Particle, method for manufacturing particle, inspection particle, reagent, kit, and detection method

Abstract

To provide a particle for a latex agglutination method that has a reactive functional group for chemically coupling a ligand and has small nonspecific adsorption, and that can be used for inspection particles excellent in dispersion stability when stored standing, that is, excellent in standing storage stability.SOLUTION: A particle has a polymer including a structural unit A represented by a formula (1) and a structural unit B represented by a formula (2) (for the formula (1) and the formula (2), refer to the specifications). The specific gravity of the particle is within a range of 1.00 or more and 1.10 or less. The composition ratio of a C element to an O element quantified by XPS measurement is within a range of 2.1 or more and 3.3 or less.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to particles, a method for producing the particles, test particles, a reagent, a kit, and a detection method.

Background Art

[0002] As a simple and rapid immunoassay method, the latex agglutination method can be mentioned. In this method, a dispersion of test particles formed by binding a ligand having an affinity for a target substance to latex particles is mixed with a sample that may contain the target substance. At this time, if the target substance is contained in the sample, the test particles cause an agglutination reaction. Therefore, the disease can be diagnosed by optically detecting this agglutination reaction as a change amount such as scattered light intensity, transmitted light intensity, absorbance, etc.

[0003] In immunoassay items in clinical tests, many have important diagnostic points in the region where the target substance is present at a low concentration, and it is required to be able to determine the presence or absence of the target substance even at a low concentration. As a means for improving such detection sensitivity, it is known to increase the change amount such as absorbance detected by using latex particles with a large particle diameter, and improve the measurement sensitivity in the low concentration range.

[0004] In addition, it is desired that the particles used in the latex agglutination method and the test particles characterized by having a ligand for the target substance on the particle surface have small adsorption characteristics with substances other than the target substance, so-called non-specific adsorption. As particles with small non-specific adsorption, particles having polyglycidyl methacrylate arranged on the surface are known. It is considered that polyglycidyl methacrylate arranged on the surface of this particle reduces non-specific adsorption by causing a part of the glycidyl group to open the ring and exhibit glycol. Patent Document 1 discloses an example of applying, to bioseparation, a particle in which a ligand is chemically bonded via a polyethylene glycol chain to the surface of a particle having polyglycidyl methacrylate arranged in copolymer particles of styrene and glycidyl methacrylate.

[0005] The glycidyl group of glycidyl methacrylate can also be used as a reactive functional group for chemically bonding a ligand to the particle surface. By using a chemical bond for immobilizing the ligand on the particle surface, concerns such as the detachment of the ligand from the particle surface during long-term storage of the reagent are reduced. Patent Document 2 discloses an example in which the glycidyl group is carboxylated to impart a carboxy group for chemically bonding a ligand to the particle surface.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] The inventors of the present invention conducted studies for the purpose of improving the detection sensitivity in a low concentration range. As a result, in the copolymer particles of styrene and glycidyl (meth) acrylate as described in Patent Document 1, when the particle diameter was further increased, the sedimentation of the inspection particles during static storage was accelerated. This is presumably mainly due to the combination of the blending amount of glycidyl (meth) acrylate having a specific gravity greater than 1 and the particle diameter.

[0008] Depending on the inspection institution, the dispersion of the inspection particles may be statically stored in units of several weeks. Therefore, devising a way to maintain the dispersed state of the inspection particles in the dispersion is an important technical issue. If particle sedimentation occurs during storage, the dispensing accuracy at the time of inspection will decrease, making accurate measurement difficult.

[0009] In order to maintain the dispersion state of the inspection particles, the particle specific gravity is important. Generally, the dispersion medium of in vitro diagnostic reagents is water. Therefore, the difference between the specific gravity of water (1.0) and the particle specific gravity greatly affects the sedimentation level. In this case, when comparing particles with a specific gravity of 1.1 and particles with a specific gravity of 1.2, there is a two-fold difference in the sedimentation rate. In order to reduce sedimentation and maintain dispersion stability, components that increase the specific gravity and viscosity of the dispersion medium are added, but this may affect the detection sensitivity, and a technique for maintaining the balance of characteristics is required.

[0010] While it is preferable to use glycidyl (meth) acrylate as a particle component for the purpose of reducing non-specific adsorption and chemically bonding a ligand to the particle surface, in order to reduce sedimentation and maintain dispersion stability, it is considered necessary to reduce the blending ratio of glycidyl (meth) acrylate in the particles. However, simply changing the blending ratio also changes the abundance ratio on the surface of the copolymer particles, which may limit the detection sensitivity due to a decrease in the amount of ligand that can bind, or may significantly deteriorate the non-specific adsorption property due to a decrease in the surface hydroxy groups.

[0011] The present invention has been made in view of these background technologies and problems. An object of the present invention is to provide particles for latex aggregation methods that have reactive functional groups for chemically bonding ligands and have low non-specific adsorption, and that are excellent in dispersion stability when stored statically, that is, particles for latex aggregation methods that can be used for inspection particles having excellent static storage stability. Further, the present invention aims to provide a method for producing such particles. Furthermore, the present invention aims to provide an inspection particle reagent, a kit, and a detection method.

Means for Solving the Problems

[0012] In order to solve the above problems, the present invention has a polymer containing a structural unit A represented by formula (1) and a structural unit B represented by formula (2), wherein the specific gravity is in the range of 1.00 or more and 1.10 or less, Particles in which the composition ratio of carbon element to oxygen element quantified by XPS measurement is in the range of 2.1 or more and 3.3 or less.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Advantages of the Invention

[0013] According to the present invention, it is possible to provide particles for latex agglutination methods having reactive functional groups for chemically bonding ligands, low non-specific adsorption, and excellent stability upon standing storage, and a method for producing the same. Furthermore, it is possible to provide test particles formed by chemically bonding ligands, and reagents and kits for in vitro diagnostic agents containing the same, as well as a method for detecting a target substance.

Brief Description of the Drawings

[0014]

Figure 1

Modes for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described in detail, but the technical scope of the present invention is not limited to these embodiments.

[0016] 〔First Embodiment〕 The first embodiment relates to particles. The particles of the present invention have a polymer containing a structural unit A represented by formula (1) and a structural unit B represented by formula (2), and have a specific gravity in the range of 1.00 or more and 1.10 or less, The composition ratio of carbon element to oxygen element quantified by XPS measurement is in the range of 2.1 or more and 3.3 or less. [Chemical formula] [Chemical formula] (In formula (1), R 1 represents a methyl group or a hydrogen atom. L 1 represents an alkylene group having 1 to 4 carbon atoms. R 2 is a group containing a sulfide group or a secondary amine and a hydroxy group. In formula (2), R 3 represents a methyl group or a hydrogen atom. L 4 represents an alkylene group having 1 to 4 carbon atoms. R 4 is a group containing a sulfide group or a secondary amine and a carboxy group.)

[0017] L in formula (1) 1 , and L in formula (2) 4 are preferably a linear or branched alkylene group such as a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, etc., and more preferably a methylene group from the viewpoint of the balance between hydrophilicity and hydrophobicity. For this purpose, it is more preferable to use glycidyl (meth) acrylate as the epoxy group-containing monomer used in the formation of mother particles in the stage prior to the production of the particles of the present invention.

[0018] When a glycidyl group-containing monomer such as glycidyl (meth) acrylate is used in the formation of mother particles, a specific compound can be reacted with the glycidyl group present on the surface layer of the mother particles. Thereby, R in formula (1) 2 , and R in formula (2) 4 can be added to the particle surface layer as side chains, and particles having a polymer containing structural units A and B can be obtained.

[0019] In particular, the side chain of structural unit A is a terminal hydroxy group and R 2Having three or more hydroxy groups combined from two or more hydroxy groups contained therein has a high effect of reducing non-specific adsorption of particles.

[0020] Preferably, in the particle of the present invention, the structural unit A contains a structure represented by the formula (5).

Chemical formula

[0021] L in the formula (5) 1 is preferably a linear or branched alkylene group such as a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, or an isobutylene group. L in the formula (5) 2 , L 3 is preferably a single bond or a linear or branched alkylene group such as a methylene group, an ethylene group, or an n-propylene group. L in the formula (5) 1 , L 2 , L 3 are more preferably each a methylene group. When each is a methylene group, the balance between hydrophilicity and hydrophobicity is good, and furthermore, since the side chain of the structural unit A does not become longer than the side chain of the structural unit B, the possibility of hindering the reactivity of the carboxy group of the structural unit B can be further reduced.

[0022] In formula (5), X can be either a sulfur atom or a nitrogen atom. When it is a sulfur atom, it is more preferable from the perspective of improving sensitivity. When it is a nitrogen atom, it is more preferable from the perspective of suppressing non-specific adsorption. Since the sulfide bond has a weak hydrophobic tendency, it can moderately weaken the water-binding force of the highly hydrophilic side chain and is expected to suppress the osmotic aggregation that may occur when mixed with a high-concentration analyte, contributing to an improvement in sensitivity. On the other hand, since the amino group has a hydrophilic tendency, it can contribute to a reduction in non-specific adsorption.

[0023] When 3-mercapto-1,2-propanediol is reacted with the glycidyl group derived from glycidyl (meth)acrylate, L in formula (5) 1 , L 2 , L 3 each becomes a methylene group, and a structural unit A in which X is a sulfur atom is formed, which is more preferable from the perspective of improving sensitivity as described above.

[0024] Also, when 3-amino-1,2-propanediol is reacted with the glycidyl group derived from glycidyl (meth)acrylate, L in formula (5) 1 , L 2 , L 3 each becomes a methylene group, and a structural unit A in which X is a nitrogen atom is formed, which is more preferable from the perspective of reducing non-specific adsorption as described above.

[0025] The side chain of structural unit B has a carboxy group for chemically bonding with a ligand. In particular, having two or more carboxy groups improves the reaction efficiency with the ligand. It is preferable that the particle of the present invention contains a structural unit B having a structure represented by formula (6).

Chemical formula

[0026] When carboxyl groups are present in extremely close proximity to each other as in formula (6), they interact with each other to suppress dissociation and destabilize the dispersibility of the particles. Therefore, when used in the immuno-latex agglutination measurement method, it is advantageous for detecting the target substance with high sensitivity.

[0027] L in formula (6) 4 is preferably a linear or branched alkylene group such as a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, or an isobutylene group. L in formula (6) 5 is preferably a single bond or a linear or branched alkylene group such as a methylene group, an ethylene group, or an n-propylene group. L in formula (6) 4 and L 5 are more preferably methylene groups. If the carbon number becomes too large, there is a possibility of promoting non-specific adsorption to the particles due to an increase in hydrophobicity.

[0028] X in formula (6) may be either a sulfur atom or a nitrogen atom, but is more preferably a sulfur atom. Since the sulfide bond is a structure showing a weak hydrophobic tendency, it is expected to moderately weaken the water restraint force of the side chain and suppress the osmotic pressure aggregation that may occur when mixed with a high-concentration analyte.

[0029] When mercaptosuccinic acid is reacted with the glycidyl group derived from glycidyl (meth)acrylate, L 4 and L 5 each become a structural unit B in which the group is a methylene group and X is a sulfur atom, and thus, as described above, it is more preferable.

[0030] The molar fraction of "structural unit A" / "structural unit B" is preferably 0.2 or more and 20 or less. When it is less than 0.2, the proportion of carboxy groups contained in structural unit B is excessive, which may impair the dispersion stability of the particles. In addition, when a ligand is chemically bonded, unreacted carboxy groups may interact with the ligand and modify the ligand. When it is greater than 20, the proportion of carboxy groups is small, which may reduce the reaction efficiency when chemically bonding the ligand or reduce the electrostatic repulsion contributing to the dispersion stability of the particles.

[0031] The particles of the present invention further have a structural unit C represented by formula (3) or formula (4). From the viewpoint of the mechanical strength of the particles, it is preferable that the total amount of structural unit A and structural unit B in the total amount of structural unit A, structural unit B, and structural unit C is 43 mol% or less, and more preferably 5 mol% or more and 43 mol% or less.

Chemical formula

Chemical formula

[0032] Examples of the compound from which the component represented by the formula (3) is derived include styrenes such as styrene, α-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, p-n-butylstyrene, p-tert-butylstyrene, p-n-hexylstyrene, p-n-octylstyrene, and p-n-nonylstyrene. Examples of the compound from which the component represented by the formula (4) is derived include (meth)acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, iso-propyl acrylate, n-butyl acrylate, iso-butyl acrylate, tert-butyl acrylate, n-amyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate, cyclohexyl 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, and n-nonyl methacrylate.

[0033] In any case, the invention is not limited to these as long as the object of the invention can be achieved. Also, two or more kinds of components may be used in combination. The compound from which the structural unit C is derived is preferably a styrene represented by the formula (3), more preferably styrene. Further, in the particles of the present invention, the structural unit C is preferably at least one selected from the group of styrenes, and more preferably styrene. Styrenes are preferable from the viewpoints of reducing the particle specific gravity and improving the refractive index and sensitivity of the particles because the specific gravity of the monomer is small and the refractive index is excellent.

[0034] In the particles of the present invention, the amount of structural unit C is preferably 57 mol% or more and 95 mol% or less, more preferably 68 mol% or more and 93 mol% or less, in the total amount of the amount of structural unit A, the amount of structural unit B, and the amount of structural unit C. (That is, as described above, the total amount of structural unit A and structural unit B is preferably 5 mol% or more and 43 mol% or less in the total amount of the amount of structural unit A, the amount of structural unit B, and the amount of structural unit C.)

[0035] <Specific gravity of the particles of the present invention> The particles of the present invention have a specific gravity in the range of 1.00 or more and 1.10 or less. By having a specific gravity of 1.10 or less, excellent dispersion stability can be maintained even when the dispersion of the test particles, which are the test reagent, is stored standing. Since particle sedimentation is reduced, the usable period of the test reagent can be extended. Specifically, by maintaining the dispensing accuracy when stored for a certain period, a decrease in detection sensitivity can be prevented.

[0036] The dominant factor determining the specific gravity of the particles is the constituent components of the particles. Further, in the case of crosslinkable particles, a crosslinking degree of a certain level or more also affects the specific gravity.

[0037] The constituent components of the particles of the present invention are mainly components derived from the reactive monomers used to form the aforementioned mother particles. Specifically, since the glycidyl group-containing monomer (more preferably glycidyl (meth) acrylate in the present invention) which is the origin of structural units A and B, and the monomer which is the origin of structural unit C (more preferably styrene in the present invention) are the main components, the specific gravity of the particles of the present invention is almost determined by the content ratio of these mother particle-forming monomers.

[0038] In other words, the side chain components and ligands added to the surface after the formation of the mother particles exist only in the vicinity of the particle surface, and the proportion as the constituent components of the particles is small, so the contribution to the specific gravity of the particles of the present invention is very small.

[0039] Among the mother particle-forming monomers, the higher the content of glycidyl group-containing monomers (more preferably glycidyl (meth)acrylate in the present invention) having a specific gravity greater than 1, the greater the particle specific gravity. Therefore, in order to reduce sedimentation and maintain dispersion stability, it is preferable to reduce the content of epoxy group-containing monomers in the formation of mother particles. Specifically, the total amount of structural unit A and structural unit B in the total amount of structural unit A, structural unit B, and structural unit C is preferably 43 mol% or less, and more preferably 32 mol% or less.

[0040] On the other hand, the structures of structural units A and B are essential for excellent non-specific adsorption properties and chemical bonding of ligands to the particle surface. If the abundance is too small, there is a possibility of deterioration of non-specific adsorption properties and inability to bind a sufficient amount of ligands. Therefore, the total amount of the amount of structural unit A and the amount of structural unit B in the total amount of the amount of structural unit A, the amount of structural unit B, and the amount of structural unit C is preferably 20 mol% or more.

[0041] <Composition ratio quantified by XPS measurement of the particles of the present invention> As described above, while it is preferable to use glycidyl (meth)acrylate as a particle component for the purpose of reducing non-specific adsorption and chemical bonding of ligands to the particle surface, in order to reduce sedimentation and maintain dispersion stability, it is considered necessary to reduce the content of glycidyl (meth)acrylate in particle formation. However, simply reducing the blending amount will not only reduce the abundance throughout the particles but also the abundance in the "surface layer" of the particles. In that case, the amount of carboxy groups and hydroxy groups imparted in the subsequent process decreases, which may result in a decrease in the amount of ligands that can be bound or a significant deterioration in non-specific adsorption properties.

[0042] The particles of the present invention are preferably particles in which the abundance ratio in the "surface layer" of the particles is appropriately controlled even when the abundance ratio of the components throughout the particles, that is, the charging ratio, is changed. (A method for producing particles for appropriate control will be described later.)

[0043] The particles of the present invention are particles in which the composition ratio of C element to O element quantified by XPS measurement is controlled within the range of 2.1 or more and 3.3 or less. In XPS measurement, since photoelectrons generated from the outermost surface to about 10 nm are detected, it is a technique capable of analyzing the components of the "surface layer" of the particles. When the composition ratio is less than 2.1, the O element in the "surface layer" of the particles is excessive, and while sufficient hydroxyl groups and carboxyl groups are added, the hydrophilicity is extremely high, making it difficult for the particles to aggregate, and in some cases, the detection sensitivity based on the principle of latex aggregation method may decrease. Also, when the composition ratio is greater than 3.3, the O element in the "surface layer" of the particles is too little, and due to insufficient hydrophilicity, not only is the dispersion stability disadvantageous when the particles are stored for a long time, but also the non-specific adsorption property deteriorates. Thus, by optimizing the abundance of the components in the "surface layer" of the particles, it is possible to provide particles with both excellent detection sensitivity and non-specific adsorption property.

[0044] <DSC Curve of the Particles of the Present Invention> The particles of the present invention, on the DSC curve obtained during the second heating in differential scanning calorimetry (DSC) measurement, draw a straight line passing through the point on the DSC curve where the temperature is 80 °C and the point on the DSC curve where the temperature is 100 °C, and when the intersection point of the straight line and the DSC curve in the temperature range of 105 °C or more and 140 °C or less is defined as intersection point A, it is preferable that the DSC curve has an endothermic peak in the temperature range between the temperature At of the intersection point A and 100 °C.

[0045] This endothermic peak is derived from the characteristics of styrenes (structural unit C of the present invention). The fact that it is observed means that there are regions in the particles where polystyrene aggregates at a high concentration, indicating that structural units A and B and polystyrene (structural unit C) are constitutively separated within the particles. Since structural units A and B of the particles of the present invention are mainly arranged on the surface layer for the aforementioned "surface layer" control, it is inferred that the other, polystyrene (structural unit C), is confined inside the particles. Since the presence of polystyrene, which is a hydrophobic material, on the particle surface layer is a major factor in deteriorating non-specific adsorption properties, by confining polystyrene firmly inside the particles in this way, particles with excellent non-specific adsorption properties can be provided.

[0046] When the structural unit C of the present invention includes a polymethyl (meth)acrylate-based structure represented by the formula (4), when a straight line is drawn through the point at 80 °C and the point at 90 °C on the DSC curve obtained during the second heating in the DSC measurement, in the region of 95 °C or higher and 140 °C or lower, it preferably has an intersection point A between the straight line and the DSC curve, and has an endothermic peak between the temperature At of the intersection point A and 90 °C.

[0047] <Zeta potential of the particles of the present invention> The particles of the present invention preferably have a zeta potential of -50 mV or more and -10 mV or less. When it is less than -50 mV, it indicates that there are many carboxy groups on the particle surface layer. When a ligand is bound to the particles to form test particles, there is a possibility of a decrease in the reactivity of the ligand due to the electrostatic interaction between the ligand and the particle surface, and a possibility that the charge bias on the surface of the test particles due to the zeta potential difference between the ligand and the particle surface may lead to heteroaggregation. Therefore, it is preferably -50 mV or more in order to reduce them. Also, when it is greater than -10 mV, it indicates that there are few carboxy groups on the particle surface layer. In order to bind a sufficient amount of ligand to the particles and achieve excellent detection sensitivity, it is preferably -10 mV or less.

[0048] <Particle diameter of the particles of the present invention> The particles of the present invention preferably have a volume average particle diameter in the aqueous dispersion of 50 nm or more and 500 nm or less, more preferably 280 nm or more and 400 nm or less, and even more preferably 280 nm or more and 350 nm or less.

[0049] When the particle diameter is 280 nm or more, the amount of change such as absorbance detected by the latex aggregation method increases, and the detection sensitivity particularly when the target substance is present at a low concentration is improved. Conventionally, when attempting to use particles having a particle diameter of 280 nm or more and containing a component derived from glycidyl (meth) acrylate as a test reagent, problems in actual use tend to occur during long-term storage of the test reagent due to particle sedimentation caused by the particle size and the specific gravity of glycidyl (meth) acrylate. Since the particle specific gravity of the particles of the present invention is controlled, using these particles can improve the dispersion stability of the test reagent and particle sedimentation during long-term storage even for particles of 280 nm or more. Also, from the same viewpoint, it is more preferably 400 nm or less, and even more preferably 350 nm or less.

[0050] 〔Second Embodiment〕 The second embodiment relates to a method for producing particles. As described above, while it is preferable to use glycidyl (meth) acrylate as a particle component, in order to reduce sedimentation and maintain dispersion stability, it is considered necessary to reduce the content of glycidyl (meth) acrylate in particle formation. However, simply reducing the blending amount will reduce not only the abundance throughout the particles but also the abundance in the "surface layer" of the particles.

[0051] The particles of the present invention are preferably particles in which the abundance ratio in the "surface layer" of the particles is appropriately controlled even when the abundance ratio of the components throughout the particles, that is, the charging ratio, is changed. The method for producing the particles of the present invention for achieving this includes the following steps. Step 1: A step of mixing a compound represented by formula (7), a compound represented by formula (8), water, and a radical polymerization initiator to initiate polymerization Step 2: After Step 1, a step of further adding the compound represented by the formula (7) to the reaction system. Here, the ratio of the mass of the compound represented by the formula (7) added in Step 2 to the total mass of the compound represented by the formula (7) and the compound represented by the formula (8) mixed in Step 1 is 0.16 or more and 0.30 or less.

Chemical formula

Chemical formula

[0052] L in formula (7) 6 is preferably a linear or branched alkylene group such as a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, or an isobutylene group. R in formula (8) 11 is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, a tert-pentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, or an n-nonyl group.

[0053] The compound represented by the formula (7) added in Step 2, that is, the glycidyl group-containing monomer, is considered to dissolve immediately after being added to the water in the reaction system and to be used in the polymerization reaction and adhere to the particle surface layer.

[0054] In Step 2, by setting the addition amount of the compound represented by the formula (7), that is, the glycidyl group-containing monomer, to 16% by mass or more, even when the total usage amount of the glycidyl group-containing monomer used for forming the mother particles is reduced, a sufficient amount of glycidyl groups can be present in the "surface layer" of the mother particles. As a result, in the subsequent steps, a sufficient amount of hydroxy groups and carboxy groups can be added to the particle surface layer. The composition ratio of carbon element to oxygen element quantified by XPS measurement of the particles is 3.3 or less, providing particles with high hydrophilicity and excellent non-specific adsorption properties. Further, by setting the addition amount of the glycidyl group-containing monomer in Step 2 to 30% by mass or less, the glycidyl groups on the surface layer of the mother particles do not become excessive, and the addition of an excessive amount of hydroxy groups and carboxy groups to the particle surface layer can be prevented. The composition ratio of carbon element to oxygen element quantified by XPS measurement of the particles is 2.1 or more, providing particles with appropriate hydrophobicity, ensuring the aggregability required for the latex aggregation method, and providing particles with excellent detection sensitivity.

[0055] In the method for producing the particles of the present invention, in Step 2, it is preferable to add the compound represented by the formula (7), that is, the glycidyl group-containing monomer, in multiple portions. In order to more accurately control the amount of glycidyl groups on the surface layer of the mother particles, it is necessary to consider the amount of monomer remaining in the reaction system during the addition in Step 2. When the monomer addition in Step 2 is divided into multiple portions, in the first addition, the monomers remaining in the reaction system (especially styrenes with low solubility in water) can be efficiently dissolved in water, promoting the polymerization reaction. As a result, the amount of residual monomer in the reaction system can be reduced at the time of the last addition in Step 2, and the dissolution of the residual monomer in the last addition is reduced, so that the ratio of glycidyl groups finally present in the "surface layer" of the particles can be efficiently increased. "Dividing the monomer addition into multiple portions" means that the time interval between the first addition and the last addition is 1 minute or more. As a specific method, a certain amount may be added multiple times at intervals, or continuous dropping may be performed without intervals.

[0056] The method for producing the particles of the present invention preferably includes the following step 3 after step 2. Step 3: A step of preparing a mixed solution by mixing an aqueous dispersion of the obtained granular copolymer, 3-mercapto-1,2-propanediol, and mercaptosuccinic acid, and reacting an epoxy group derived from the compound represented by the formula (7) with a thiol group derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid.

[0057] By reacting 3-mercapto-1,2-propanediol and mercaptosuccinic acid with the epoxy group of the obtained granular copolymer (mother particles), the side chains represented by the structural unit A represented by the formula (5) and the structural unit B represented by the formula (6) can be imparted to the particle surface layer. The reasons why 3-mercapto-1,2-propanediol and mercaptosuccinic acid are more preferable are as described above.

[0058] In the method for producing the particles of the present invention, other typical production methods will be described. However, the method for producing the particles of the present invention is not limited within the range capable of achieving the object of the present invention.

[0059] The radical polymerization initiator in the step 1 is preferably a water-soluble polymerization initiator. The water-soluble polymerization initiator is not particularly limited, but water-soluble azo compounds and water-soluble peroxides are preferably used.

[0060] The water-soluble azo compound is preferably 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.

[0061] As the water-soluble peroxide, potassium persulfate, ammonium persulfate, sodium persulfate, tertiary butyl hydroperoxide, cumene hydroperoxide, paramethane hydroperoxide, or diisopropylbenzene hydroperoxide is preferably used.

[0062] In the step 1, a crosslinkable radically polymerizable monomer can be further used. Examples of the crosslinkable radically polymerizable monomer 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, 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 thereto as long as the object of the present invention can be achieved. Further, two or more kinds of crosslinkable radically polymerizable monomers may be used in combination.

[0063] The amount of the crosslinkable radically polymerizable monomer is preferably from 0.1% by mass to 5% by mass of the total monomers used for the polymerization. When it is more than 5% by mass, the particle density may increase and the specific gravity of the particles may increase.

[0064] When forming the granular copolymer (parent particles) by radical polymerization, it is preferable to use emulsion polymerization, soap-free emulsion polymerization, or suspension polymerization, and more preferably emulsion polymerization or soap-free emulsion polymerization. Even more preferably, soap-free emulsion polymerization is used. Generally, compared with suspension polymerization, emulsion polymerization and soap-free emulsion polymerization can obtain a granular polymer with a sharp particle size distribution. Also, when binding particles to a ligand to form test particles, there is a concern that an anionic surfactant or a cationic surfactant generally used in emulsion polymerization may exist as a residue and denature the ligand. For this reason, when forming the granular polymer (parent particles) by emulsion polymerization, it is preferable to use a nonionic surfactant.

[0065] In the step 3, it includes a step of using an organic base having no primary amine and adjusting its pH to the alkaline region. In the step 3, the epoxy groups of the granular copolymer (parent particles) are reacted in the depth direction from the surface of the granular copolymer (parent particles) using thiol groups derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid. In order to perform sufficient reaction in the depth direction, it is preferable to select triethylamine having permeability to the granular copolymer (parent particles) as the organic base.

[0066] The method for producing the particles of the present invention preferably includes the following step 3' after the step 2. Step 3': A step of preparing a mixed solution by mixing an aqueous dispersion of the obtained granular copolymer, 3-amino-1,2-propanediol, and mercaptosuccinic acid, and reacting an epoxy group derived from the compound represented by the formula (7), an amino group derived from 3-amino-1,2-propanediol, and a thiol group derived from mercaptosuccinic acid.

[0067] 〔Application Examples〕 The application examples of the present invention are inspection particles, reagents, kits, and detection methods. Each item will be described below.

[0068] <Inspection Particles> The inspection particles of the present invention are preferably formed by chemically bonding the above-described particles with a ligand, and more preferably formed by bonding the above particles with a ligand having an affinity for a target substance. 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 determined and has selective or specifically high affinity. For example, examples of ligands include antigens and antibodies, enzyme proteins and their substrates, signal substances represented by hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc., but are not limited to these within the scope capable of achieving the object of the present invention. Specifically, ligands include antigens, antibodies, antigen-binding fragments (for example, Fab, F(ab’)2, F(ab’), Fv, scFv, etc.), naturally-derived nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, and the like. The inspection particles of the present invention preferably have a ligand that is an antibody or an antigen.

[0069] In the present invention, as a method of a chemical reaction for bonding a carboxy group or carboxylate derived from structural unit B with a ligand, a conventionally known method can be applied within the scope capable of achieving the object of the present invention. For example, carbodiimide-mediated reactions and NHS ester activation reactions are suitable examples of chemical reactions. Also, avidin may be bound to the carboxy group and a biotin-modified ligand may be bound. However, the method of the chemical reaction for bonding the carboxy group or carboxylate derived from structural unit B with the ligand is not limited to these within the scope capable of achieving the object of the present invention.

[0070] As described above, it is reasonable to judge that the specific gravity of the particles of the present invention does not change whether they are the particles before binding the ligand or the test particles with the ligand chemically bonded, because the ratio of the ligand to the particles is sufficiently small.

[0071] <Reagent> The reagent of the present invention is a reagent used for detecting a target substance in a specimen by in vitro diagnosis, and preferably contains the above test particles. In the present invention, when an antibody (antigen) is used as the ligand and an antigen (antibody) is used as the target substance, as a method for detecting the target substance in a specimen in in vitro diagnosis, it can be very preferably applied to the latex agglutination method in immunoassays widely used in areas such as clinical examinations and biochemical research. When general particles are used as particles for the latex agglutination method, antigens (antibodies) as target substances or foreign substances in serum or plasma may non-specifically adsorb on the particle surface, which may cause unintended aggregation between particles and thus affect the accuracy of immunoassays.

[0072] The reagent of the present invention is preferably used for detecting a target substance in a specimen by an agglutination method, and preferably contains particles for the latex agglutination method. The amount of the particles for the latex agglutination method contained in the reagent of the present invention is preferably from 0.001% by mass to 20% by mass, more preferably from 0.01% by mass to 10% by mass. The reagent of the present invention may contain a third substance such as a solvent or a blocking agent in addition to the particles for the latex agglutination method within the range capable of achieving the object of the present invention. Examples of the solvent used in the present invention include various aqueous buffers such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, HEPES buffer, MES buffer, and ammonia buffer, but the solvent contained in the reagent of the present invention is not limited thereto.

[0073] <Kit> The kit of the present invention is a kit for detecting a target substance in a specimen by in vitro diagnosis, and preferably includes at least the reagent described above. As the kit of the present invention, in addition to the reagent of the present invention (hereinafter, reagent 1), it is preferable to further include a reaction buffer containing albumin (hereinafter, reagent 2). Examples of the albumin include serum albumin, and it may be protease-treated. The amount of albumin contained in reagent 2 is preferably in the range of 0.001% by mass to 5% by mass, but the kit of the present invention is not limited thereto. Either both or one of reagent 1 and reagent 2 may contain a sensitizer for latex agglutination measurement. Examples of the sensitizer for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, alginic acid, etc., but the kit of the present invention is not limited thereto. Further, the kit of the present invention may include a positive control, a negative control, a serum diluent, etc. in addition to reagent 1 and reagent 2. As the medium for the positive control and the negative control, in addition to serum and physiological saline that do not contain a measurable target substance, a solvent may also be used. The kit of the present invention can be used in the method for detecting the target substance of the present invention in the same manner as a kit for detecting a target substance in a specimen by ordinary in vitro diagnosis. Also, the concentration of the target substance can be measured by a conventionally known method, and in particular, it is suitable for detecting the target substance in a specimen by the latex agglutination method.

[0074] <Detection method> A method for detecting a target substance in a sample by in vitro diagnosis, preferably mixing the test particles described above with a sample that may contain the target substance, and more preferably a method for detecting the target substance in the sample by aggregation. Further, the mixing of the test particles of the present invention and the sample is preferably performed in the range of pH 3.0 to pH 11.0. The mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 1 minute to 20 minutes. Further, in this detection method, it is preferable to use a solvent. Further, the concentration of the test particles of the present invention in the detection method of the present invention 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 of the present invention is characterized by optically detecting the aggregation between particles generated as a result of the mixing of the test particles of the present invention and the sample. By optically detecting the aggregation between the particles, the target substance in the sample is detected, and further, the concentration of the target substance can also be measured. As a method for optically detecting the aggregation reaction, the change amount of these values may be measured using an optical instrument capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc.

[0075] [Method for Measuring Specific Gravity of Particles] The method for measuring the specific gravity of the particles in the present invention will be described. The specific gravity of the particles in the present invention is calculated by measuring the sedimentation rate when a predetermined centrifugal force is applied in a state where the particles are dispersed in ion-exchanged water to a concentration of 0.5% by mass, and using the values of the specific gravity, viscosity of the dispersion medium, and the volume average particle diameter of the particles. As the ion-exchanged water, that having an electric conductivity of 10 μS / cm or less is used. Specifically, using a lumisizer (MS Scientific Co., Ltd.), the sedimentation rate determined by the change in transmitted light when a centrifugal force of 4000 rpm is applied to the particle dispersion liquid is measured, and the specific gravity, viscosity of the ion-exchanged water as the dispersion medium, and the value of the volume average particle diameter of the particles, the measurement method of which will be described later, are used to calculate the specific gravity of the particles from the Stokes' equation shown below. The measurement is performed three times, and the specific gravity is the average value of the three measurement values.

[0076] (Stokes' Equation) Sedimentation rate Vs = D p 2 (ρ p -ρf ) g / 18η (Vs is the sedimentation velocity (m / s), D p is the particle diameter (m), ρ p is the specific gravity of the particles (kg / m 3 ), ρf is the specific gravity of the dispersion medium (kg / m 3 ), g is the acceleration due to gravity (m / s 2 ), and η is the viscosity of the dispersion medium (Pa·s))

[0077] [Method for Measuring C / O Ratio of Particles] The method for quantifying the composition ratio by XPS measurement of the particles in the present invention will be described. The composition ratio by XPS measurement of the particles in the present invention is measured using a sample in which the particles are fixed to an indium foil in a freeze-dried state. The measurement apparatus and measurement conditions are as follows. · Measurement apparatus: X-ray photoelectron spectrometer: Quantum2000 (trade name, manufactured by ULVAC-PHI, Inc.) · X-ray source: Monochromatic Al Kα · Xray Setting: 100μmφ (25W (15KV)) · Angle for extracting photoelectrons: 45 degrees · Neutralization condition: Combined use of a neutralization gun and an ion gun · Analysis area: 300×200μm · Pass Energy: 58.70 eV · Step size: 0.125 eV · Analysis software: Maltipak (PHI)

[0078] The number of integrations during measurement is C1 S , and O1 S is 15 times, and N1 S is 30 times. Using the quantitative values of the obtained elemental amounts (the composition ratio (atomic%) of the C element and the composition ratio (atomic%) of the O element with respect to the total amount of the three elements), the composition ratio of the C element to the O element is determined.

[0079] [Method for Obtaining DSC Curve of Particles] A method for obtaining the DSC curve of the particles in the present invention will be described. The lyophilized particles are used as samples, and the measurement is carried out using a differential scanning calorimeter (DSC) (trade name "Discovery DSC2500", manufactured by TA Instruments). The sample enclosed in an aluminum pan is measured using the temperature program described below. First, as the first heating, the temperature is raised from 20°C to 180°C at a heating rate of 10°C / min, then held at 180°C for 10 minutes, and then cooled to 10°C at a cooling rate of 10°C / min, and then held at 10°C for 10 minutes. Subsequently, as the second heating, the temperature is raised again to 180°C at 10°C / min. The DSC curve during the second heating is obtained.

[0080] [Method for measuring the zeta potential of particles] A method for measuring the zeta potential of the particles in the present invention will be described. The zeta potential in the present invention is measured in a state where the particles are dispersed to 0.001% by mass in a 0.01N potassium aqueous solution at pH 7.8. The 0.01N potassium aqueous solution at pH 7.8 is obtained 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 adjusted using ion-exchanged water with an electric conductivity of 10 μS / cm or less. The measuring device is a zetasizer (Nano-ZS: Spectris Co., Ltd.), and the measurement is carried out at 25°C. As the analysis parameter, latex (n ≈ 1.59) is selected as the refractive index of the particles, and pure water is selected as the dispersion medium. The measurement is carried out 10 times, and the zeta potential is the average value of the 10 measurement values.

[0081] [Method for measuring the volume average particle diameter in the aqueous dispersion of particles] A method for measuring the volume average particle diameter in the aqueous dispersion of the particles in the present invention will be described. The volume average particle diameter in the aqueous dispersion in the present invention is measured in a state where the particles are dispersed in ion-exchanged water to a concentration of 0.001% by mass. As the ion-exchanged water, that having an electric conductivity of 10 μS / cm or less is used. For the measurement of the particle size in water, the dynamic light scattering method is applied. Specifically, it is measured at 25 °C using a Zetasizer (Nano-ZS: Spectris Co., Ltd.). As the analysis parameters, latex (n≈1.59) is selected as the refractive index of the particles, and pure water is selected as the dispersion medium. The measurement is carried out 10 times, and the particle size in water is the average value of the 10 measurement values.

[0082] [Method for measuring the antibody sensitization rate of test particles] A method for measuring the antibody sensitization rate of test particles prepared using the particles of the present invention will be described. The antibody sensitization rate (%) of the test particles is 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 (the amount of antibody charged).

[0083] First, 7 mL of Solution A and 140 μL of Solution B of a Protein Assay BCA Kit (Wako Pure Chemical Industries, Ltd.) are mixed, and the prepared solution is designated as the AB solution. Next, 200 μL of the AB solution is added to 25 μL (particle amount 25 μg) of the dispersion of the test particles (0.1% solution), and the mixture is incubated at 60 °C for 30 minutes. The solution is centrifuged at 15000 rpm (20400 g) for 5 minutes at 4 °C, and 200 μL of the supernatant is pipetted into a 96-well microplate. The absorbance at 562 nm is measured with a microplate reader together with standard samples (several points in the range of 0 to 200 μg / mL of antibody in 10 mM HEPES), and the amount of antibody is calculated from the standard curve. The amount of antibody sensitized to the particles (the amount of antibody bound per particle mass (μg / mg)) is obtained by dividing the calculated amount of antibody by the particle mass (here, 0.025 mg). Finally, the sensitization rate is calculated. When the amount of antibody charged is 25 μg per 1 mg of particles and the amount of antibody sensitized is 12.5 μg / mg, the sensitization rate is 50%. [Examples]

[0084] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[0085] [Example 1-1] (Synthesis of granular copolymer (master particle) 1) In a 2 L four-neck separable flask, 27.8 g of styrene (St: Kishida Chemical Co., Ltd.), 17.3 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.), 0.70 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.), and 1305.5 g of ion-exchanged water were weighed and mixed to form a mixed solution. Then, while stirring this mixed solution at 200 rpm, it was maintained at 70 °C, and nitrogen flow was carried out at a flow rate of 200 ml / min to purge the oxygen in the four-neck separable flask. Next, a solution prepared separately by dissolving 0.68 g of V-50 (Fuji Film Wako Pure Chemical Corporation) in 18 g of ion-exchanged water was added to the mixed solution to initiate soap-free emulsion polymerization.

[0086] Two hours after the start of polymerization, as an additional GMA step, 5.2 g of GMA was added to the four-neck separable flask, and 5.2 g of GMA was added after another 30 minutes (GMA 5.2 g was added twice). A water dispersion containing granular copolymer 1 was obtained by maintaining at 70 °C while stirring at 200 rpm for 22 hours. After slowly cooling the dispersion to room temperature, a part of it was collected, and when the polymerization conversion rate was evaluated using proton NMR and gas chromatography, it was confirmed that it was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in granular copolymer 1 is 42.2 mol%. Also, the mass ratio of the additional GMA amount to the monomer amount at the start of polymerization is 0.23. The volume average particle diameter of granular copolymer 1 was 240 nm. Granular copolymer 1 was stored at 4 °C under light-shielded conditions.

[0087] [Example 1-2] (Synthesis of granular copolymer (master particle) 2) The amount of St in Example 1-1 was changed from 27.8 g to 35.0 g, the amount of GMA was changed from 17.3 g to 9.7 g, and the amount of additional GMA was changed from 5.2 g added twice to 6.2 g added twice. A water dispersion of granular copolymer 2 was obtained in the same manner as in Example 1-1. After slowly cooling the dispersion to room temperature, a part thereof was collected, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. As a result, it was confirmed that the conversion rate was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in granular copolymer 2 was 32.7 mol%. Also, the mass ratio of the amount of additional GMA to the amount of monomer at the start of polymerization was 0.27. The volume average particle diameter of granular copolymer 2 was 245 nm.

[0088] [Example 1-3] (Synthesis of granular copolymer (parent particle) 3) The amount of GMA in Example 1-1 was changed from 17.3 g to 19.3 g, and the amount of additional GMA was changed from 5.2 g added twice to 4.1 g added twice. A water dispersion of granular copolymer 3 was obtained in the same manner as in Example 1-1. After slowly cooling the dispersion to room temperature, a part thereof was collected, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. As a result, it was confirmed that the conversion rate was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in granular copolymer 3 was 41.9 mol%. Also, the mass ratio of the amount of additional GMA to the amount of monomer at the start of polymerization was 0.17. The volume average particle diameter of granular copolymer 3 was 234 nm.

[0089] [Example 1-4] (Synthesis of granular copolymer (parent particle) 4) The amount of 1305.5 g of ion-exchanged water in Example 1-1 was changed to 1958.3 g, and the amount of 0.68 g of V-50 was changed to 1.02 g. A water dispersion of granular copolymer 4 was obtained in the same manner as in Example 1-1. After slowly cooling the dispersion to room temperature, a part thereof was sampled, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. As a result, it was confirmed that it was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in granular copolymer 4, and the mass ratio of the additional GMA amount to the monomer amount at the start of polymerization were 42.2 mol% and 0.23, respectively, in the same manner as in Example 1-1. The volume average particle diameter of granular copolymer 4 was 212 nm.

[0090] [Example 1-5] (Synthesis of granular copolymer (parent particle) 5) A water dispersion of granular copolymer 5 was obtained in the same manner as in Example 1-1, except that St in Example 1-1 was changed to methyl methacrylate (MMA: Tokyo Chemical Industry Co., Ltd.). After slowly cooling the dispersion to room temperature, a part thereof was sampled, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. As a result, it was confirmed that it was substantially 100%. That is, the molar ratio of GMA in the total amount of methyl methacrylate and GMA in granular copolymer 5 was 41.2 mol%. Also, the mass ratio of the additional GMA amount to the monomer amount at the start of polymerization was 0.23. The volume average particle diameter of granular copolymer 5 was 262 nm.

[0091] [Example 1-6] (Synthesis of granular copolymer (parent particle) 6) The amount of GMA in Example 1-1 was changed to 6.0 g, and the amount of additional GMA was changed from 5.2 g added twice to 4.2 g added twice. A water dispersion of granular copolymer 6 was obtained in the same manner as in Example 1-1, except that the additional GMA was added 6 hours after the start of polymerization. After slowly cooling the dispersion to room temperature, a part thereof was collected, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. It was confirmed that the conversion rate was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in granular copolymer 6 is 27.5 mol%. Also, the mass ratio of the amount of additional GMA to the amount of monomer at the start of polymerization is 0.27. The volume average particle diameter of granular copolymer 2 was 232 nm.

[0092] [Example 2-1] (Synthesis of Particle 1) Into a 100 ml round-bottom flask, 24 g of a 2.5 wt% aqueous dispersion of granular copolymer 1, 3.3 g of ion-exchanged water, 80 mg (0.53 mmol) of mercaptosuccinic acid (MSA: Fujifilm Wako Pure Chemical Corporation), and 230 mg (2.12 mmol) of 3-mercapto-1,2-propanediol (MPD: Fujifilm Wako Pure Chemical Corporation) were weighed, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 11.3. Next, the contents of the round-bottom flask were heated to 70° C. while stirring at 200 rpm, and further held in this state for 18 hours to obtain a dispersion of Particle 1. Particle 1 was separated from the dispersion by a centrifuge, and the operation of redispersing Particle 1 in ion-exchanged water was repeated 8 times to purify Particle 1, and finally, it was stored in the state of an aqueous dispersion adjusted to 1.0 wt%. The storage conditions were 4° C. under light-shielded conditions. The particle physical properties are shown in Table 1.

[0093] [Example 2-2] (Synthesis of Particle 2) A water dispersion of Particle 2 was obtained in the same manner as in Example 2-1, except that granular copolymer 1 in Example 2-1 was changed to granular copolymer 2. The particle physical properties are shown in Table 1.

[0094] [Example 2-3] (Synthesis of Particle 3) Aqueous dispersion of Particle 3 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 in Example 2-1 was changed to granular copolymer 3. The physical properties of the particles are shown in Table 1.

[0095] [Example 2-4] (Synthesis of Particle 4) An aqueous dispersion of Particle 4 was obtained in the same manner as in Example 2-1, except that pH = 11.3 in Example 2-1 was changed to 10.0. The physical properties of the particles are shown in Table 1.

[0096] [Example 2-5] (Synthesis of Particle 5) Aqueous dispersion of Particle 5 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 in Example 2-1 was changed to granular copolymer 4. The physical properties of the particles are shown in Table 1.

[0097] [Example 2-6] (Synthesis of Particle 6) An aqueous dispersion of Particle 6 was obtained in the same manner as in Example 2-1, except that 80 mg (0.53 mmol) of mercaptosuccinic acid in Example 2-1 was changed to 78 mg (0.53 mmol) of 2-aminopentanedioic acid (APA: L-glutamic acid, Fujifilm Wako Pure Chemical Corporation). The physical properties are shown in Table 1.

[0098] [Example 2-7] (Synthesis of Particle 7) An aqueous dispersion of Particle 7 was obtained in the same manner as in Example 2-1, except that 230 mg (2.12 mmol) of 3-mercapto-1,2-propanediol in Example 2-1 was changed to 193 mg (2.12 mmol) of 2-amino-1,3-propanediol (2APD: Tokyo Chemical Industry Co., Ltd.). The physical properties are shown in Table 1.

[0099] [Example 2-8] (Synthesis of Particle 8) Aqueous dispersion of Particle 8 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 in Example 2-1 was changed to granular copolymer 5. The physical properties of the particles are shown in Table 1.

[0100] [Example 2-9] (Synthesis of Particle 9) Aqueous dispersion of Particle 9 was obtained in the same manner as in Example 2-1, except that 80 mg (0.53 mmol) of mercaptosuccinic acid in Example 2-1 was changed to 20 mg (0.13 mmol), and 230 mg (2.12 mmol) of 3-mercapto-1,2-propanediol was changed to 273 mg (2.52 mg). Physical properties are shown in Table 1.

[0101] [Example 2-10] (Synthesis of Particle 10) Aqueous dispersion of Particle 10 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 in Example 2-1 was changed to granular copolymer 6, 80 mg (0.53 mmol) of mercaptosuccinic acid was changed to 480 mg (3.18 mmol), and 230 mg (2.12 mmol) of 3-mercapto-1,2-propanediol was changed to 345 mg (3.18 mmol) of 3-amino-1,2-propanediol (3APD: Tokyo Chemical Industry Co., Ltd.). Physical properties are shown in Table 1.

[0102] [Comparative Example 1-1] (Synthesis of Comparative Granular Copolymer (Mother Particle) 1) Aqueous dispersion of Comparative Granular Copolymer 1 was obtained in the same manner as in Example 1-1, except that the amount of St in Example 1-1 was changed from 27.8 g to 22.0 g, the amount of GMA was changed from 17.3 g to 28.0 g, the amount of additional GMA was 5.8 g, and the timing of addition was changed to once 2 hours after the start of polymerization. After the dispersion was slowly cooled to room temperature, a part of it was collected, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. It was confirmed that it was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in Comparative Granular Copolymer 1 is 53.0 mol%. Also, the mass ratio of the amount of additional GMA to the amount of monomer at the start of polymerization is 0.11. The volume average particle diameter of Comparative Granular Copolymer 1 was 238 nm.

[0103] [Comparative Example 1-2] (Synthesis of Comparative Granular Copolymer (Mother Particle) 2) The amount of GMA in Example 1-1 was changed from 17.3 g to 21.8 g, and the amount and timing of the additional GMA were changed to 5.8 g at one time 2 hours after the start of polymerization. Otherwise, a water dispersion of Comparative Granular Copolymer 2 was obtained in the same manner as in Example 1-1. After the dispersion was slowly cooled to room temperature, a part of it was collected, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. It was confirmed that it was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in Comparative Granular Copolymer 2 is 42.2 mol% as in Example 1-1, but the mass ratio of the additional GMA amount to the monomer amount at the start of polymerization is 0.12. The volume average particle diameter of Comparative Granular Copolymer 2 was 239 nm.

[0104] [Comparative Example 1-3] (Synthesis of Comparative Granular Copolymer (Mother Particle) 3) The amount of GMA in Example 1-1 was changed from 17.3 g to 13.4 g, the amount of the additional GMA was 4.8 g per time, and the additional timing was changed to three times, 2 hours after the start of polymerization, 30 minutes after that, and another 30 minutes after that. Otherwise, a water dispersion of Comparative Granular Copolymer 4 was obtained in the same manner as in Example 1-1. After the dispersion was slowly cooled to room temperature, a part of it was collected, and the polymerization conversion rate was evaluated in the same manner as in Example 1-1. It was confirmed that it was substantially 100%. That is, the molar ratio of GMA in the total amount of St and GMA in Comparative Granular Copolymer 3 is 42.2 mol% as in Example 1-1, but the mass ratio of the additional GMA amount to the monomer amount at the start of polymerization is 0.34. The volume average particle diameter of Comparative Granular Copolymer 4 was 243 nm.

[0105] [Comparative Example 2-1] (Synthesis of Comparative Particle 1) A water dispersion of Comparative Particle 1 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 in Example 2-1 was changed to Comparative Granular Copolymer 1. The particle physical properties are shown in Table 2.

[0106] [Comparative Example 2-2] (Synthesis of Comparative Particle 2) A aqueous dispersion of Comparative Particle 2 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 of Example 2-1 was changed to Comparative Granular Copolymer 2. The physical properties of the particles are shown in Table 2.

[0107] [Comparative Example 2-3] (Synthesis of Comparative Particle 3) Aqueous dispersion of Comparative Particle 3 was obtained in the same manner as in Comparative Example 2-2, except that 80 mg (0.53 mmol) of mercaptosuccinic acid in Comparative Example 2-2 was changed to 20 mg (0.13 mmol), and 230 mg (2.12 mmol) of 3-mercapto-1,2-propanediol was changed to 273 mg (2.52 mg). The physical properties of the particles are shown in Table 2.

[0108] [Comparative Example 2-4] (Synthesis of Comparative Particle 4) Aqueous dispersion of Comparative Particle 4 was obtained in the same manner as in Example 2-1, except that the granular copolymer 1 of Example 2-1 was changed to Comparative Granular Copolymer 3. The physical properties of the particles are shown in Table 2.

[0109] [Evaluation 1] Evaluation of non-specific adsorption to particles Dispersion liquids (P liquids) were prepared by dispersing Particles 1 to 10 and Comparative Particles 1 to 4 in a phosphate buffer solution (containing 0.01% Tween 20) to a concentration of 0.1 wt%. Next, 55 μL of a sample dilution liquid (Q liquid) composed of human normal sample (serum sample, 5 μL) and phosphate buffer solution (50 μL) was added to 50 μL of each dispersion liquid, and the absorbance at a wavelength of 572 nm was measured for the mixed liquid immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, after allowing each mixed liquid to stand 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. The results are summarized in Table 1 and Table 2. The larger the value, the more non-specific adsorption occurs. When used for sample testing as particles for the latex agglutination method, there is a concern that normal samples may be misinterpreted as false positives. The obtained values of the change in absorbance ΔABS × 10000 were ranked according to the following criteria based on the noise risk when detecting a low-concentration target substance by the latex agglutination method. A: Less than ±50 B: 50 or more and less than 100 C: 100 or more and less than 500 D: 500 or more

[0110] From Table 1, it can be seen that particles 1 to 10, in which the amount of C element relative to the O element on the particle surface layer is 2.1 or more and 3.3 or less, are excellent in the ability to suppress non-specific adsorption.

[0111] On the other hand, it can be seen that comparative particles 2 and 3, in which the amount of C element relative to the O element exceeds 3.3, have a large non-specific adsorption. Similar to the examples, comparative particles 2 and 3 have a GMA ratio in the mother particles below a certain level in order to cope with long-term storage. However, in comparative particles 2 and 3, mother particles synthesized with an additional GMA amount ratio of less than 16% by mass are used. Therefore, compared with the particles of the present invention, the small amount of GMA on the surface layer of the mother particles cannot be covered, and it is expected that the styrene ratio on the surface layer of the mother particles is high. Even in the particles after adding hydroxy groups and carboxy groups, since the styrene on the surface layer of the mother particles remains unchanged and exists on the surface layer, the amount of C element relative to the O element on the particle surface layer is high, and it is expected that comparative particles 2 and 3 have a large non-specific adsorption.

[0112] Also, since no specific endothermic peak (derived from polystyrene) was detected in the DSC curve, it is expected that in the particles of comparative particles 2 and 3, there is no high-concentration polystyrene region in the particles, and compared with the particles of the present invention, polystyrene is not firmly confined inside the particles.

[0113] Although the endothermic peak in the DSC curve was not detected for comparative particle 1 either, on the other hand, its non-specific adsorption property is good. This is presumably because the GMA ratio of comparative particle 1 is higher than the scope of the present invention and the polystyrene in the original particle composition is small, so the behavior derived from polystyrene is difficult to observe.

[0114] [Evaluation 2] Preparation of test particles by sensitizing particles with an antibody, and evaluation of the antibody sensitization rate (Preparation of test particles) For particles 1 to 10 and comparative particles 1 to 4, 180 μL of a 1.7 wt% water-suspension was taken into a 1.5 mL microtube, and 90 μL of a 5.0 wt% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 5.0 wt% aqueous solution of N-hydroxysulfosuccinimide sodium were added thereto, followed by stirring at room temperature for 30 minutes to obtain a particle dispersion having carboxyl groups activated (activated particle dispersion).

[0115] After centrifugal washing, 270 μL of a phosphate buffer-saline (hereinafter referred to as PBS) at pH 7.2 was added, and the particles having carboxyl groups activated were dispersed by ultrasonic waves. 5 μL of a 15.0 mg / mL dispersion of monoclonal mouse anti-human C-reactive protein (hereinafter referred to as CRP antibody), clone C5 (Funakoshi Co., Ltd.) was added thereto, followed by stirring at room temperature for 3 hours to obtain test particles by sensitizing the particles with the antibody. After centrifugal washing of these test particles, 1 mL of PBS was added and they were stored in a dispersed state.

[0116] Regarding the test particles of particle 1, as a result of evaluating the particle specific gravity, it was 1.079 with respect to the particle specific gravity of 1.082 before sensitization with the antibody. From this, it can be judged that the particle specific gravity of the test particles that actually become components of the test reagent can be regarded as equivalent to the particle specific gravity before sensitization without any problem.

[0117] (Evaluation of antibody sensitization rate of test particles) Next, the antibody sensitization rate (%) was determined by the method for measuring the antibody sensitization rate of the test particles described above. The results are summarized in Tables 1 and 2. From Table 1, it can be seen that particles 1 to 10, in which the composition ratio of C element to O element on the particle surface layer is 2.1 or more and 3.3 or less, all show a sufficient antibody sensitization rate. In particular, when the zeta potential is lower than -10 mV (the absolute value is large), it is expected that the particle surface layer has many carboxyl groups, and therefore the antibody sensitization rate is considered to be particularly high.

[0118] Among the comparative particles, in particular, comparative particle 3, in which the composition ratio of C element to O element is higher than 3.3 and the zeta potential is higher than -10 mV (the absolute value is smaller), has a poor antibody sensitization rate. As described in [Evaluation 1], comparative particle 3 is a particle using mother particles synthesized with an additional GMA amount ratio of less than 16% by mass. Since the amount of GMA on the surface layer of the mother particles is small, it is difficult to originally impart a sufficient amount of carboxyl groups. Furthermore, since the amount of mercaptosuccinic acid used in comparative particle 3 is also small, it is considered that there are few carboxyl groups as expected from the small absolute value of the zeta potential, and sufficient antibodies cannot be bound.

[0119] [Evaluation 3] Evaluation of latex aggregation sensitivity and non-specific adsorption of test particles (Evaluation of standard serum) The standard serum for CRP was diluted with PBS to a concentration of 0.15 mg / dL to obtain a CRP sample solution. A mixed solution (hereinafter referred to as R1+) prepared by mixing 1 μL of the CRP sample solution and 50 μL of a buffer solution (PBS containing 0.01% Tween 20) was incubated at 37°C. Also, as a control, a mixed solution (hereinafter referred to as R1-) prepared by mixing 1 μL of physiological saline and 50 μL of a buffer solution (PBS containing 0.01% Tween 20) was prepared and incubated at the same 37°C.

[0120] Next, 50 μL of a dispersion of each of the test particles that had been sufficiently dispersed again by ultrasonic waves before use (particle concentration 0.1 wt%, referred to as R2) was mixed with R1+ or R1-. The absorbance at a wavelength of 572 nm was measured for the mixed solution (volume 101 μL) immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, after allowing this mixed solution to stand 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. The results are summarized in Table 1 and Table 2.

[0121] Regarding the value of R- (reactivity with physiological saline), a large value means that aggregation due to non-specific adsorption to the test particles or osmotic pressure aggregation has occurred. In that case, when using the particles for latex agglutination method in specimen testing, there is a concern that normal specimens may be misinterpreted as false positives due to noise. However, it was confirmed that no such aggregation was observed in all the particles evaluated in this example.

[0122] The larger the value of R+ (reactivity with 0.15 mg / dl of human CRP) of the test particles, the more expected it is that the target substance can be detected with high sensitivity when used for specimen testing as particles for the latex agglutination method. In addition, the obtained R+ values were ranked according to the following criteria. A: 1500 or more B: 1200 or more and less than 1500 C: 1000 or more and less than 1200 D: Less than 1000

[0123] From Table 1, it can be seen that the test particles of Particles 1 to 10 with a composition ratio of C element to O element on the particle surface layer of 2.1 or more and 3.3 or less can detect human CRP (0.15 mg / dl) at a lower concentration than the reference value with high sensitivity by the latex agglutination method. In particular, when the volume average particle diameter is larger than 280 nm, when the zeta potential is lower than -10 mV (the absolute value is larger) and there is a sufficient antibody sensitization rate, and when using a granular copolymer of styrene and glycidyl methacrylate as the mother particle, the detection sensitivity is excellent.

[0124] On the other hand, Comparative Particle 4 with a composition ratio of C element to O element smaller than 2.1 has inferior sensitivity in the latex agglutination method. Comparative Particle 4 is a particle using a mother particle synthesized with an additional GMA amount ratio of more than 30% by mass. Therefore, while the amount of GMA on the surface layer of the mother particle is large and a sufficient amount of hydroxy groups, carboxy groups, and antibody sensitization rate can be obtained, the high hydrophilicity makes it difficult for the particles to aggregate, and the detection sensitivity based on the principle of the latex agglutination method is considered to have decreased. Also, it is expected that sufficient detection sensitivity could not be obtained for Comparative Particle 3 due to the low amount of antibody sensitization described in [Evaluation 2].

[0125] [Evaluation 4] (Evaluation of latex aggregation sensitivity after static storage of the test particle dispersion) [In Evaluation 2], each test particle prepared was used as a dispersion with a particle concentration of 0.1 wt% in a 10 mM HEPES, pH 7.9, 0.01% Tween 20 solution. 1 mL portions were aliquoted into 1.5 mL microtubes. After static storage at 4°C for 1 week, the dispersion was visually inspected to observe particle sedimentation. Also, at this time, 50 μL of the dispersion was carefully collected from a position 3 mm deep from the liquid surface of the dispersion. Using this liquid, the latex aggregation sensitivity to human CRP (measurement of R+) was evaluated in the same manner as in [Evaluation 3]. The results are summarized in Tables 1 and 2.

[0126] Note that from the value of ΔABS×10000 obtained in [Evaluation 4] (Δ(2)), the value of ΔABS×10000 obtained in [Evaluation 3] (Δ(1)) was subtracted, and the ratio (%) of the resulting value divided by (Δ(1)) was calculated as the change rate of the R+ value. Then, the obtained change rate of the R+ value was ranked according to the following criteria. A: -5% or more B: -10% or more and less than -5% C: -15% or more and less than -10% D: Less than -15%

[0127] From Table 1, for test particles 1 to 10 with a particle specific gravity of 1.10 or less and a composition ratio of C element to O element on the particle surface of 2.1 or more and 3.3 or less, no particle sedimentation was visually confirmed even after 1 week of static storage. Also, in the dispersion collected from the upper part of the dispersion after static storage, there was no significant difference in latex aggregation sensitivity in [Evaluation 3], which is the evaluation immediately after ultrasonic dispersion. In particular, particles with a volume average particle diameter of 350 nm or less naturally settled more slowly due to their particle size compared to particles with a diameter of 350 nm or more, and are considered to have particularly excellent dispersion stability.

[0128] On the one hand, for Comparative Particle 1 with a particle specific gravity greater than 1.10, a transparent liquid phase could be confirmed near the liquid surface of the dispersion, and it was predicted that particle sedimentation was progressing. Also, the dispersion collected from the upper part of the dispersion after static storage had a significantly reduced latex aggregation sensitivity. Among the comparative particles with a particle specific gravity less than 1.10, for Comparative Particles 2 and 3 with a composition ratio of carbon element to oxygen element of 3.3 or more, a slight transparent liquid phase was visually confirmed. Since the hydrophilicity of the particle surface was insufficient, it was predicted that sedimentation was slightly accelerated. Accordingly, the decrease in the latex aggregation sensitivity of the dispersion collected after static storage also became larger.

[0129]

Table 1

[0130]

Table 2

[0131] The disclosure regarding the embodiments according to the present invention includes the following configurations and methods. (Configuration 1) Having a polymer containing a structural unit A represented by formula (1) and a structural unit B represented by formula (2), with a specific gravity in the range of 1.00 or more and 1.10 or less, and a particle in which the composition ratio of carbon element to oxygen element quantified by XPS measurement is in the range of 2.1 or more and 3.3 or less.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

1. It has a polymer containing a structural unit A represented by formula (1) and a structural unit B represented by formula (2), The specific gravity is in the range of 1.00 or more and 1.10 or less, Particles in which the composition ratio of C element to O element quantified by XPS measurement is in the range of 2.1 or more and 3.3 or less. 【Chemical 1】 [Chemical 2] R in formula (1) １ represents a methyl group or a hydrogen atom. L １ represents an alkylene group having 1 to 4 carbon atoms. R ２ is a group containing a sulfide group or a secondary amine and a hydroxy group. R in formula (2) ３ represents a methyl group or a hydrogen atom. L ４ represents an alkylene group having 1 to 4 carbon atoms. R ４ is a group containing a sulfide group or a secondary amine and a carboxy group.)

2. Furthermore, it has a structural unit C represented by formula (3) or formula (4), The particles according to claim 1, wherein the total amount of the structural unit A and the structural unit B in the total amount of the structural unit A, the structural unit B, and the structural unit C is 5 mol% or more and 43 mol% or less. [Chemical 3] 【Chemical Formula 4】 (R in formula (3) ５ represents a methyl group or a hydrogen atom. R in formula (3) ６ represents a linear or branched alkyl group having 1 to 9 carbon atoms, or a hydrogen atom. n in formula (3) represents an integer of 1 to 5. R in formula (4) ７ represents a methyl group or a hydrogen atom. R ８ represents a linear or branched alkyl group having 1 to 12 carbon atoms.)

3. The particles according to claim 1, wherein the zeta potential is -50 mV or more and -10 mV or less.

4. The particles according to claim 2, wherein the structural unit C is at least one selected from the group of styrenes.

5. The particles according to claim 2, wherein the amount of the structural unit C in the total amount of the structural unit A, the structural unit B, and the structural unit C is 57 mol% or more and 95 mol% or less.

6. On the DSC curve obtained during the second heating in differential scanning calorimetry (DSC) measurement, a straight line is drawn through the point on the DSC curve where the temperature is 80 °C and the point on the DSC curve where the temperature is 100 °C. When the intersection point of the straight line and the DSC curve in the temperature range of 105 °C or more and 140 °C or less is defined as intersection point A, the DSC curve has an endothermic peak in the temperature range between the temperature At of the intersection point A and 100 °C. The particles according to claim 1.

7. The particles according to claim 1, wherein the structural unit A contains a structure represented by formula (5). [Chemical Formula 5] (R in formula (5) １ represents a methyl group or a hydrogen atom. L １ represents an alkylene group having 1 to 4 carbon atoms. L ２ and L ３ each independently represent either a single bond or an alkylene group having 1 to 3 carbon atoms. X represents S or NH.)

8. The particles according to claim 1, wherein the structural unit B contains a structure represented by formula (6). 【Chemical Formula 6】 (R in formula (6) ３ represents a methyl group or a hydrogen atom. L ４ represents an alkylene group having 1 to 4 carbon atoms. L ５ represents either a single bond or an alkylene group having 1 to 3 carbon atoms. X represents S or NH.)

9. The particles according to claim 2, wherein the structural unit C is styrene.

10. The particles according to claim 1, wherein the volume average particle diameter in the aqueous dispersion is 280 nm or more and 400 nm or less.

11. A method for producing particles including the following steps. Step 1: A step of mixing a compound represented by formula (7), a compound represented by formula (8), water, and a radical polymerization initiator to initiate polymerization, Step 2: A step of further adding the compound represented by formula (7) to the reaction system after Step 1. Here, the ratio of the mass of the compound represented by the formula (7) added in Step 2 to the total mass of the compound represented by the formula (7) and the compound represented by the formula (8) mixed in Step 1 is 0.16 or more and 0.30 or less. 【Chemical Formula 7】 [Chemical 8] (R in formula (7) ９ represents a methyl group or a hydrogen atom. L ６ represents an alkylene group having 1 to 4 carbon atoms. R in formula (8) １０ represents a methyl group or a hydrogen atom. R １１ represents a linear or branched alkyl group having 1 to 9 carbon atoms, or a hydrogen atom.)

12. The method for producing particles according to claim 11, including the following Step 3 after Step 2. Step 3: A step of preparing a mixed solution by mixing an aqueous dispersion of the obtained granular copolymer with 3-mercapto-1,2-propanediol and mercaptosuccinic acid, and reacting an epoxy group derived from the compound represented by the formula (7) with a thiol group derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid.

13. The method for producing particles according to claim 11, including the following Step 3' after Step 2. Step 3': A step of preparing a mixed solution by mixing an aqueous dispersion of the obtained granular copolymer with 3-amino-1,2-propanediol and mercaptosuccinic acid, and reacting an epoxy group derived from the compound represented by the formula (7) with an amino group derived from 3-amino-1,2-propanediol and a thiol group derived from mercaptosuccinic acid.

14. The method for producing particles according to any one of claims 11 to 13, wherein in Step 2, the addition of the compound represented by the formula (7) is carried out in multiple portions.

15. The inspection particles according to any one of claims 1 to 10, which are formed by chemically bonding with a ligand.

16. The inspection particles according to claim 15, wherein the ligand is an antibody or an antigen.

17. A reagent for detecting a target substance in a specimen by in vitro diagnosis, the reagent containing the inspection particles according to claim 15.

18. The reagent according to claim 17, which is used for detecting a target substance in a specimen by an agglutination method.

19. A kit for detecting a target substance in a specimen by in vitro diagnosis, the kit comprising at least the reagent according to claim 17.

20. A method for detecting a target substance in a specimen by in vitro diagnosis, the method comprising mixing the inspection particles according to claim 15 with a specimen that may contain the target substance.

21. The detection method according to claim 20, which is a method for detecting a target substance in a specimen by agglutination.

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