0165] The radical polymerization initiator preferably used in step 1 is any one of 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]tetrahydrate, 2,2'-azobis(2-methylpropionamidine)dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, and 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate. Among them, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, 2,2'-azobis(2-methylpropionamidine)dihydrochloride, or 2,2'-azobis[2-(2-imidazolin-2-yl)propane] is more preferably used. This is because the epoxy group derived from glycidyl (meth)acrylate is not ring-opened in step 1 of obtaining an aqueous dispersion of a granular polymer. For example, when potassium persulfate is used as a radical polymerization initiator, the radical polymerization reaction field becomes acidic due to the influence of the initiator residue, and the epoxy group derived from glycidyl (meth)acrylate may react with water to form glycol. In addition, when ammonium persulfate is used as a radical polymerization initiator, the epoxy group derived from glycidyl (meth)acrylate may react with ammonia. In addition, when an anionic radical polymerization initiator having a carboxy group is used as the radical polymerization initiator, the epoxy group derived from glycidyl (meth)acrylate reacts with the carboxy group derived from the polymerization initiator, causing the granular copolymer to aggregate. In order to avoid this aggregation, it is possible to form the granular copolymer at a temperature significantly lower than the 10-hour half-life temperature of the radical polymerization initiator, but this requires a large amount of radical polymerization initiator and the radical polymerization time becomes long, making it unsuitable for industrialization.

[0166] It is also preferable to further contain a crosslinkable radical polymerizable monomer in addition to glycidyl (meth)acrylate and styrene or methyl (meth)acrylate in step 1. By containing a crosslinkable radical polymerizable monomer, the resulting granular copolymer becomes physically strong, and there is no risk of cracking or chipping even if centrifugal operations are repeated during purification.

[0167] Examples of crosslinkable radical polymerizable monomers that can be used in the third embodiment are listed below, but the third embodiment is not limited to these. Two or more oil-based radical polymerizable monomers may be used. Among the radical polymerizable monomers listed above, divinylbenzene is preferred because it is easy to handle during the radical polymerization reaction, although the reason is unclear.

[0168] Crosslinkable radical polymerizable monomers: diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene glycol diacrylate, 2,2'-bis(4-(acryloxydiethoxy)phenyl)propane, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, Examples of suitable vinyl ethers include ethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, 1,6-hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis(4-(methacryloxydiethoxy)phenyl)propane, 2,2'-bis(4-(methacryloxypolyethoxy)phenyl)propane, trimethylolpropane trimethacrylate, tetramethylolmethane tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether.

[0169] In addition, in the step 1, it is preferable to further include a step of mixing glycidyl (meth)acrylate in the process of forming the granular polymer and coating the surface of the granular copolymer with polyglycidyl (meth)acrylate. Here, coating with a polymer may refer to any bonding state. In other words, it is not necessary for the particles to be entirely covered, but it may be partial, and the layer of the coated polymer may not be uniform.

[0170] Next, a description will be given of step 2. Step 2 is a step in which an epoxy group derived from the glycidyl (meth)acrylate of the granular polymer is reacted with a thiol group derived from mercaptosuccinic acid or mercaptopropionic acid to introduce a carboxyl group via a sulfide group.

[0171] In step 2, a basic component can be added to adjust the pH. The basic component is not particularly limited, but an organic base having a secondary or tertiary amine is preferably used. As the organic base having a secondary or tertiary amine, for example, pyridine, triethylamine, diazabicycloundecene, 1,8-bis(dimethylamino)naphthalene, etc. are preferably used, and triethylamine is more preferably used. Two or more kinds of organic bases may be used in combination.

[0172] In step 2, the amount of carboxylic acid can be controlled by adjusting the pH of the mixed solution. In particular, by adjusting the pH to a range greater than 9.0, a large amount of carboxylic acid can be added to the particles to be produced, and by adjusting the pH to greater than 10.0, an even larger amount of carboxylic acid can be added to the particles to be produced, which is more preferable.

[0173] <Quantitative analysis of carboxylic acids> First, a method for measuring the amount of carboxy groups in particles will be described. Using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC / HCl) as a catalyst, the carboxy groups of the particles are activated esterified with N-hydroxysuccinimide (NHS). After that, this activated ester is reacted with aminoethanol to release NHS, and the amount of free NHS is quantified by a high-performance liquid chromatograph to calculate the amount of carboxyl groups per unit particle mass. Note that the unit of the amount of carboxyl groups per unit particle mass is nmol / mg. The specific method will be described below.

[0174] <Activated esterification> Add an aqueous dispersion of particles containing 2.5 mg equivalent of particles to a 1.5 mL microtube, separate the particles from the above dispersion by a centrifuge, and perform the operation of redispersing the particles in dimethylformamide (DMF) three times. Further, with the particles sedimented in the microtube by a centrifuge, remove the DMF from the microtube. Next, add 400 μL of DMF, 19.2 mg of EDC·HCl, and 100 μL of a 1 mol / L DMF solution of N-hydroxysuccinimide to this microtube, and shake at 25°C for 2 hours to activate esterify the carboxy groups of the particles.

[0175] <Release of NHS> To remove excess NHS from the DMF dispersion of the above-activated esterified particles, separate the above-activated esterified particles from the above dispersion by a centrifuge, and perform the operation of redispersing the particles in DMF three times. Further, with the above-activated esterified particles sedimented in the microtube by a centrifuge, remove the DMF from the microtube. Next, add 500 μL of a 1 mol / L DMF solution of aminoethanol to this microtube, and shake at 25°C for 2 hours to release NHS from the particles.

[0176] <Quantification of NHS> The DMF dispersion of the particles containing the free NHS is centrifuged, the particles are allowed to settle in a microtube, and the DMF solution containing the free NHS is collected. The amount of free NHS contained in the DMF solution is quantified using a high performance liquid chromatograph. The carboxyl group mass per unit particle mass is calculated from this quantified value. The instruments and reagents used for quantifying the amount of carboxyl groups are as follows.

[0177] <Equipment> High-performance liquid chromatograph: LC20A-FL (Shimadzu Corporation) Column: Kinetex 5μm C18 100A LC Column 150×4.6mm (phenomenex) Eluent: 4mmol / L ammonium acetate aqueous solution Flow rate: 1.0mL / min Oven temperature: 40℃ Sample injection volume: 1 μL To quantify the amount of free NHS contained in the DMF solution, a calibration curve of the NHS amount and the peak area derived from NHS is prepared in the range of 0.1 mmol / L to 10 mmol / L, and the amount of free NHS is quantified from the NHS peak area.

[0178] <Reagents> EDC / HCl (Dojindo Chemical Research Institute Co., Ltd.) NHS (Kishida Chemical Co., Ltd.) Aminoethanol (Tokyo Chemical Industry Co., Ltd.) DMF (FUJIFILM Wako Pure Chemical Corporation) Ammonium acetate (Tokyo Chemical Industry Co., Ltd.) EXAMPLES

[0179] EXAMPLES The present invention will be described in detail below by giving examples corresponding to the first embodiment, but the present invention is not limited to these examples.

[0180] [Example 1-1] (Synthesis of Granular Copolymer 1-1) 22.7g of styrene (St: Kishida Chemical Industry Co., Ltd.), 33.9g of glycidyl methacrylate (GMA: Tokyo Chemical Industry Co., Ltd.), 0.86g of divinylbenzene (DVB: Kishida Chemical Industry Co., Ltd.), and 2168.6g of ion-exchanged water were weighed into a 2L four-necked separable flask to prepare a mixed solution, and the mixed solution was stirred at 200 rpm while being held at 70°C, and nitrogen was flowed at a flow rate of 200 ml / min to deoxygenate the inside of the three-necked separable flask. Next, a solution of 1.13g of V-50 (FUJIFILM Wako Pure Chemical Co., Ltd.) that had been separately prepared and dissolved in 30g of ion-exchanged water was added to the mixed solution to start soap-free emulsion polymerization. Two hours after the start of polymerization, 5.8g of GMA was added to the four-necked separable flask, and the mixture was further stirred at 200 rpm while being held at 70°C for 22 hours to obtain an aqueous dispersion containing granular copolymer 1. The dispersion was gradually cooled to room temperature, and a portion of the dispersion was collected. The polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, and it was confirmed that the conversion was substantially 100%. The dry particle size of the granular copolymer 1-1 was 196.6 nm, and the particle size in water was 206.9 nm. The granular copolymer 1-1 was concentrated by ultrafiltration or diluted with ion-exchanged water to obtain a 2.5 wt% aqueous dispersion, and stored at 4°C under light-shielded conditions.

[0181] [Example 1-2] (Synthesis of Granular Copolymer 1-2) The 22.7gSt in Example 1-1 was changed to 12.0g, the 33.9g GMA was changed to 17.9g, the 0.86g divinylbenzene was changed to 0.45g, and the amount of GMA added to the three-necked separable flask 2 hours after the start of polymerization was changed from 5.8g to 3.1g, except that the same procedure was followed as in Example 1-1 to obtain an aqueous dispersion of granular copolymer 2. The dispersion was gradually cooled to room temperature, and a portion of it was collected, and the polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was substantially 100%. The dry particle size of the granular copolymer 1-2 was 151.4nm, and the particle size in water was 160.2nm. The granular copolymer 1-2 was concentrated by ultrafiltration or diluted with ion-exchanged water to obtain a 2.5wt% aqueous dispersion, and stored at 4°C under light-shielded conditions.

[0182] [Examples 1-3] (Synthesis of Granular Copolymer 1-3) In Example 1-1, 22.7 gSt was changed to 5.0 g, 33.9 g GMA was changed to 7.5 g, 0.86 g divinylbenzene was changed to 0.19 g, and the amount of GMA added to the three-necked separable flask 2 hours after the start of polymerization was changed from 5.8 g to 1.3 g, except that an aqueous dispersion of granular copolymer 1-3 was obtained in the same manner as in Example 1-1. The dispersion was gradually cooled to room temperature, and a part of it was collected, and the polymerization conversion rate was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, and it was confirmed to be substantially 100%. The dry particle size of the granular copolymer 1-3 was 96.8 nm, and the particle size in water was 105.2 nm. The granular copolymer 1-3 was concentrated by ultrafiltration to a 2.5 wt% aqueous dispersion, or diluted by adding ion-exchanged water, and stored at 4 ° C. under light-shielded conditions.

[0183] [Examples 1-4] (Synthesis of particle 1-1) 24g of 2.5wt% aqueous dispersion of granular copolymer 1, 3.3g of ion-exchanged water, 40mg (0.26mmol) of mercaptosuccinic acid (FUJIFILM Wako Pure Chemical Co., Ltd.), and 0.214mL (2.34mmol) of 3-mercapto-1,2-propanediol (FUJIFILM Wako Pure Chemical Co., Ltd.) were weighed into a 100ml round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200rpm, and the mixture was kept in this state for another 4 hours to obtain a dispersion of particles 1-1. The particles 1-1 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 1-1 in ion-exchanged water was repeated 8 times to purify the particles 1-1, and the particles were stored in the state of an aqueous dispersion adjusted to 1.0wt% of the particles 1-1. The storage conditions were a dark environment at 4° C. Table 1-1 shows the particle properties of Particle 1.

[0184] [Examples 1-5] (Synthesis of particles 1-2) A 1.0 wt % aqueous dispersion of particles 1-2 was obtained in the same manner as in Example 1-4, except that the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 6 hours. Table 1-1 shows the particle properties of particles 1-2.

[0185] [Examples 1-6] (Synthesis of particles 1-3) A 1.0 wt % aqueous dispersion of particles 1-3 was obtained in the same manner as in Example 1-4, except that the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 8 hours. Table 1-1 shows the particle properties of particles 1-3.

[0186] [Examples 1-7] (Synthesis of particles 1-4) A 1.0 wt % aqueous dispersion of particles 1-4 was obtained in the same manner as in Example 1-4, except that the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 12 hours. The particle properties of particles 1-4 are summarized in Table 1-1.

[0187] [Examples 1-8] (Synthesis of particles 1-5) A 1.0 wt % aqueous dispersion of particles 1-5 was obtained in the same manner as in Example 1-4, except that the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 18 hours. The particle properties of particles 1-5 are summarized in Table 1-1.

[0188] [Examples 1-9] (Synthesis of particles 1-6) A 1.0 wt % aqueous dispersion of particles 1-6 was obtained in the same manner as in Example 1-8, except that the particulate copolymer 1-1 in Example 1-8 was changed to particulate copolymer 1-2. Table 1-1 shows the particle properties of particles 1-6.

[0189] [Examples 1-10] (Synthesis of particles 1-7) A 1.0 wt % aqueous dispersion of particles 1-7 was obtained in the same manner as in Example 1-8, except that the particulate copolymer 1-1 in Example 1-8 was changed to particulate copolymer 1-3. Table 1-1 shows the particle properties of particles 1-7.

[0190] [Examples 1-11] (Synthesis of particles 1-8) 24g of 2.5wt% aqueous dispersion of granular copolymer 1, 3.3g of ion-exchanged water, 0.046mL (0.52mmol) of mercaptopropionic acid (FUJIFILM Wako Pure Chemical Co., Ltd.), and 0.190mL (2.08mmol) of 3-mercapto-1,2-propanediol (FUJIFILM Wako Pure Chemical Co., Ltd.) were weighed into a 100mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and the mixture was kept in this state for 18 hours to obtain a dispersion of particles 1-8. The particles 1-8 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 1-8 in ion-exchanged water was repeated eight times to purify the particles 1-8, and the particles were stored in the state of an aqueous dispersion adjusted to 1.0wt% of the particles 1-8. The storage conditions were a dark environment at 4° C. Table 1-1 shows the particle properties of Particles 1-8.

[0191] [Examples 1-12] (Synthesis of particles 1-9) A 1.0 wt % aqueous dispersion of particles 1-9 was obtained in the same manner as in Example 1-4, except that the pH of Example 1-4 was changed from pH 10 to pH 11, and the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 18 hours. The particle properties of particles 1-9 are summarized in Table 1-1.

[0192] [Examples 1-13] (Synthesis of particles 1-10) A 1.0 wt % aqueous dispersion of particles 1-10 was obtained in the same manner as in Example 1-4, except that the pH of Example 1-4 was changed from pH 10 to pH 11, and the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 30 hours. Table 1-1 shows the particle properties of particles 1-10.

[0193] [Examples 1-14] (Synthesis of particles 1-11) A 1.0 wt % aqueous dispersion of particles 1-11 was obtained in the same manner as in Example 1-4, except that the pH of Example 1-4 was changed from pH 10 to pH 11, and the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 48 hours. Table 1-1 shows the particle properties of particles 1-11.

[0194] [Examples 1-15] (Synthesis of particles 1-12) A 1.0 wt% aqueous dispersion of particles 1-12 was obtained in the same manner as in Example 1-11, except that the mercaptopropionic acid in Example 1-11 was changed to 323 mg (2.08 mmol) of mercaptosuccinic acid and the 3-mercapto-1,2-propanediol was changed to 0.047 mL (0.52 mmol). Table 1-1 shows the particle properties of particles 1-12.

[0195] [Examples 1-16] (Synthesis of particles 1-13) A 1.0 wt% aqueous dispersion of particles 1-13 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 282 mg (1.82 mmol) and the amount of 3-mercapto-1,2-propanediol was changed to 0.071 mL (0.78 mmol). Table 1-1 shows the particle properties of particles 1-13.

[0196] [Example 1-17] (Synthesis of particles 1-14) A 1.0 wt% aqueous dispersion of particles 1-14 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 242 mg (1.56 mmol) and the amount of 3-mercapto-1,2-propanediol was changed to 0.095 mL (1.04 mmol). Table 1-1 shows the particle properties of particles 1-14.

[0197] [Example 1-18] (Synthesis of particles 1-15) A 1.0 wt% aqueous dispersion of particles 1-15 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 202 mg (1.30 mmol) and the amount of 3-mercapto-1,2-propanediol was changed to 0.119 mL (1.30 mmol). Table 1-1 shows the particle properties of particles 1-15.

[0198] [Example 1-19] (Synthesis of particles 1-16) A 1.0 wt% aqueous dispersion of particles 1-16 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 81 mg (0.52 mmol) and the amount of 3-mercapto-1,2-propanediol was changed to 0.191 mL (2.08 mmol). Table 1-1 shows the particle properties of particles 1-16.

[0199] [Example 1-20] (Synthesis of particles 1-17) A 1.0 wt% aqueous dispersion of particles 1-17 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 20 mg (0.13 mmol) and the amount of 3-mercapto-1,2-propanediol was changed to 0.225 mL (2.47 mmol). Table 1-1 shows the particle properties of particles 1-17.

[0200] [Comparative Example 1-1] (Synthesis of particles 1-18) A 1.0 wt % aqueous dispersion of particles 1-18 was obtained in the same manner as in Example 4, except that the time for which the content of the round-bottom flask in Example 1-4 was kept at 70° C. after heating was changed from 4 hours to 3 hours. Table 1-1 shows the particle properties of particles 1-18.

[0201] [Comparative Example 1-2] (Synthesis of Particles 1-19) A 1.0 wt % aqueous dispersion of particles 1-19 was obtained in the same manner as in Example 1-4, except that the pH of Example 1-4 was changed from pH 10 to pH 11, and the time for which the content of the round-bottom flask was heated to 70° C. and then held therein was changed from 4 hours to 60 hours. Table 1-1 shows the particle properties of particles 1-19.

[0202] [Comparative Example 1-3] (Synthesis of particles 1-20) A 1.0 wt % aqueous dispersion of particles 1-20 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 403 mg (2.60 mmol) and 3-mercapto-1,2-propanediol was not used. Table 1-1 shows the particle properties of particles 1-20.

[0203] [Comparative Example 1-4] (Synthesis of Particles 1-21) A 1.0 wt% aqueous dispersion of particles 1-21 was obtained in the same manner as in Example 1-15, except that the amount of mercaptosuccinic acid in Example 1-15 was changed to 8 mg (0.05 mmol) and the amount of 3-mercapto-1,2-propanediol was changed to 0.232 mL (2.55 mmol). Table 1-1 shows the particle properties of particles 1-21.

[0204] [Comparative Example 1-5] (Synthesis of particles 1-22) The 2.5 wt% aqueous dispersion of the granular copolymer 1-1 obtained in Example 1-1 was concentrated to a 10 wt% aqueous dispersion using a centrifuge to obtain a concentrated dispersion. 20 g of the concentrated dispersion was weighed into a 200 mL round-bottom flask, and while maintaining at 4°C, 50 times the molar amount of 28% aqueous ammonia (Kishida Chemical Co., Ltd.) was added relative to the theoretical amount of epoxy groups derived from GMA contained in the 20 g concentrated dispersion, and the pH was adjusted to 11 using a 2N aqueous hydrochloric acid solution and a 2N aqueous sodium hydroxide solution. Thereafter, the contents of the round-bottom flask were heated to 70°C while stirring at 100 rpm, and maintained as is for 24 hours to obtain a dispersion of particles 1-22-1. The particles 1-22-1 were separated from the dispersion liquid by a centrifuge, and the operation of redispersing the particles 1-22-1 in ion-exchanged water was repeated eight times to purify the particles 1-22-1, and the particles 1-22-1 were finally stored in the form of an aqueous dispersion adjusted to 10 wt % of the particles 1-22-1. The storage conditions were 4°C under light-shielded conditions.

[0205] 6.3 g of a 10 wt% aqueous dispersion of particles 1-22-1 and 3.81 g of ethylene glycol diglycidyl ether (Tokyo Chemical Industry Co., Ltd.) were weighed into a 50 ml round-bottom flask, and the pH was adjusted to 9 using a 0.1 N aqueous hydrochloric acid solution and a 0.1 N aqueous sodium hydroxide solution. The contents of the round-bottom flask were then heated to 30°C while stirring at 100 rpm, and held for 24 hours to obtain a dispersion of particles 1-22-2. The particles 1-22-2 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 1-22-2 in ion-exchanged water was repeated eight times to purify the particles 1-22-2, and the particles 1-22-2 were finally stored in the form of an aqueous dispersion adjusted to 10 wt%. The storage conditions were 4°C under light-shielded conditions.

[0206] A 10 wt% aqueous dispersion of 6.0 g of particles 1-22-2 in a 50 ml round-bottom flask and the theoretical amount of epoxy groups derived from ethylene glycol diglycidyl ether possessed by the particles contained in the aqueous dispersion (assuming that 100% of the epoxy groups derived from glycidyl methacrylate possessed by the granular copolymer 1-1 reacted with ammonia to form primary amines, and further assuming that 100% of the primary amines reacted with one end of the epoxy groups at both ends possessed by ethylene glycol diglycidyl ether) were added in an amount of 10 times the molar amount of 28% aqueous ammonia, and the pH was adjusted to 11 using an aqueous 1N hydrochloric acid solution and an aqueous 1N sodium hydroxide solution. Thereafter, the contents of the round-bottom flask were heated to 70° C. while stirring at 100 rpm, and the solution was kept as it was for 24 hours to obtain a dispersion of particles 1-22-3. The particles 1-22-3 were separated from the dispersion liquid by a centrifuge, and the operation of redispersing the particles 1-22-3 in ion-exchanged water was repeated eight times to purify the particles 1-22-3, and the particles 1-22-3 were finally stored in the form of an aqueous dispersion adjusted to 10 wt % of the particles 1-22-3. The storage conditions were 4°C under light-shielded conditions.

[0207] The particle 1-22-3 was separated from a 10 wt% aqueous dispersion of the particle 1-22-3 by a centrifuge, and the operation of redispersing the particle 1-22-3 in methanol was repeated three times to prepare a methanol dispersion of the particle 1-22-3 adjusted to 1 wt%. 63 g of the methanol dispersion of the particle 1-22-3 and 2.77 g of succinic anhydride (Tokyo Chemical Industry Co., Ltd.) were weighed into a 200 ml round-bottom flask, and the contents of the round-bottom flask were then heated to 30°C while stirring at 100 rpm, and held as is for 5 hours to obtain a dispersion of the particle 1-22. The particle 1-22 was separated from the dispersion by a centrifuge, and the operation of redispersing the particle 1-22 in ion-exchanged water was repeated eight times to purify the particle 1-22, and the particle 1-22 was finally stored in the state of an aqueous dispersion adjusted to 1.0 wt% of the particle 1-22.

[0208] [Comparative Example 1-6] (Synthesis of particles 1-23) The 2.5 wt% aqueous dispersion of the granular copolymer 1-1 obtained in Example 1-1 was concentrated to a 10 wt% aqueous dispersion using a centrifuge to obtain a concentrated dispersion. 20 g of the concentrated dispersion was weighed into a 200 ml round-bottom flask, and 10 times the molar amount of glycine (Kishida Chemical Co., Ltd.) was added to the theoretical amount of epoxy groups derived from GMA contained in the 20 g concentrated dispersion while keeping it at 4°C, and the pH was adjusted to 11 using a 0.1 N aqueous hydrochloric acid solution and a 0.1 N aqueous sodium hydroxide solution. Thereafter, the contents of the round-bottom flask were heated to 70°C while stirring at 100 rpm, and kept as is for 24 hours to obtain a dispersion of particles 1-23. The particles 1-23 were separated from the dispersion using a centrifuge, and the operation of redispersing the particles 1-23 in ion-exchanged water was repeated eight times to purify the particles 1-23, and the particles 1-23 were finally stored in the state of an aqueous dispersion adjusted to 10 wt%. The storage conditions were dark and at 4°C.

[0209] [Example 1-21] (Evaluation of non-specific adsorption to particles) Particles 1-1 to 1-23 were each dispersed in phosphate buffer (containing 0.01% Tween 20) to prepare a dispersion (P solution) at 0.1 wt%. Next, 51 μL of a specimen dilution solution (Q solution) consisting of a normal human specimen (serum specimen, 1 μL) and phosphate buffer (50 μL) was added to 50 μL of each dispersion, and the absorbance at a wavelength of 572 nm was measured for the mixed solution immediately after stirring. The absorbance was measured using a Biochrom spectrophotometer GeneQuAnt1300. Then, each mixed solution was left to stand at 37°C for 5 minutes, and the absorbance at a wavelength of 572 nm was measured again, and the value of the change in absorbance ΔABS×10000 was calculated. The results are summarized in Table 1-1. The larger the value, the more nonspecific adsorption is interpreted to occur, but particles with a high carboxyl group density or particles with a high [particle size in water / particle size in dry] value may have a high value due to osmotic pressure aggregation rather than aggregation caused by nonspecific adsorption. In any case, particles with a large value of the absorbance change ΔABS×10000 in this evaluation may be interpreted as a normal sample being a false positive due to noise when used as particles for latex agglutination testing. A Q solution containing 1 μL of human normal sample is written as human normal sample concentration x1. A Q solution containing 2.5 μL of human normal sample is written as human normal sample concentration x2.5. A Q solution containing 5 μL of human normal sample is written as human normal sample concentration x5. The normal sample volume is 1x, 2.5x, and 5x, respectively. The thresholds of excellent, good, fair, and bad in Table 1-1 are standards determined from the noise risk when trying to detect low concentrations of target substances by latex agglutination.

[0210] [Example 1-22] (Preparation of affinity particles by antibody sensitization to particles) For particles 1-1 to 1-23, 0.1 mL (1 mg of particles) of each particle dispersion (1.0 wt% solution, 10 mg / mL) was transferred to a microtube (volume 1.5 mL), 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C. After centrifugation, the supernatant was discarded. 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was redispersed by ultrasonication. Centrifugation and redispersion were repeated once.

[0211] Next, 60 μL of WSC solution (50 mg of WSC dissolved in 1 mL of activation buffer, WSC means 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] hydrochloride) and Sulfo NHS solution (50 mg of Sulfo NHS dissolved in 1 mL of activation buffer, Sulfo NHS means sulfo-N-hydroxysuccinimide) were added and dispersed by ultrasonic waves. The particles were stirred at room temperature for 30 minutes to convert the carboxyl groups of the particles to active esters. The mixture was centrifuged at 15000 rpm (20400 g) for 5 min at 4° C., and the supernatant was discarded. 0.2 mL of immobilization buffer (25 mM MES, pH 5.0) was added and dispersed by ultrasonic waves. The mixture was centrifuged at 15000 rpm (20400 g) for 5 min at 4° C., and the supernatant was discarded. 50 μL of immobilization buffer was added, and the particles with activated carboxyl groups were dispersed by ultrasonic waves.

[0212] 50 μL of antibody solution (anti-CRP antibody diluted with immobilization buffer to 25 μg / 50 μL) was added to 50 μL of particle solution with activated carboxyl groups, and the particles were dispersed by ultrasonic waves. The amount of antibody added was 25 μg per 1 mg of particles (25 μg / mg). The final antibody concentration was 0.25 mg / mL, and the final particle concentration was 10 mg / mL. The tube was stirred at room temperature for 60 minutes to bind the antibody to the carboxyl groups of the particles. Then, the tube was centrifuged at 15000 rpm (20400 g) for 5 minutes at 4°C, and the supernatant was discarded. 0.24 mL of masking buffer (1 M Tris, pH 8.0 containing 0.1% Tween 20) was added, and the mixture was dispersed by ultrasonic waves. The mixture was stirred at room temperature for 1 hour, and then left to stand overnight at 4°C to bind Tris to the remaining activated carboxyl groups. Next, the mixture was centrifuged at 15000 rpm (20400 g) for 5 min at 4°C, and the supernatant was discarded. 0.2 mL of washing buffer (10 mM HEPES, pH 7.9) was added, and the mixture was dispersed by ultrasonic waves. A washing operation (centrifugation and redispersion) using washing buffer (10 mM HEPES, pH 7.9) was repeated once. A washing operation was performed once with 0.2 mL of storage buffer (10 mM HEPES, pH 7.9, containing 0.01% Tween 20). 1.0 mL of storage buffer was added, and the mixture was dispersed by ultrasonic waves. The final particle concentration was 0.1 wt% (1 mg / mL). The mixture was stored in a refrigerator. Hereinafter, the name of the affinity particle will be expressed as affinity particle 1-1, following the particle name as it is.

[0213] [Example 1-23] (Evaluation of antibody sensitization rate of affinity particles) The antibody sensitization rate (%) of the affinity particles prepared in Examples 1-21 was determined by protein quantification. Here, the antibody sensitization rate (%) means the ratio of the amount of antibody bound to the particles to the amount of antibody used in the reaction (antibody charge amount). An example of protein quantification evaluation is shown below.

[0214] First, 7 mL of solution A and 140 μL of solution B from the Protein Assay BCA Kit (Wako Pure Chemical Industries) were mixed, and the resulting solution was designated as solution AB. Next, 25 μL (particle amount 25 μg) of the affinity particle dispersion (0.1% solution) was taken and placed in a 1.5 mL tube. Next, 200 μL of solution AB was added to the dispersion (25 μL) and incubated at 60 ° C for 30 minutes. The solution was centrifuged at 15000 rpm (20400 g) for 5 min at 4 ° C, and 200 μL of the supernatant was placed into a 96-well microwell with a pipetter. The absorbance at 562 nm was measured with a microplate reader together with a standard sample (several points of antibody in the range of 0 to 200 μg / mL with 10 mM HEPES). The amount of antibody was calculated from the standard curve. The amount of antibody sensitization to the particles (amount of antibody bound per particle weight (μg / mg)) was calculated by dividing the calculated amount of antibody by the particle weight (here 0.025 mg). Finally, the sensitization rate was calculated. If the amount of antibody charged was 25 μg per 1 mg of particles and the amount of antibody sensitization was 12.5 μg / mg, the sensitization rate would be 50%. The results are summarized in Table 1-1.

[0215] [Example 1-24] (Evaluation of latex agglutination sensitivity of affinity particles) A mixture (hereinafter referred to as R1+) was prepared by mixing 1 μL of human CRP (Denka Seiken, C-reactive protein derived from human plasma, 40 μg / ml) with 50 μL of a buffer solution (PBS containing 0.01% Tween 20) and was kept at 37° C. As a control, a mixture (hereinafter referred to as R1-) was prepared by mixing 1 μL of physiological saline with 50 μL of a buffer solution (PBS containing 0.01% Tween 20) and was also kept at 37° C. Next, 50 μL of a solution containing each affinity particle prepared in Example 1-21 (particle concentration 0.1 wt%, referred to as R2) was mixed with R1+ or R1-, and the absorbance at a wavelength of 572 nm was measured for the mixture (volume 101 μL) immediately after stirring. The absorbance was measured using a Biochrom spectrophotometer GeneQuant1300. Then, after leaving this mixture at 37°C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the value of the change in absorbance ΔABS×10000 was calculated. This series of evaluations was performed for R2 immediately after preparation in Example 1-21, R2 24 hours after preparation, and R2 72 hours after preparation. The results are summarized in Table 1-1. Note that the larger the R- value in Table 1-1, the more aggregation due to non-specific adsorption or osmotic aggregation has occurred in the affinity particles, and there is a concern that normal samples may be interpreted as false positives due to noise when used as particles for latex agglutination in specimen testing. On the other hand, it is expected that the larger the R+ value in Table 1-1 of an affinity particle is, the more sensitive the target substance can be detected when used as particles for latex agglutination in specimen testing. The thresholds for excellent, good, fair, and bad in R- in Table 1-1 are standards determined based on the noise risk when trying to detect low-concentration target substances by latex agglutination. The thresholds for excellent, good, fair, and bad in R+ are standards determined based on the following values ​​obtained using CRP-L Auto "TBA", a CRP detection kit from Denka Seiken Co., Ltd., and CRP standard solution "TBA" for latex. ΔABS of 0 μg / ml (physiological saline) × 10000: -80 5μg / ml ΔABS×10000:1410 20μg / ml ΔABS×10000:3530 40μg / ml ΔABS×10000:5130 160μg / ml ΔABS×10000:9750 320μg / ml ΔABS×10000:12150

[0216] [Table 1-1] TIFF0007659498000010.tif70153 TIFF0007659498000011.tif80125

[0217] [Example 1-25] (Preparation of affinity particles for KL-6) Particles 1-5, 1-6, and 1-7 were sensitized with anti-KL-6 antibody in the same manner as in Example 1-22, except that the antibody in Example 1-22 was changed from anti-CRP antibody to anti-KL-6 antibody and the amount of antibody added was changed from 25 to 100. The obtained affinity particles for KL-6 are referred to as affinity particle 1-5K, affinity particle 1-6K, and affinity particle 1-7K, respectively. The antibody sensitization rates of affinity particles 1-5K, 1-6K, and 1-7K were evaluated in the same manner as in Example 1-23, and the antibody sensitization rates were 93%, 100%, and 94%, respectively.

[0218] [Example 1-26] (Evaluation of latex agglutination sensitivity to KL-6) The latex agglutination sensitivity of affinity particles 1-5K, affinity particles 1-6K, and affinity particles 1-7K was evaluated in the same manner as in Example 1-24, except that the human CRP in Example 1-24 was changed to human KL-6.

[0219] The absorbance change (ΔABSx10000) obtained with affinity particles 1-5K is as follows. 0 U / mL (saline): -140 500 U / mL: 410 1000 U / mL: 1380 2000 U / mL: 4840 5000 U / mL:7520

[0220] The absorbance change (ΔABS x 10000) obtained with affinity particles 1-6K is as follows. 0 U / mL (saline): -240 500 U / mL:-90 1000 U / mL: 480 2000 U / mL:3320 5000 U / mL:6160

[0221] The absorbance change (ΔABSx10000) obtained with affinity particles 1-7K is as follows. 0 U / mL (saline): -320 500 U / mL: -50 1000 U / mL: -340 2000 U / mL:550 5000 U / mL:1200

[0222] All particles showed a linear change in absorbance relative to the KL-6 concentration. When the KL-6 concentration was constant, the larger the particle size, the greater the change in absorbance. It is possible to control the latex agglutination sensitivity by controlling the particle size.

[0223] [Example 1-27] (Effect of sensitizers on latex agglutination sensitivity) The latex agglutination sensitivity of affinity particles 1-5K and affinity particles 1-6K was evaluated in the same manner as in Examples 1-24, except that the human CRP in Examples 1-24 was changed to human KL-6 and the buffer solution (PBS containing 0.01% Tween 20) was changed to a buffer solution containing a sensitizer (PBS containing 0.01% Tween 20 containing 0.58% PVP K90 or 0.68% sodium alginate 80-120).

[0224] The change in absorbance (ΔABSx10000) obtained with affinity particles 1-5K when alginic acid was used as the sensitizer is as follows: 0 U / mL (saline): -240 500 U / mL: 1090 1000 U / mL: 2780 2000 U / mL:9960 5000 U / mL: 11520

[0225] The change in absorbance (ΔABSx10000) obtained with affinity particles 1-6K when alginic acid was used as the sensitizer is as follows: 0 U / mL (saline): -50 500 U / mL: 420 1000 U / mL: 1260 2000 U / mL:6590 5000 U / mL:8720

[0226] The absorbance change (ΔABSx10000) obtained with affinity particles 1-5K when PVP was used as the sensitizer is as follows: 0 U / mL (saline): -70 500 U / mL: 480 1000 U / mL:1020 2000 U / mL: 3860 5000 U / mL: 5970

[0227] The change in absorbance (ΔABS x 10000) obtained with affinity particles 1-6K when PVP was used as the sensitizer is as follows: 0 U / mL (saline): -280 500 U / mL: -60 1000 U / mL: 670 2000 U / mL:4080 5000 U / mL:6820

[0228] The sensitizing effects of PVP and alginic acid were examined for affinity particles 1-5K and 1-6K. In particular, the sensitizing effect of alginic acid was found to be high.

[0229] [Example 1-28] (Preparation of affinity particles for IgE) Particles 1-5 were sensitized with anti-IgE antibody in the same manner as in Example 1-22, except that the antibody in Example 1-22 was changed from anti-CRP antibody to anti-IgE antibody, and the amount of antibody added was changed from 25 to 100. The obtained affinity particles for IgE are referred to as affinity particles 1-5I. The antibody sensitization rate of affinity particles 1-5I was evaluated in the same manner as in Example 1-23, and the antibody sensitization rate was found to be 80%.

[0230] [Example 1-29] (Assessment of latex agglutination sensitivity to IgE) The latex agglutination sensitivity of affinity particles 1-5I was evaluated in the same manner as in Example 1-24, except that human CRP in Example 1-24 was changed to human IgE. The sample volume was 1.5 μL, the sample diluent (Goodt's buffer, LT Autowako IgE, Wako Pure Chemical Industries) was 75 μL, and the affinity particle dispersion was 25 μL.

[0231] The absorbance change (ΔABS×10000) obtained with affinity particles 1-5I is as follows: 0 IU / mL (saline): -60 100 IU / mL:-50 300 IU / mL:100 1000 IU / mL:200 2000 IU / mL:510 3000 IU / mL: 1790

[0232] Affinity particles 5I gave a linear change in absorbance relative to the IgE concentration. Furthermore, in the same manner as in Example 1-27, the latex agglutination sensitivity of affinity particles 1-5I was evaluated using a buffer solution containing a sensitizer (Good's buffer solution containing 0.045% alginic acid was used as a sample diluent).

[0233] The change in absorbance (ΔABSx10000) obtained with affinity particles 1-5K when alginic acid was used as the sensitizer is as follows: 0 IU / mL (saline): 30 100 IU / mL:140 300 IU / mL:300 1000 IU / mL: 1130 2000 IU / mL:3870 3000 IU / mL:5910 For affinity particles 1-5I, the sensitizing effect of alginic acid was confirmed.

[0234] The present invention will be described in detail below by giving examples corresponding to the second embodiment, but the present invention is not limited to these examples.

[0235] (Example 2-1: Synthesis of granular copolymer) The following materials were weighed into a 2 L four-neck separable flask to prepare a mixture. 22.7g of styrene (St, Kishida Chemical Industry Co., Ltd.) 33.9 g of glycidyl methacrylate (GMA, manufactured by Tokyo Chemical Industry Co., Ltd.) 0.86 g of divinylbenzene (DVB, Kishida Chemical Industry Co., Ltd.) 2168.6g of deionized water

[0236] After that, the mixture was stirred at 200 rpm and held at 70°C, and nitrogen was flowed at a flow rate of 200 mL / min to deoxygenate the inside of the three-necked separable flask. Next, a solution of 1.13 g of a polymerization initiator (product name: V-50, Fujifilm Wako Pure Chemical Industries, Ltd.) separately prepared and dissolved in 30 g of ion-exchanged water was added to the mixture to initiate soap-free emulsion polymerization. Two hours after the start of polymerization, 5.8 g of GMA was added to the four-necked separable flask, and the mixture was further stirred at 200 rpm and held at 70°C for 22 hours to obtain an aqueous dispersion containing granular copolymer 1.

[0237] The dispersion was gradually cooled to room temperature, and a portion of the dispersion was sampled and the polymerization conversion was evaluated by proton NMR, gas chromatography, and gel permeation chromatography, which confirmed that the conversion was essentially 100%. The particle size of the granular copolymer in dry form was 196.6 nm, and the particle size in water was 206.9 nm.

[0238] The granular copolymer was concentrated by ultrafiltration or diluted with ion-exchanged water to give a 2.5% by mass aqueous dispersion, and stored at 4° C. in the dark.

[0239] (Example 2-2: Synthesis of Particles 2-1) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 202 mg (1.30 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.119 mL (1.30 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0240] Next, the contents of the round-bottom flask were heated to 70° C. while stirring at 200 rpm, and were maintained in this state for 18 hours to obtain a dispersion of particles 2-1. The particles 2-1 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-1 in ion-exchanged water was repeated eight times to purify the particles 2-1, and the particles 2-1 were finally stored in the form of an aqueous dispersion adjusted to 1.0% by mass. The storage conditions were 4° C. under light-shielded conditions. Table 2-1 shows the particle properties of particles 2-1.

[0241] (Example 2-3: Synthesis of Particles 2-2) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 40mg (0.26mmol) mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0242] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained at this temperature for another 6 hours to obtain a dispersion of particles 2-2. The particles 2-2 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-2 in ion-exchanged water was repeated 8 times to purify the particles 2, and the particles 2-2 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass%. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of particles 2-2.

[0243] (Example 2-4: Synthesis of Particles 2-3) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 40mg (0.26mmol) mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0244] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained at this temperature for another 8 hours to obtain a dispersion of particles 2-3. The particles 2-3 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-3 in ion-exchanged water was repeated 8 times to purify the particles 2-3, and the particles 2-3 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass%. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of particles 2-3.

[0245] (Example 2-5: Synthesis of Particles 2-4) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 1 3.3g deionized water 40mg (0.26mmol) mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0246] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained at this temperature for 12 hours to obtain a dispersion of particles 2-4. The particles 2-4 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-4 in ion-exchanged water was repeated eight times to purify the particles 2-4, and the particles 2-4 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass% of the particles 2-4. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of the particles 2-4.

[0247] (Example 2-6: Synthesis of Particles 2-5) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 40mg (0.26mmol) mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0248] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained at this temperature for 18 hours to obtain a dispersion of particles 2-5. The particles 2-5 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-5 in ion-exchanged water was repeated eight times to purify the particles 2-5, and the particles 2-5 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass% of the particles 2-5. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of the particles 2-5.

[0249] (Example 2-7: Synthesis of Particles 2-6) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 11. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 40mg (0.26mmol) mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0250] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained in this state for another 30 hours to obtain a dispersion of particles 2-6. The particles 2-6 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-6 in ion-exchanged water was repeated eight times to purify the particles 2-6, and the particles 2-6 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass% of the particles 2-6. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of the particles 2-6.

[0251] (Example 2-8: Synthesis of Particles 2-7) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 81 mg (0.52 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.191 mL (2.08 mmol) of 3-mercapto-1,2-propanediol (FUJIFILM Wako Pure Chemical Industries, Ltd.)

[0252] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained at this temperature for 18 hours to obtain a dispersion of particles 2-7. The particles 2-7 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-7 in ion-exchanged water was repeated eight times to purify the particles 2-7, and the particles 2-7 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass%. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of particles 2-7.

[0253] (Example 2-9: Synthesis of Particles 2-8) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 1 3.3g deionized water 323 mg (2.08 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.047 mL (0.52 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0254] Next, the contents of the round-bottom flask were heated to 70°C while stirring at 200 rpm, and were maintained in this state for 18 hours to obtain a dispersion of particles 2-8. The particles 2-8 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-8 in ion-exchanged water was repeated eight times to purify the particles 2-8, and the particles 2-8 were finally stored in the form of an aqueous dispersion adjusted to 1.0 mass%. The storage conditions were 4°C under light-shielded conditions. Table 2-1 shows the particle properties of particles 2-8.

[0255] (Example 2-10: Synthesis of Particles 2-9) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 282 mg (1.82 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.071 mL (0.78 mmol) of 3-mercapto-1,2-propanediol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0256] Next, the contents of the round-bottom flask were heated to 70° C. while stirring at 200 rpm, and were maintained in this state for 18 hours to obtain a dispersion of particles 2-9. The particles 2-9 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-9 in ion-exchanged water was repeated eight times to purify the particles 2-9, and the particles 2-9 were finally stored in the form of an aqueous dispersion adjusted to 1.0% by mass. The storage conditions were 4° C. under light-shielded conditions. Table 2-1 shows the particle properties of the particles 2-9.

[0257] (Example 2-11: Synthesis of Particles 2-10) The following materials were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. 24 g of 2.5% by weight aqueous dispersion of granular copolymer 2-1 3.3g deionized water 242 mg (1.56 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) 0.095 mL (1.04 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0258] Next, the contents of the round-bottom flask were heated to 70° C. while stirring at 200 rpm, and were maintained at this temperature for 18 hours to obtain a dispersion of particles 2-10. The particles 2-10 were separated from the dispersion by a centrifuge, and the operation of redispersing the particles 2-10 in ion-exchanged water was repeated eight times to purify the particles 2-10, and the particles 2-10 were finally stored in the form of an aqueous dispersion adjusted to 1.0% by mass. The storage conditions were 4° C. under light-shielded conditions. Table 2-1 shows the particle properties of the particles 2-10.

[0259] (Example 2-12: Preparation of affinity particles 2-1 to 2-6 by antibody sensitization to particles) For particles 2-1 to 2-6, 0.1 mL (1 mg of particles) of each particle dispersion (1.0 mass% solution, 10 mg / mL) was transferred to a microtube (volume 1.5 mL). Then, 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C. After centrifugation, the supernatant was discarded. 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was redispersed by ultrasonication. Centrifugation and redispersion were repeated once.

[0260] Next, 60 μL of each of the following materials was added and dispersed by ultrasonic waves. 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] hydrochloride (WSC) solution (50 mg of WSC dissolved in 1 mL of activation buffer) Sulfo-N-hydroxysuccinimide (SulfoNHS) solution (50 mg of SulfoNHS dissolved in 1 mL of activation buffer)

[0261] The particles were stirred at room temperature for 30 minutes to convert the carboxyl groups to active esters. The mixture was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded. 0.2 mL of immobilization buffer (25 mM MES, pH 5.0) was added, and the mixture was dispersed by ultrasonication. The mixture was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded. 50 μL of immobilization buffer was added, and the particles with activated carboxyl groups were dispersed by ultrasonication.

[0262] 50μL of antibody solution (anti-C-reactive protein (CRP) antibody diluted with immobilization buffer to 25μg / 50μL) was added to 50μL of particle solution with activated carboxy groups, and the particles were dispersed by ultrasonication. The amount of antibody added was 25μg per 1mg of particles (25μg / mg). The final antibody concentration was 0.25mg / mL, and the final particle concentration was 10mg / mL. The tube was stirred at room temperature for 60 minutes to bind the antibody to the carboxy groups of the particles.

[0263] Then, the mixture was centrifuged at 15000 rpm (20400 g) for 5 minutes at 4°C, and the supernatant was discarded. 0.24 mL of masking buffer (1 M Tris, pH 8.0 containing 0.1% Tween 20) was added, and the mixture was dispersed by ultrasonic waves. The mixture was stirred at room temperature for 1 hour, and then left to stand overnight at 4°C to bind trishydroxymethylaminomethane (Tris) to the remaining activated carboxyl groups. Next, the mixture was centrifuged at 15000 rpm (20400 g) for 5 minutes at 4°C, and the supernatant was discarded. 0.2 mL of washing buffer (10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES), pH 7.9) was added, and the mixture was dispersed by ultrasonic waves. The washing operation (centrifugation and redispersion) with washing buffer (10 mM HEPES, pH 7.9) was repeated once. A washing operation was performed once with 0.2 mL of storage buffer (10 mM HEPES, pH 7.9, containing 0.01% Tween 20). 1.0 mL of storage buffer was added, and dispersed by ultrasonic waves. The final particle concentration was 0.1% by mass (1 mg / mL). The obtained particle dispersion was stored in a refrigerator.

[0264] Hereinafter, the affinity particles obtained here will be referred to as affinity particle 2-1 when the particle used is particle 2-1. When the particle used is particle 2-2, it will be referred to as affinity particle 2-2. When the particle used is particle 2-3, it will be referred to as affinity particle 2-3. When the particle used is particle 2-4, it will be referred to as affinity particle 2-4. When the particle used is particle 2-5, it will be referred to as affinity particle 2-5. When the particle used is particle 2-6, it will be referred to as affinity particle 2-6. The particle properties of affinity particles 2-1 to 2-6 are summarized in Table 2-1.

[0265] (Example 2-13: Preparation of affinity particles 2-7 to 2-12 by antibody sensitization to particles) The particles used were particle 2-5, and the amount of antibody charged in Example 2-12 was changed from 25 μg / mg to 48 μg / mg, 41 μg / mg, 31 μg / mg, 25 μg / mg, 18 μg / mg, and 11 μg / mg, respectively. Otherwise, 0.1 mass% aqueous dispersions of affinity particles 2-7, 2-8, 2-9, 2-10, 2-11, and 2-12 were obtained in the same manner as in Example 2-12. Table 2-1 shows the particle properties of affinity particles 2-7 to 2-12.

[0266] (Example 2-14: Preparation of affinity particles 2-13 by antibody sensitization to particles) A 0.1% by mass aqueous dispersion of affinity particles 2-13 was obtained in the same manner as in Example 2-12, except that the amount of antibody used in Example 2-12 was changed from 25 μg / mg to 54 μg / mg, using particles 2-7. Table 2-1 shows the particle properties of affinity particles 2-13.

[0267] (Example 2-15: Preparation of affinity particles 2-14 by antibody sensitization to particles) The particles used were particles 2-5, the antibody in Example 2-12 was changed from anti-CRP antibody to anti-prostate specific antigen (PSA) antibody, and the amount of antibody added in Example 2-12 was changed from 25 μg / mg to 20 μg / mg. Otherwise, a 0.1% by mass aqueous dispersion of affinity particles 2-14 was obtained in the same manner as in Example 2-12. Table 2-1 shows the particle properties of affinity particles 2-14.

[0268] (Example 2-16: Preparation of affinity particles 2-15 by antibody sensitization to particles) The particles used were particles 2-5, the antibody in Example 2-12 was changed from anti-CRP antibody to anti-bovine serum albumin (BSA) antibody, and the amount of antibody added in Example 2-12 was changed from 25 μg / mg to 23 μg / mg. Otherwise, a 0.1% by mass aqueous dispersion of affinity particles 2-15 was obtained in the same manner as in Example 2-12. Table 2-1 shows the particle properties of affinity particles 2-15.

[0269] (Comparative Example 2-1: Preparation of affinity particles 2-16 to 2-18 by antibody sensitization to particles) Except for using particles 2-8, 2-9, and 2-10, respectively, in Example 2-12, 0.1 mass% aqueous dispersions of affinity particles 2-16, 2-17, and 2-18 were obtained in the same manner as in Example 2-12. The particle properties of affinity particles 2-16 to 2-18 are summarized in Table 2-1.

[0270] (Comparative Example 2-2: Preparation of affinity particles 2-19 by antibody sensitization to particles) A 0.1% by mass aqueous dispersion of affinity particles 2-19 was obtained in the same manner as in Example 2-12, except that the amount of antibody used in Example 2-12 was changed from 25 μg / mg to 5 μg / mg, using particles 2-5. Table 2-1 shows the particle properties of affinity particles 2-19.

[0271] (Comparative Example 2-3: Preparation of affinity particles 2-20 and 2-21 by antibody sensitization to particles) Particles 2-5 were used, and the amount of antibody added in Example 2-12 was changed from 25 μg / mg to 64 μg / mg and 56 μg / mg, respectively, in the same manner as in Example 2-12, to obtain 0.1% by mass aqueous dispersions of affinity particles 2-20 and 2-21. Table 2-1 shows the particle properties of affinity particles 2-20 and 2-21.

[0272] (Example 2-17: Evaluation of antibody sensitization rate of affinity particles) The antibody sensitization rate (%) of affinity particles 2-1 to 2-21 prepared in Examples 2-12 to 2-16 and Comparative Examples 2-1 to 2-3 was determined by protein quantification. Here, the antibody sensitization rate (%) means the ratio of the amount of antibody bound to the particles to the amount of antibody used in the reaction (antibody charge amount). An example of protein quantification evaluation is shown below.

[0273] First, 7 mL of solution A and 140 μL of solution B of Protein Assay BCA Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed, and the prepared solution was designated as solution AB. Next, 25 μL (particle amount 25 μg) of the affinity particle dispersion solution (0.1% solution) was taken and placed in a 1.5 mL tube. Next, 200 μL of solution AB was added to the dispersion solution (25 μL) and incubated at 60 ° C for 30 minutes. The solution was centrifuged at 15000 rpm (20400 g) for 5 min at 4 ° C, and 200 μL of the supernatant was placed into a 96-well microwell with a pipetter. The absorbance at a wavelength of 562 nm was measured with a microplate reader together with a standard sample (several points of antibody in the range of 0 to 200 μg / mL with 10 mM HEPES). The amount of antibody was calculated from the standard curve. The amount of antibody sensitization to the particles (amount of antibody bound per particle weight (μg / mg)) was calculated by dividing the calculated amount of antibody by the particle weight (0.025 mg in this case). Finally, the sensitization rate was calculated. If the amount of antibody charged was 25 μg per 1 mg of particles and the amount of antibody sensitization was 12.5 μg / mg, the sensitization rate would be 50%. The results are summarized in Table 2-1.

[0274] (Example 2-18: Evaluation of non-specific adsorption to affinity particles using chyle) A dispersion (liquid A) was prepared by dispersing each of affinity particles 2-1 to 2-21 in a phosphate buffer solution to a concentration of 0.1% by mass. Next, 60 μL of chyle (liquid B) consisting of triolein, lecithin, free fatty acid, bovine albumin, and Tris buffer solution was added to 30 μL of each dispersion, and the absorbance at a wavelength of 572 nm was measured for the mixed solution immediately after stirring. The absorbance was measured using a spectrophotometer GeneQuAnt1300 manufactured by Biochrom. Then, each mixed solution was left to stand at 37° C. for 5 minutes, and the absorbance at a wavelength of 572 nm was measured again, and the value of the change in absorbance ΔABS×10000 was calculated. The results are summarized in Table 2-1.

[0275] The larger the value, the more non-specific adsorption is interpreted as occurring, but it is possible that the value is not agglutination caused by non-specific adsorption but osmotic aggregation. In any case, when affinity particles with a large value of the absorbance change ΔABS × 10000 in this evaluation are used as particles for latex agglutination in specimen testing, there is a concern that normal specimens may be interpreted as false positives due to noise. The threshold values ​​of excellent, good, fair, and bad in Table 2-1 are standards determined from the noise risk when attempting to detect low concentrations of target substances by latex agglutination.

[0276] (Example 2-19: Evaluation of dispersion stability of affinity particles) Affinity particles 2-1 to 2-21 were each thoroughly dispersed by ultrasonic waves and then stored at 4°C. Sedimentation of the particles was observed visually over time. The results are summarized in Table 2-1. As described in the embodiment, particles with a small zeta potential difference and affinity particles with an appropriate range of antibody occupation area ratio showed good dispersion stability. The thresholds of excellent, good, fair, and bad in Table 2-1 are standards determined based on the storage period and particle size of the reagent used in the latex agglutination method (since natural sedimentation of particles is unavoidable, albeit slowly, in the solution composition of this embodiment).

[0277] (Example 2-20: Evaluation of latex agglutination sensitivity of affinity particles 1) A mixture (hereinafter referred to as R1+) was prepared by mixing 1 μL of human CRP (manufactured by Denka Seiken Co., Ltd., derived from human plasma, 40 μg / ml) with 50 μL of a buffer solution (phosphate buffered saline (PBS) containing 0.01% Tween 20) and was kept at 37° C. As a control, a mixture (hereinafter referred to as R1-) was prepared by mixing 1 μL of saline with 50 μL of a buffer solution (PBS containing 0.01% Tween 20) and was also kept at 37° C. Next, 50 μL of a solution containing affinity particles 2-1 to 2-13 and affinity particles 2-16 to 2-21 prepared in each Example and Comparative Example (particle concentration 0.1% by mass, referred to as R2) was mixed with R1+ or R1- and stirred. The absorbance at a wavelength of 572 nm was measured for the mixture (volume 101 μL) immediately after mixing and stirring. The absorbance was measured using a Biochrom spectrophotometer GeneQuant1300. After leaving the mixture at rest for 5 minutes at 37° C., the absorbance at a wavelength of 572 nm was measured again, and the change in absorbance ΔABS×10000 was calculated. This series of evaluations was performed for R2 immediately after preparation in each Example and Comparative Example, R2 24 hours after preparation, and R2 72 hours after preparation. The results are summarized in Table 2-1. Note that the larger the R- value in Table 2-1, the more aggregation due to nonspecific adsorption or osmotic aggregation has occurred in the affinity particles, and when the particles are used in specimen testing as particles for latex agglutination, there is a concern that normal specimens may be interpreted as false positives due to noise. On the other hand, affinity particles with a larger R+ value in Table 2-1 are expected to be able to detect target substances with higher sensitivity when used as affinity particles for latex agglutination in specimen testing. The thresholds for excellent, good, fair, and bad in R- in Table 2-1 are standards determined based on the noise risk when attempting to detect low concentrations of target substances by latex agglutination. The thresholds for excellent, good, fair, and bad in R+ are standards determined based on the following values ​​obtained using CRP-L Auto "TBA", a CRP detection kit manufactured by Denka Seiken Co., Ltd., and CRP standard solution "TBA" for Latex. ΔABS×10000 at 0 μg / ml (physiological saline): -80 5 μg / ml ΔABS×10000:1410 20 μg / ml ΔABS×10000:3530 40 μg / ml ΔABS×10000:5130 160 μg / ml ΔABS×10000:9750 320 μg / ml ΔABS×10000:12150

[0278] (Example 2-21: Evaluation of latex agglutination sensitivity of affinity particles 2) The latex agglutination sensitivity of affinity particle 2-14 was evaluated in the same manner as in Example 2-20, except that human CRP in Example 2-20 was changed to PSA and the affinity particle was changed to affinity particle 2-14. The results are shown in Table 2-1. It was found that affinity particle 2-14 is an affinity particle with high sensitivity to PSA, similar to affinity particles 2-1 to 2-13 with high sensitivity to CRP.

[0279] (Example 2-22: Evaluation of latex agglutination sensitivity of affinity particles 3) The latex agglutination sensitivity of affinity particle 2-15 was evaluated in the same manner as in Example 2-20, except that the human CRP in Example 2-20 was changed to BSA and the affinity particle was changed to affinity particle 2-15. The results are shown in Table 2-1. It was found that affinity particle 2-15 is an affinity particle with high sensitivity to BSA, similar to affinity particles 2-1 to 2-13 with high sensitivity to CRP.

[0280] [Table 2-1] TIFF0007659498000013.tif104116 TIFF0007659498000014.tif90128

[0281] (Example 2-23: Synthesis of Particle 23) The 22.7gSt in Example 2-1 was changed to 12.0g, the 33.9g GMA was changed to 17.9g, the 0.86g divinylbenzene was changed to 0.45g, and the amount of GMA added to the three-necked separable flask 2 hours after the start of polymerization was changed from 5.8g to 3.1g, except that the same procedure was followed as in Example 2-1 to obtain an aqueous dispersion of granular copolymer 2-2. The dispersion was gradually cooled to room temperature, and a portion of it was collected, and the polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was substantially 100%. The dry particle size of the granular copolymer 2-2 was 151.4nm, and the particle size in water was 160.2nm. The granular copolymer 2-2 was concentrated by ultrafiltration or diluted with ion-exchanged water to obtain a 2.5wt% aqueous dispersion, and stored at 4°C under light-shielded conditions. A 1.0 wt% aqueous dispersion of particles 2-23 was obtained in the same manner as in Example 2-6, except that the particulate copolymer 2-1 in Example 2-6 was replaced with the particulate copolymer 2-2. The dry particle size of particles 2-23 was 161.6 nm, the particle size in water was 202 nm, and the zeta potential was -16 mV.

[0282] (Example 2-24: Synthesis of Particles 2-24) In Example 2-1, 22.7 gSt was changed to 5.0 g, 33.9 g GMA was changed to 7.5 g, 0.86 g divinylbenzene was changed to 0.19 g, and the amount of GMA added to the three-necked separable flask 2 hours after the start of polymerization was changed from 5.8 g to 1.3 g, except that an aqueous dispersion of granular copolymer 2-3 was obtained in the same manner as in Example 2-1. The dispersion was gradually cooled to room temperature, and a part of it was collected, and the polymerization conversion rate was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, and it was confirmed to be substantially 100%. The dry particle size of the granular copolymer 2-3 was 96.8 nm, and the particle size in water was 105.2 nm. The granular copolymer 2-3 was concentrated by ultrafiltration to a 2.5 wt% aqueous dispersion, or diluted with ion-exchanged water, and stored at 4 ° C. under light-shielded conditions. A 1.0 wt% aqueous dispersion of particles 2-24 was obtained in the same manner as in Example 2-6, except that the particulate copolymer 2-1 in Example 2-6 was replaced with the particulate copolymer 2-3. The dry particle size of particles 2-24 was 119 nm, the particle size in water was 144 nm, and the zeta potential was -15 mV.

[0283] (Example 2-25: Preparation of affinity particles 2-5K to 2-7K by sensitizing particles with anti-KL-6 antibody) The particles used in Example 2-12 were particle 2-5, particle 2-23, and particle 2-24, respectively, and the antibody in Example 2-12 was changed from anti-CRP antibody to anti-KL-6 antibody, but the same procedure was followed as in Example 2-12 to sensitize particle 2-5, particle 2-23, and particle 2-24 with anti-KL-6 antibody. The affinity particles for KL-6 obtained are referred to as affinity particle 2-5K, affinity particle 2-6K, and affinity particle 2-7K, respectively. The antibody sensitization rates of affinity particle 2-5K, affinity particle 2-6K, and affinity particle 2-7K were evaluated in the same manner as in Example 2-17, and the antibody sensitization rates were 93%, 100%, and 94%, respectively. The antibody occupation area rates were 20.3%, 17.3%, and 12.4%, respectively. The zeta potential of the anti-KL-6 antibody was −14 mV. Therefore, the zeta potential differences (mV) of the affinity particles 2-5K, 2-6K, and 2-7K were 2, 2, and 1, respectively.

[0284] (Example 2-26: Evaluation of latex agglutination sensitivity of affinity particles for KL-6) The latex agglutination sensitivity of affinity particles 2-5K, affinity particles 2-6K, and affinity particles 2-7K was evaluated in the same manner as in Example 2-20, except that the human CRP in Example 2-20 was changed to human KL-6.

[0285] The absorbance change (ΔABSx10000) obtained with affinity particles 2-5K is as follows. 0 U / mL (saline): -140 500 U / mL: 410 1000 U / mL: 1380 2000 U / mL: 4840 5000 U / mL:7520

[0286] The absorbance change (ΔABS x 10000) obtained with affinity particles 2-6K is as follows. 0 U / mL (saline): -240 500 U / mL:-90 1000 U / mL: 480 2000 U / mL:3320 5000 U / mL:6160

[0287] The absorbance change (ΔABSx10000) obtained with affinity particles 2-7K is as follows. 0 U / mL (saline): -320 500 U / mL: -50 1000 U / mL: -340 2000 U / mL:550 5000 U / mL:1200

[0288] All particles showed a linear change in absorbance relative to the KL-6 concentration. When the KL-6 concentration was constant, the larger the particle size, the greater the change in absorbance. It is possible to control the latex agglutination sensitivity by controlling the particle size.

[0289] (Example 2-27: Effect of sensitizer on latex agglutination sensitivity) The latex agglutination sensitivity of affinity particles 2-5K and affinity particles 2-6K was evaluated in the same manner as in Example 2-20, except that the human CRP in Example 2-20 was changed to human KL-6 and the buffer solution (PBS containing 0.01% Tween 20) was changed to a buffer solution containing a sensitizer (PBS containing 0.01% Tween 20 containing 0.58% PVP K90 or 0.68% sodium alginate 80-120).

[0290] The change in absorbance (ΔABSx10000) obtained with affinity particles 2-5K when alginic acid was used as the sensitizer is as follows. 0 U / mL (saline): -240 500 U / mL: 1090 1000 U / mL: 2780 2000 U / mL:9960 5000 U / mL: 11520

[0291] The change in absorbance (ΔABSx10000) obtained with affinity particles 2-6K when alginic acid was used as the sensitizer is as follows: 0 U / mL (saline): -50 500 U / mL: 420 1000 U / mL: 1260 2000 U / mL:6590 5000 U / mL:8720

[0292] The change in absorbance (ΔABSx10000) obtained with affinity particles 2-5K when PVP was used as the sensitizer is as follows: 0 U / mL (saline): -70 500 U / mL: 480 1000 U / mL:1020 2000 U / mL: 3860 5000 U / mL: 5970

[0293] The change in absorbance (ΔABSx10000) obtained with affinity particles 2-6K when PVP was used as the sensitizer is as follows: 0 U / mL (saline): -280 500 U / mL: -60 1000 U / mL: 670 2000 U / mL:4080 5000 U / mL:6820

[0294] The sensitizing effects of PVP and alginic acid were examined for affinity particles 2-5K and 2-6K. In particular, the sensitizing effect of alginic acid was found to be high.

[0295] (Example 2-28: Preparation of affinity particles 2-5I by sensitizing particles with anti-IgE antibodies) Particles 2-5 were sensitized with anti-IgE antibody in the same manner as in Example 2-12, except that the antibody in Example 2-12 was changed from anti-CRP antibody to anti-IgE antibody. The obtained affinity particles for IgE are referred to as affinity particles 2-5I. The antibody sensitization rate of affinity particles 2-5I was evaluated in the same manner as in Example 2-17, and the antibody sensitization rate was 80%. The antibody occupied area rate was 18%. The zeta potential of the anti-IgE antibody was -6 mV. Therefore, the zeta potential difference (mV) of affinity particles 2-5I was 10.

[0296] (Example 2-29: Evaluation of latex agglutination sensitivity of affinity particles for IgE) The latex agglutination sensitivity of affinity particles 2-5I was evaluated in the same manner as in Example 2-20, except that human CRP in Example 2-20 was changed to human IgE. The sample volume was 1.5 μL, the sample diluent (Goodt's buffer, LT Autowako IgE, Wako Pure Chemical Industries) was 75 μL, and the affinity particle dispersion was 25 μL.

[0297] The absorbance change (ΔABS×10000) obtained with affinity particles 2-5I is as follows: 0 IU / mL (saline): -60 100 IU / mL:-50 300 IU / mL:100 1000 IU / mL:200 2000 IU / mL:510 3000 IU / mL: 1790 The affinity particles 2-5I gave a linear change in absorbance relative to the IgE concentration.

[0298] Furthermore, in the same manner as in Example 2-27, the latex agglutination sensitivity of affinity particles 2-5I was evaluated using a buffer solution containing a sensitizer (Good's buffer solution containing 0.045% alginic acid was used as a sample diluent).

[0299] The change in absorbance (ΔAPS x 10,000) obtained with affinity particles 2-5K when alginic acid was used as the sensitizer is as follows. 0 IU / mL (saline): 30 100 IU / mL:140 300 IU / mL:300 1000 IU / mL: 1130 2000 IU / mL:3870 3000 IU / mL:5910 For affinity particles 2-5I, the sensitizing effect of alginic acid was confirmed.

[0300] (Example 2-30: Preparation and evaluation of affinity particles 2-22 by antibody sensitization to particles) Particles 2-5 were sensitized with anti-CRP antibodies in the same manner as in Example 2-12, except that the 1M Tris in the masking buffer in Example 2-12 was changed to 1M ethanolamine. The obtained affinity particles for CRP are referred to as affinity particles 2-22. The difference from affinity particle 2-5 is that some of the carboxyl groups in repeating unit A are Tris in affinity particle 2-5, whereas they are ethanolamine in affinity particle 2-22. Therefore, the antibody sensitization rate and antibody occupied area rate are the same as those of affinity particle 2-5. The latex agglutination sensitivity of affinity particles 2-22 was evaluated in the same manner as in Example 2-20.

[0301] The relative values ​​of the absorbance change (ΔABS×10000) obtained with affinity particles 2-22 to those of affinity particles 2-5 are as follows: 5 μg / mL: 1.11 20 μg / mL: 1.17 40 μg / mL: 1.13 160 μg / mL: 1.09 320 μg / mL: 1.10 It was found that the change in absorbance was greater for affinity particles 2-22 than for affinity particles 2-5. By using ethanolamine as the masking chemical structure, it was possible to control the latex agglutination sensitivity to CRP.

[0302] The present invention will be described in detail below by giving examples corresponding to the third embodiment, but the present invention is not limited to these examples.

[0303] (Example 3-1: Synthesis of Particles 3-1) In a 200 ml flask, 1.2 g of styrene (Kishida Chemical Co., Ltd.), 1.8 g of glycidyl methacrylate (Kishida Chemical Co., Ltd.), 0.04 g of divinylbenzene (Kishida Chemical Co., Ltd.), and 100 g of ion-exchanged water were weighed and taken to obtain a mixed solution. After that, this mixed solution was stirred at 200 rpm while being held at 40°C, and nitrogen bubbling was performed for 30 minutes. Next, the nitrogen bubbling was switched to nitrogen flow. Then, a solution of 0.06 g of V-50 (Wako Pure Chemical Industries, Ltd.) that had been separately prepared and dissolved in 3 g of ion-exchanged water was added to the above mixed solution to start radical polymerization (soap-free emulsion polymerization). Two hours after the start of polymerization, 0.3 g of glycidyl methacrylate was added to the radical polymerization reaction field, and the mixture was further stirred at 200 rpm while being held at 70°C for 8 hours, and then gradually cooled to room temperature to obtain a particle dispersion (step 1). At this point, the contents of the 200 ml flask were sampled, and the radical polymerization conversion rate was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, and it was confirmed that it was substantially 100%. The particle dispersion obtained in this step 1 was cooled in an ice bath and stirred, and an aqueous solution was prepared by adding 2.2 g of mercaptosuccinic acid (Wako Pure Chemical Industries, Ltd.: the total molar amount of mercaptosuccinic acid is equal to the molar amount of the above glycidyl methacrylate) and 3.0 g of triethylamine to 40 g of ion-exchanged water, and was dropped into the particle dispersion. After the dropwise addition, the pH of the reaction solution was adjusted to 10.3 using triethylamine and a 2N aqueous hydrochloric acid solution. Thereafter, the temperature was raised to 70°C and kept for 4 hours while stirring, and the epoxy group derived from the glycidyl methacrylate and the thiol group derived from the mercaptosuccinic acid were chemically reacted to obtain particles 3-1 having a carboxyl group as a reactive functional group (step 2). During the chemical reaction, no aggregates or the like were generated. Particle 3-1 was purified by centrifugation, replaced with pure water, and stored (the dispersion medium was also replaced by centrifugation). Particle 3-1 was evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 214 nm. The amount of carboxyl groups per unit mass was quantified and found to be 130 [nmol / mg].

[0304] (Example 3-2: Synthesis of Particles 3-2) Particles 3-2 having carboxy groups as reactive functional groups were obtained by the same experimental procedure as in Example 3-1, except that the pH of the reaction solution in step 2 in Example 3-1 was changed from 10.3 to 10.7. The purification and storage methods were also the same. The particle size of particles 3-2 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was 226 nm. In addition, the amount of carboxy groups per unit mass was quantified, and was found to be 200 [nmol / mg].

[0305] (Example 3-3: Synthesis of Particles 3-3) Particles 3-3 having a carboxy group as a reactive functional group were obtained by the same experimental operation as in Example 3-1, except that 2.2 g of mercaptosuccinic acid in step 2 of Example 3-1 was changed to 1.6 g of mercaptopropionic acid (Wako Pure Chemical Industries, Ltd.: the number of moles of mercaptopropionic acid was equal to the number of moles of the above glycidyl methacrylate) and the amount of triethylamine added to the aqueous solution was changed to 1.6 g. As in Example 3-1, no aggregates or the like were generated during the chemical reaction. The purification and storage methods were also the same. When the particle size of particles 3-3 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), the number average particle size was 211 nm. In addition, the amount of carboxy groups per unit mass was quantified and found to be 120 [nmol / mg].

[0306] (Example 3-4: Synthesis of Particles 3-4) Particles 3-4 having a carboxy group as a reactive functional group were obtained by the same experimental procedure as in Example 3-1, except that in step 2 of Example 3-1, the reaction was carried out without adding triethylamine. The purification and storage methods were also the same. When the particle size of particles 3-4 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), the number average particle size was 209 nm. In addition, the amount of carboxy groups per unit mass was quantified to be 20 [nmol / mg].

[0307] (Example 3-5: Synthesis of Particles 3-5) Particles 3-5 having a carboxy group as a reactive functional group were obtained by the same experimental procedure as in Example 1, except that the pH of the reaction solution in step 2 of Example 3-1 was changed from 10.3 to 9.9. The purification and storage methods were also the same. The particle size of particles 3-5 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was 216 nm. In addition, the amount of carboxy groups per unit mass was quantified and found to be 80 [nmol / mg].

[0308] (Example 3-6: Synthesis of Particles 3-6) While cooling and stirring the particles 3-5 obtained in Example 3-5 in an ice bath, an aqueous solution was prepared by adding 0.9 g of aminoethanol (Wako Pure Chemical Industries, Ltd.: the total molar amount of aminoethanol is equal to the molar amount of the above glycidyl methacrylate) and 0.6 g of sodium hydroxide (Kishida Chemical Industries, Ltd.: the total molar amount of aminoethanol is equal to the molar amount of the above glycidyl methacrylate) to 40 g of ion-exchanged water, and the aqueous solution was dropped into the particle dispersion. After the dropwise addition, the pH of the reaction solution was adjusted to 10.0 using sodium hydroxide and 1N hydrochloric acid. Thereafter, the temperature was raised to 70°C and the mixture was kept for 4 hours while stirring, and the epoxy group derived from the glycidyl methacrylate and the amino group derived from the aminoethanol were chemically reacted, and the epoxy group was opened to obtain particles 3-6. During the chemical reaction, no aggregates were generated. The particles 3-6 were purified by centrifugation, replaced with pure water, and stored (the replacement of the dispersion medium was also performed by centrifugation). Particles 3-5 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 214 nm. The amount of carboxyl groups per unit mass was quantified and found to be 80 nmol / mg.

[0309] (Example 3-7: Synthesis of Particles 3-7) The particles 3-5 obtained in Example 3-5 were cooled in an ice bath and stirred, and an aqueous solution was prepared by adding 1.4 g of 3-mercapto-1-propanol (Wako Pure Chemical Industries, Ltd.: the total molar amount of mercaptopropanol is equal to the molar amount of the above glycidyl methacrylate) and 1.5 g of triethylamine (Kishida Chemical Industries, Ltd.: the total molar amount of 3-mercapto-1-propanol is equal to the molar amount of the above glycidyl methacrylate) to 40 g of ion-exchanged water, and the aqueous solution was dropped into the particle dispersion. After the dropwise addition, the pH of the reaction solution was adjusted to 10.0 using triethylamine and 1N hydrochloric acid. Thereafter, the temperature was raised to 70°C and the mixture was kept for 4 hours while stirring, and the epoxy group derived from the glycidyl methacrylate and the mercapto group derived from the mercaptopropanol were chemically reacted, and the epoxy group was opened to obtain particles 3-7. During the chemical reaction, no aggregates were generated. The particles 3-7 were purified by centrifugation, replaced with pure water, and stored (the replacement of the dispersion medium was also performed by centrifugation). Particle 3-7 was evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 220 nm. The amount of carboxyl groups per unit mass was quantified and found to be 80 nmol / mg.

[0310] (Comparative Example 3-1: Synthesis of SG Particle Modified 3-1) SG particles modified 3-1 having a carboxy group as a reactive functional group were obtained by the same experimental operation as in Example 3-1, except that 2.2 g of mercaptosuccinic acid in step 2 of Example 3-1 was changed to 1.1 g of glycine (Wako Pure Chemical Industries, Ltd.: the number of moles of glycine was equal to the number of moles of the above glycidyl methacrylate) and the amount of triethylamine added to the aqueous solution was changed to 1.6 g. As in Example 3-1, no aggregates were generated during the chemical reaction. The purification and storage methods were also the same. When the particle size of the SG particles modified 3-1 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), the number average particle size was 211 nm. In addition, the amount of carboxy groups per unit mass was quantified to be 120 [nmol / mg].

[0311] (Synthesis of affinity particles: antibody binding) Particles 3-1 to 3-7 and SG particle modified 3-1 were each dispersed in MES buffer to a concentration of 1.0 wt% to prepare 1 μl of each dispersion. To each of these dispersions, a solution of 0.055 mg of 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] (Wako Pure Chemical Industries, Ltd.) dissolved in 10 μl of phosphate buffer was added. Then, 5 μl of a 4.9 mg / ml dispersion of clone C5 (Funakoshi Co., Ltd.) of monoclonal mouse anti-human C-reactive protein (hereinafter, CRP antibody) and 5 μl of a 5.8 mg / ml dispersion of clone C6 (Funakoshi Co., Ltd.) were added. Then, affinity particles were synthesized by shaking for 180 hours at room temperature. Next, the affinity particles were purified by performing centrifugation purification (15,000 rpm) three times, and finally stored in a state of being dispersed in 1 ml of phosphate buffer. Hereinafter, the affinity particles obtained from particles 3-1 to 3-7 will be referred to as affinity particles 3-1 to 3-7, respectively. Also, the affinity particles obtained from modified SG particle 3-1 will be referred to as modified CRP-immobilized SG particle 3-1. The sensitization rates of affinity particles 3-1 to 3-7 and modified CRP-immobilized SG particles 3-1 were evaluated using the following indices, and the results are shown in Table 3-1. A: 80% or more B: 60% or more but less than 80% C: Less than 60%

[0312] (Evaluation of antigen-antibody reactivity against human CRP antigen) 1 μl of human CRP (Sigma C4063, C-reactive protein derived from human plasma, 32 mg / dl) was mixed with 50 μl of buffer solution (Denka Seiken CRP-L Auto "TBA" buffer solution (R-1)) to prepare a mixture, which was then heated at 37° C. for 5 minutes. Next, 50 μl of the dispersion of affinity particles obtained in Examples 3-1 to 3-7 and Comparative Example 3-1 was mixed with the above mixture, and the absorbance at a wavelength of 572 nm was measured for the dispersion immediately after stirring. The absorbance was measured using a spectrophotometer GeneQuant 1300 manufactured by Biochrom. Then, this dispersion was left to stand at 37° C. for 5 minutes, and the absorbance at a wavelength of 572 nm was measured again, and the change in absorbance ΔABS×10000 was calculated. The results of evaluating the change in absorbance ΔABS×10,000 for each of the affinity particles 3-1 to 3-7 and the modified CRP-immobilized SG particles 3-1 using the following indices are shown in Table 3-1. A: The change is 10,000 or more B: The change is between 5,000 and 10,000 C: Change is less than 5000

[0313] (Non-specific adsorption evaluation) Moreover, instead of adding 1 μl of human CRP (Sigma C4063, C-reactive protein derived from human plasma, 32 mg / dl), physiological saline was used for the evaluation in the same manner, non-specific adsorption was evaluated, and the change in absorbance ΔABS × 10000 was calculated. The results of evaluation of the change in absorbance ΔABS × 10000 for each of affinity particles 3-1 to 3-7 and CRP-immobilized SG particles modified 3-1 using the following indexes are shown in Table 3-1. A: The change is less than 1000 B: Change is 1000 or more

[0314] [Table 3-1]

[0315] As described above, by having a structure having a carboxy group via a sulfide group in the side chain, it is possible to provide particles that have excellent ability to suppress nonspecific adsorption, have high sensitivity in the immunolatex agglutination method, and chemically bond ligands to the particle surface with high yield.

[0316] (Example 3-8: Synthesis of particles 3-8 with small particle size) A particle dispersion was obtained in the same manner as in step 1 of Example 3-1, except that the amount of styrene in Example 3-1 was changed to 0.5 g, the amount of glycidyl methacrylate was changed to 0.8 g, the amount of divinylbenzene was changed to 0.02 g, and the amount of GMA added to the three-necked separable flask 2 hours after the start of polymerization was changed to 0.13 g. Particles 3-8 having a carboxy group as a reactive functional group were obtained by the same experimental operation as in step 2 of Example 3-1, except that the pH of the reaction solution in step 2 of Example 3-1 was changed from 10.3 to 10.7. The particles were centrifugal purified, replaced with pure water, and stored. When particles 3-8 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.), the number average particle size was 184 nm. In addition, the amount of carboxy groups per unit mass was quantified to be 215 [nmol / mg].

[0317] (Example 3-9: Synthesis of small particles 3-9 having different carboxyl group amounts) Particles 3-9 having a carboxy group as a reactive functional group were obtained by the same experimental procedure as in Example 3-8, except that the pH of the reaction solution in step 2 in Example 3-8 was changed from 10.3 to 9.98. The purification and storage methods were also the same. The particle size of particles 3-9 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was 153 nm. In addition, the amount of carboxy groups per unit mass was quantified to be 96 [nmol / mg].

[0318] (Example 3-10: Synthesis of small particles 3-10 with different carboxyl group amounts) Particles 3-10 having carboxyl groups as reactive functional groups were obtained by the same experimental procedure as in Example 3-8, except that the pH of the reaction solution in step 2 in Example 3-8 was changed from 10.3 to 2.3 (triethylamine was not used). The purification and storage methods were also the same. The particle size of particles 3-10 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was 149 nm. The amount of carboxyl groups per unit mass was quantified and found to be 22 [nmol / mg].

[0319] (Example 3-11: Synthesis of particles 3-11 with large particle size) A particle dispersion was obtained in the same manner as in step 1 of Example 3-1, except that the amount of styrene in Example 3-1 was changed to 2.27 g, the amount of glycidyl methacrylate was changed to 3.4 g, the amount of divinylbenzene was changed to 0.08 g, and the amount of GMA added to the three-necked separable flask 2 hours after the start of polymerization was changed to 0.56 g. Particles 3-11 having carboxy groups as reactive functional groups were obtained by the same experimental operation as in step 2 of Example 3-1, except that the pH of the reaction solution in step 2 of Example 3-1 was changed from 10.3 to 11.3. The particles were purified by centrifugation, replaced with pure water, and stored. When particles 3-11 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.), the number average particle size was 309 nm. In addition, the amount of carboxy groups per unit mass was quantified to be 266 [nmol / mg].

[0320] (Example 3-12: Synthesis of large-sized particles 3-12 having different amounts of carboxy groups) Particles 3-12 having a carboxy group as a reactive functional group were obtained by the same experimental procedure as in Example 3-11, except that the pH of the reaction solution in step 2 in Example 3-11 was changed from 11.3 to 11.0. The purification and storage methods were also the same. The particle size of particles 3-12 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was 280 nm. In addition, the amount of carboxy groups per unit mass was quantified to be 239 [nmol / mg].

[0321] (Example 3-13: Synthesis of large-sized particles 3-13 with different amounts of carboxy groups) Particles 3-13 having carboxyl groups as reactive functional groups were obtained by the same experimental procedure as in Example 3-11, except that the pH of the reaction solution in step 2 in Example 3-11 was changed from 10.3 to 2.4 (without using triethylamine). The purification and storage methods were also the same. The particle size of particles 3-13 was evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was 247 nm. The amount of carboxyl groups per unit mass was quantified and found to be 16 [nmol / mg].

[0322] (Example 3-14: Synthesis of affinity particles) For particles 3-8 to 3-13, 0.1 mL (1 mg of particles) of each particle dispersion (1.0 wt% solution, 10 mg / mL) was transferred to a microtube (volume 1.5 mL), 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C. After centrifugation, the supernatant was discarded. 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was redispersed by ultrasonication. Centrifugation and redispersion were repeated once.

[0323] Next, 60 μL of WSC solution (50 mg of WSC dissolved in 1 mL of activation buffer, WSC means 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] hydrochloride) and Sulfo NHS solution (50 mg of Sulfo NHS dissolved in 1 mL of activation buffer, Sulfo NHS means sulfo-N-hydroxysuccinimide) were added and dispersed by ultrasonic waves. The particles were stirred at room temperature for 30 minutes to convert the carboxyl groups of the particles to active esters. The mixture was centrifuged at 15000 rpm (20400 g) for 5 min at 4° C., and the supernatant was discarded. 0.2 mL of immobilization buffer (25 mM MES, pH 5.0) was added and dispersed by ultrasonic waves. The mixture was centrifuged at 15000 rpm (20400 g) for 5 min at 4° C., and the supernatant was discarded. 50 μL of immobilization buffer was added, and the particles with activated carboxyl groups were dispersed by ultrasonic waves.

[0324] 50 μL of antibody solution (anti-CRP antibody diluted with immobilization buffer to 25 μg / 50 μL) was added to 50 μL of particle solution with activated carboxyl groups, and the particles were dispersed by ultrasonic waves. The amount of antibody added was 25 μg per 1 mg of particles (25 μg / mg). The final antibody concentration was 0.25 mg / mL, and the final particle concentration was 10 mg / mL. The tube was stirred at room temperature for 60 minutes to bind the antibody to the carboxyl groups of the particles. Then, the tube was centrifuged at 15000 rpm (20400 g) for 5 minutes at 4°C, and the supernatant was discarded. 0.24 mL of masking buffer (1 M Tris, pH 8.0 containing 0.1% Tween 20) was added, and the mixture was dispersed by ultrasonic waves. The mixture was stirred at room temperature for 1 hour, and then left to stand overnight at 4°C to bind Tris to the remaining activated carboxyl groups. Next, the mixture was centrifuged at 15000 rpm (20400 g) for 5 min at 4°C, and the supernatant was discarded. 0.2 mL of washing buffer (10 mM HEPES, pH 7.9) was added, and the mixture was dispersed by ultrasonic waves. A washing operation (centrifugation and redispersion) using washing buffer (10 mM HEPES, pH 7.9) was repeated once. A washing operation was performed once with 0.2 mL of storage buffer (10 mM HEPES, pH 7.9, containing 0.01% Tween 20). 1.0 mL of storage buffer was added, and the mixture was dispersed by ultrasonic waves. The final particle concentration was 0.1 wt% (1 mg / mL). The mixture was stored in a refrigerator. Hereinafter, the affinity particles obtained from particles 3-8 to 3-13 are referred to as affinity particles 3-8 to 3-13, respectively.

[0325] (Example 3-15: Evaluation of antigen-antibody reactivity of affinity particles 3-8 to 3-13) Antigen-antibody reactivity to human CRP antigen was evaluated in the same manner as in the above Example. The change in absorbance ΔABS×10000 of affinity particles 3-8 to 3-13 to human CRP (32 mg / dl) was 3430, 4330, 6930, 2250, 6750, and 12400, respectively. Antigen-antibody reactivity to human CRP antigen was confirmed for all particles.

[0326] (Example 3-16: Evaluation of non-specific adsorption of affinity particles 3-8 to 3-13) Non-specific adsorption was evaluated using physiological saline in the same manner as in the above examples. The change in absorbance ΔABS×10000 of affinity particles 3-8 to 3-13 was 40, 80, 120, −240, 0, and 260, respectively. No non-specific adsorption was observed in any of the particles.

[0327] (Example 3-17: Synthesis of affinity particles with increased antibody binding amount) Except for the amount of antibody added being 200 μg per 1 mg of particles (200 μg / mg), an affinity particle 3-14 was synthesized by binding an anti-CRP antibody to the particle 3-8 in the same manner as in Example 3-14, in which the amount of antibody binding was increased. The antigen-antibody reactivity of the obtained affinity particle 3-14 to the human CRP antigen was evaluated, and the value of the amount of change in absorbance of the affinity particle 3-14 to human CRP (32 mg / dl), ΔABS×10000, was 10440. The result of evaluating the non-specific adsorption using physiological saline was that the value of the amount of change in absorbance of the affinity particle 3-14, ΔABS×10000, was 600. It was found that the amount of change in absorbance, i.e., the sensitivity, can be improved by increasing the amount of antibody bound to the particle.

[0328] (Example 3-18: Evaluation of non-specific adsorption of human serum samples to particles) 51 μL of specimen diluent consisting of human serum specimen (1 μL, NHS-9, Tennessee Blood Services) and phosphate buffer (50 μL) was added to 50 μL of each dispersion of particles 3-8 to 3-12, and the absorbance of the mixture immediately after stirring was measured at a wavelength of 572 nm. The absorbance was measured using a Biochrom spectrophotometer GeneQuAnt1300. Then, each mixture was left to stand at 37°C for 5 minutes, and the absorbance at a wavelength of 572 nm was measured again, and the value of the change in absorbance ΔABS × 10000 was calculated. It was interpreted that the larger the value, the more nonspecific adsorption occurred. The values ​​of the change in absorbance ΔABS × 10000 for particles 3-8 to 3-12 were -70, -20, 60, -50, and 40, respectively. No nonspecific adsorption of human serum specimens was observed in any of the particles.

[0329] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0330] This application claims priority based on Japanese Patent Application No. 2019-158952 filed on August 30, 2019, Japanese Patent Application No. 2019-158962 filed on August 30, 2019, and Japanese Patent Application No. 2019-158138 filed on August 30, 2019, the entire contents of which are incorporated herein by reference.

Claims

1. A particle having a surface layer including a copolymer having a repeating unit A and a repeating unit B, the repeating unit A has a side chain A, and the side chain A has a carboxy group at a side chain terminal, the repeating unit B has a side chain B, and the side chain B has a hydroxyl group at a side chain terminal, the particle surface layer is configured to absorb water and form a swollen layer when the particle is dispersed in ion-exchanged water, the density of carboxy groups contained in the swelling layer satisfies formula (1-1), The particles are dispersed in ion-exchanged water at a concentration of 5% by mass, the resulting dispersion is dropped onto aluminum foil, dried at 25° C. for 48 hours, and then dried using a vacuum dryer for 24 hours. The dried particle size is measured after this, and the particle size in water is measured by dispersing the particles in ion-exchanged water having an electrical conductivity of 10 μS / cm or less to a concentration of 0.001% by mass, at a temperature of the dispersion of 25° C., using a dynamic light scattering method, and the particle size in water satisfies formula (1-2). 0.04≦[Carboxy group density (pieces / nm ３ )]≦0.15 ...Formula (1-1) 1.10≦[Particle size in water / Dry particle size]≦1.40...Formula (1-2)

2. The particle according to claim 1, characterized in that the zeta potential of the particle satisfies equation (1-3). −30≦[Zeta potential (mV)]≦−10 ... formula (1-3)

3. 3. The particle according to claim 1, wherein the number of moles of the repeating unit A and the number of moles of the repeating unit B satisfy formula (1-4). 0.05≦[number of moles of repeating unit A] / [number of moles of repeating unit B]≦1.00 ... Formula (1-4)

4. The particle according to any one of claims 1 to 3, wherein the repeating unit A is represented by formula (1-5). 【Chemistry 1】 R １ : Methyl group or hydrogen atom R ２ : Carboxy group or hydrogen atom L １ Optionally substituted alkylene or oxyalkylene group having 1 to 15 carbon atoms 5. The particle according to claim 4, wherein L 1 in the formula (1-5) is an alkylene group having one carbon atom.

6. The particle according to claim 4 or 5, wherein the repeating unit B is represented by formula (1-6). 【Chemistry 2】 R １ : Methyl group or hydrogen atom L ２ an optionally substituted alkylene or oxyalkylene group having 2 to 15 carbon atoms, [L １ [number of carbon atoms in L ２ The number of carbon atoms in the mixture satisfies the relationship shown in X: a sulfur atom or an optionally substituted nitrogen atom

7. L in the above formula (1-6) ２ The particle according to claim 6, characterized in that is an alkylene group having 2 carbon atoms.

8. X in the formula (1-6) is a sulfur atom, and L ２ 7. The particle according to claim 6, wherein: is an alkylene group having 3 carbon atoms, one of which is substituted with a hydroxyl group.

9. 9. The particle according to claim 1, wherein the particle has, as a repeating unit C, at least one selected from the group consisting of styrenes and (meth)acrylates.

10. 10. An affinity particle comprising the particle according to claim 1 bound to a ligand.

11. The affinity particle according to claim 10, wherein the ligand is an antibody or an antigen.

12. The affinity particle described in claim 10 or 11, wherein the ligand is any one of an anti-CRP antibody, an anti-KL-6 antibody, and an anti-IgE antibody.

13. An affinity particle having a ligand on the particle surface, an occupancy area ratio of the ligand to the particle surface satisfies the relationship of formula (2-1), The zeta potentials of the particles and the ligand satisfy the relationship of formula (2-2), The particles have a repeating unit A represented by formula (2-3), The occupied area ratio is represented by formula (2-7), The affinity particles are characterized in that the ligand is any one of an anti-CRP antibody, an anti-PSA antibody, an anti-BSA antibody, an anti-KL-6 antibody, and an anti-IgE antibody. 10≦[occupied area ratio (%)]≦40...Formula (2-1) 0≦[∥particle zeta potential (mV)∥−|ligand zeta potential (mV)∥]≦20 ... formula (2-2) 【Chemistry 3】 [Occupied area ratio (%)]=[Occupied area per ligand (nm 2 )]×[Number of ligands per affinity particle (units)] / [Surface area per particle (nm 2 )]×100 (Equation (2-7)) In formula (2-3), R １ R represents a methyl group or a hydrogen atom. ２ represents a carboxy group or a hydrogen atom. １ represents an alkylene group having 1 to 15 carbon atoms which may have a substituent, or an oxyalkylene group having 1 to 15 carbon atoms which may have a substituent.

14. The affinity particle according to claim 13, wherein the particle has a repeating unit B having a hydroxyl group at the end of the side chain.

15. L in formula (2-3) １ The affinity particle according to claim 13 or 14, wherein is a methylene group.

16. R in formula (2-3) ２ The affinity particle according to any one of claims 13 to 15, wherein is a carboxy group.

17. The affinity particle according to claim 14, wherein the repeating unit B is represented by formula (2-4). 【Chemistry 4】 In formula (2-4), R １ represents a methyl group or a hydrogen atom. ２ represents an alkylene group having 2 to 15 carbon atoms which may have a substituent, or an oxyalkylene group having 2 to 15 carbon atoms which may have a substituent. X represents a sulfur atom or a nitrogen atom which may have a substituent. １ and L in formula (2-4) ２ is [L １ [number of carbon atoms in L ２ The number of carbon atoms in the

18. L in formula (2-4) ２ The affinity particle according to claim 17, wherein is an alkylene group having 2 carbon atoms.

19. X in formula (2-4) is a sulfur atom, and L in formula (2-4) is ２ is an alkylene group having 3 carbon atoms, and L ２ 18. The affinity particle according to claim 17, wherein one of the hydrogen atoms of the alkylene group represented by the formula: is substituted with a hydroxyl group.

20. 15. The affinity particle according to claim 14, wherein the surface layer of the particle contains a copolymer having the repeating unit A and the repeating unit B, and when the particle is dispersed in an aqueous solution, the surface layer absorbs water and forms a swollen layer.

21. The affinity particle according to any one of claims 13 to 20, wherein the dry particle size of the particle is measured after a dispersion obtained by dispersing the particles in ion-exchanged water at a concentration of 5% by mass, dropping the dispersion onto aluminum foil, drying at 25°C for 48 hours, and drying for 24 hours using a vacuum dryer, and the particle size in water of the particle is measured by dynamic light scattering at a temperature of 25°C by dispersing the particles in ion-exchanged water having an electrical conductivity of 10 μS / cm or less to a concentration of 0.001% by mass, satisfying formula (2-5). 1.1≦[Particle size in water / Dry particle size]≦1.4 ...Formula (2-5)

22. 22. The affinity particle according to claim 13, having a chemical structure represented by formula (2-6) obtained by converting a part of the carboxyl groups of the repeating unit A using a chemical reaction. 【Chemistry 5】 In formula (2-6), R １ R represents a methyl group or a hydrogen atom. ２ represents a carboxy group or a hydrogen atom. １ represents an alkylene group having 1 to 15 carbon atoms which may have a substituent, or an oxyalkylene group having 1 to 15 carbon atoms which may have a substituent.

23. 23. The affinity particle according to any one of claims 13 to 22, wherein the particle has at least one repeating unit C selected from the group consisting of styrenes and (meth)acrylates.

24. A reagent for use in in vitro diagnosis for detecting a target substance in a specimen, comprising the affinity particle according to any one of claims 10 to 23.

25. The reagent according to claim 24, for use in detecting a target substance in a specimen by an agglutination method.

26. A kit for use in in vitro diagnosis for detecting a target substance in a specimen, comprising at least the reagent according to claim 24 or 25.

27. A method for detecting a target substance in a specimen by in vitro diagnosis, comprising mixing an affinity particle described in any one of claims 10 to 23 with a specimen that may contain the target substance.

28. The detection method according to claim 27, which is a method for detecting a target substance in a specimen by an agglutination method.

29. A particle having a copolymer containing a repeating unit A represented by the following formula (3-1): 【Chemistry 6】 R in formula (3-1) １ is a methyl group or a hydrogen atom, R ２ is a carboxy group or a hydrogen atom, L １ represents an optionally substituted alkylene group having 1 to 15 carbon atoms or an optionally substituted oxyalkylene group having 1 to 15 carbon atoms. However, among the copolymers, those containing both a repeating unit in which L 1 in formula (3-1) is an alkylene group having 1 to 3 carbon atoms and a repeating unit represented by the following general formula (1) are excluded. 【change】 In the above general formula (1), R 1 represents H or CH 3 , L1 to L3 each independently represent a direct bond or an alkylene group having 1 to 3 carbon atoms, and X represents S or NH.

30. The particle according to claim 29, characterized in that the amount of carboxylic acid per unit mass of the particle is 5 [nmol / mg] or more.

31. 31. The particle according to claim 29 or 30, characterized in that the amount of carboxylic acid per unit mass of the particle is 100 [nmol / mg] or more.

32. 32. A particle according to any one of claims 29 to 31, characterized in that the amount of carboxylic acid per unit mass of the particle is 300 [nmol / mg] or less.

33. 33. The particle according to any one of claims 29 to 32, characterized in that the number average particle size of the particles is 0.05 μm or more and 1 μm or less.

34. The amount of carboxylic acid per unit area of ​​the particles is 0.1 to 20 [pieces / nm ２ 34. A particle according to any one of claims 29 to 33, characterized in that:

35. L in the above formula (3-1) １ The particle according to any one of claims 29 to 34, characterized in that is an alkylene group having one carbon atom.

36. R in the above formula (3-1) ２ 36. The particle according to any one of claims 29 to 35, wherein is a carboxy group.

37. A particle described in any one of claims 29 to 36, characterized in that the copolymer further has a hydrophobic repeating unit C, and the repeating unit C is at least one selected from the group consisting of styrenes and (meth)acrylates.

38. An affinity particle comprising a particle according to any one of claims 29 to 37 chemically bonded to a ligand.

39. The affinity particle according to claim 38, characterized in that the ligand is an antibody or an antigen.

40. A reagent for use in in vitro diagnosis for detecting a target substance in a specimen, comprising the affinity particle according to claim 39.

41. The reagent according to claim 40, which is for use in detecting a target substance in a specimen by an agglutination method.

42. A kit for use in in vitro diagnosis for detecting a target substance in a specimen, comprising at least the reagent according to claim 40 or 41.

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