Test particle, reagent, kit, detection method and particle production method

Abstract

The invention relates to a test particle, a reagent, a kit, a detection method and a method for producing the particle. Provided is a particle for latex agglutination, which has a reactive functional group for chemical binding to a ligand, has small non-specific adsorption, and has excellent dispersion stability after standing storage, i.e., excellent static storage stability, and which can be used as a test particle for latex agglutination. The test particle has a ligand and a particle chemically bonded to the ligand, in which the particle has a polymer including a structural unit A represented by formula (1) and a structural unit B represented by formula (2) (referring to the specification), has a specific gravity in the range of 1.00-1.10, and has a composition ratio of element C to element O, as quantified by XPS measurement, in the range of 2.1-3.3.

Description

Technical Field

[0001] The present invention relates to test particles, reagents, test kits, detection methods, and production methods of particles. Background Art

[0002] Examples of simple and rapid immunological test methods include latex agglutination assays. In this method, a dispersion of test particles including latex particles having a ligand bound thereto that is affinity to a target substance is mixed with a sample that may contain the target substance. At this time, in the case where the target substance is contained in the sample, the test particles cause an agglutination reaction, which is optically detected as a change in scattered light intensity, transmitted light intensity, absorbance, etc., thereby enabling disease diagnosis.

[0003] Many immunological test items in clinical tests have important diagnostic points in regions where the target substance is present at a low concentration. Therefore, it is required to be able to determine the presence or absence of the target substance even at low concentrations. As a known means for improving test sensitivity, large-sized latex particles are used. Thereby, the change in absorbance or the like to be detected becomes larger, and thus the measurement sensitivity in the low-concentration region can be improved.

[0004] Particles used in latex agglutination assays and test particles having a ligand of a target substance on the particle surface are required to have weak and faint adsorption to substances other than the target substance, that is, to have small non-specific adsorption. As particles having faint non-specific adsorption, particles having poly(glycidyl methacrylate) disposed on the surface are known. Since a part of the glycidyl group is ring-opened to form a diol, it is presumed that poly(glycidyl methacrylate) disposed on the particle surface has faint non-specific adsorption. In the case disclosed in Japanese Patent Application Laid-Open No. 2000-351814, copolymer particles of styrene and glycidyl methacrylate (in which the surface having poly(glycidyl methacrylate) is chemically bonded to a ligand through a polyethylene glycol chain) are applied to biopurification.

[0005] The glycidyl ester group of glycidyl methacrylate can also be used as a reactive functional group to chemically bond a ligand to the particle surface. Using a chemical bond to fix a ligand to the particle surface raises concerns about problems such as desorption of the ligand from the particle surface during long-term storage of the reagent. Japanese Patent Application Laid-Open No. 2006-71297 discloses a case where the glycidyl group is carboxylated, thereby imparting a carboxyl group to the particle surface for chemical bonding with a ligand. Summary of the Invention

[0006] After the present inventors studied to improve the detection sensitivity in a low concentration region, they found that, as described in Japanese Patent Application Laid-Open No. 2000-351814, as the particle diameter of test particles made of copolymerized styrene and glycidyl (meth)acrylate further increases, during static storage, the particle sedimentation rate accelerates. It is speculated that this acceleration is mainly caused by the combination of the blending amount of glycidyl (meth)acrylate having a specific gravity greater than 1 and the particle diameter.

[0007] Since the dispersion of the test particles can be statically stored for several weeks depending on the test mechanism, a plan to maintain the dispersion of the test particles in the dispersion is technically important. The occurrence of particle sedimentation during storage results in a decrease in dispensing accuracy during testing, thus making accurate measurement difficult.

[0008] The specific gravity of the particles is important for maintaining the dispersion of the test particles. Usually, the dispersion medium for reagents used in in vitro diagnosis is water. Therefore, the difference between the specific gravity of water, i.e., 1.0 and the specific gravity of the particles has a great influence on the sedimentation level. In a comparison between particles with a specific gravity of 1.1 and particles with a specific gravity of 1.2, the difference in sedimentation rate is twofold. In order to maintain dispersion stability while reducing sedimentation, components that increase the specific gravity or viscosity of the dispersion medium can also be added. However, in some cases, the addition may affect the detection sensitivity, so a technique is needed to maintain the balance of properties.

[0009] Although it is preferable to use glycidyl (meth)acrylate as a particle component to reduce non-specific adsorption and form a chemical bond between the ligand and the particle surface, it is considered necessary to reduce the blending ratio of glycidyl (meth)acrylate in the particles to maintain dispersion stability while reducing sedimentation. However, a simple change in the blending ratio also causes a change in the quantitative ratio on the surface of the copolymerized particles, which may lead to a limitation in detection sensitivity due to a decrease in the amount of ligand that can bind, or a significant deterioration in non-specific adsorption due to a decrease in the amount of hydroxyl groups on the surface.

[0010] The present invention is made based on these background arts and problems. An object of the present invention is to provide test particles for latex agglutination methods, which have reactive functional groups chemically bonded to ligands, have little non-specific adsorption, and have excellent dispersion stability after static storage, that is, excellent static storage stability. Another object of the present invention is to provide a method for producing such particles. Another object of the present invention is to provide a reagent, a kit, and a detection method.

[0011] In order to solve the above problems, the present invention provides a test particle having:

[0012] a ligand, and

[0013] particles chemically bonded to the ligand,

[0014] wherein the particles have a polymer containing a structural unit A represented by the following formula (1) and a structural unit B represented by the following formula (2),

[0015] the specific gravity of the particles is in the range of 1.00 or more and 1.10 or less, and

[0016] the composition ratio of element C to element O quantified by XPS measurement of the particles is in the range of 2.1 or more and 3.3 or less:

[0017]

[0018]

[0019] wherein, 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, and R 2 is a group containing a sulfide group or a secondary amine and a hydroxyl group; and wherein, 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, and R 4 is a group containing a sulfide group or a secondary amine and a carboxyl group.

[0020] The present invention also provides a method for producing particles, which includes the following steps:

[0021] Step 1: Mix a compound represented by the following formula (7), a compound represented by the following formula (8), water, and a radical polymerization initiator to initiate polymerization;

[0022] Step 2: After Step 1, further add a compound represented by formula (7) to the reaction system;

[0023] wherein the ratio of the mass of the compound represented by formula (7) added in Step 2 to the sum of the mass of the compound represented by formula (7) and the mass of the compound represented by formula (8) mixed in Step 1 is 0.16 or more and 0.30 or less:

[0024]

[0025]

[0026] wherein, in formula (7), R 9 represents a methyl group or a hydrogen atom, L 6 represents an alkylene group having 1 to 4 carbon atoms; and wherein, in formula (8), R 10 represents a methyl group or a hydrogen atom, and R 11Represents a straight-chain or branched alkyl group having 1 to 9 carbon atoms or a hydrogen atom.

[0027] The present invention also provides a test particle comprising the above-described particle chemically bound to a ligand.

[0028] The present invention also provides a reagent for detecting a target substance in a specimen by in vitro diagnosis and comprising the above-described test particle.

[0029] The present invention also provides a kit for detecting a target substance in a specimen by in vitro diagnosis, which at least comprises the above-described reagent.

[0030] The present invention also provides a method for detecting a target substance in a specimen by an agglutination reaction, which comprises mixing the above-described test particle with a specimen that may contain the target substance.

[0031] With reference to the accompanying drawings, other features of the present invention will become apparent from the following description of exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shows the DSC curve of the second heating in the differential scanning calorimetry (DSC) of the particle of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0034] The embodiments of the present invention are described in more detail below, although the technical scope of the present invention is not limited to these embodiments.

[0035] [First Embodiment]

[0036] The first embodiment relates to a test particle.

[0037] The test particle of the present invention has

[0038] a ligand, and

[0039] a particle chemically bound to the ligand,

[0040] wherein the particle has a polymer containing a structural unit A represented by the following formula (1) and a structural unit B represented by the following formula (2), and the specific gravity is in the range of 1.00 or more and 1.10 or less, and the composition ratio of element C to element O quantified by XPS measurement is in the range of 2.1 or more and 3.3 or less:

[0041]

[0042] Wherein, in formula (1), R 1 represents a methyl group or a hydrogen atom, L 1represents an alkylene group having 1 to 4 carbon atoms, and R 2 represents a group containing a thioether group or a secondary amine and a hydroxyl group; and wherein, in formula (2), R 3 represents a methyl group or a hydrogen atom, and L 4 represents an alkylene group having 1 to 4 carbon atoms, and R 4 is a group containing a thioether group or a secondary amine and a carboxyl group.

[0043] L in formula (1) 1 and L in formula (2) 4 are preferably linear or branched alkylene groups such as methylene, ethylene, n-propylene, isopropyl, n-butyl, and isobutyl, and from the viewpoint of the balance between hydrophilicity and hydrophobicity, more preferably methylene. Therefore, it is more preferable to use glycidyl (meth)acrylate as the epoxy group-containing monomer for forming the mother particles in the preparatory stage of producing the particles of the present invention.

[0044] When a glycidyl group-containing monomer such as glycidyl (meth)acrylate is used to form the mother particles, a specific compound is reacted with the glycidyl group present in the surface layer of the mother particles. Thus, R in formula (1) 2 and R in formula (2) 4 can be added as side chains to the particle surface layer, and thus particles having a polymer containing structural units A and B can be obtained.

[0045] In particular, since the side chain of structural unit A has a total of 3 or more hydroxyl groups including a terminal hydroxyl group and 2 or more hydroxyl groups contained in R 2 , the effect of reducing non-specific adsorption of the particles is improved.

[0046] In the test particles of the present invention, preferably, structural unit A contains a structure represented by the following formula (5):

[0047]

[0048] wherein, R 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 represents either a single bond or an alkylene group having 1 to 3 carbon atoms, and X represents S or NH.

[0049] L in formula (5) 1 is preferably a linear or branched alkylene group such as methylene, ethylene, n-propylene, isopropyl, n-butyl, and isobutyl. L in formula (5) 2 and L 3Preferably a single bond or a linear or branched alkylene group such as methylene, ethylene, n-propylene, and isopropyl. L in formula (5) 1 、L 2 and L 3 are more preferably methylene groups respectively. In the case where each is a methylene group, the balance between hydrophilicity and hydrophobicity is good, and furthermore, the side chain of structural unit A is not longer than the side chain of structural unit B, so the possibility of hindering the reactivity of the carboxyl group of structural unit B can be further reduced.

[0050] X in formula (5) can be a sulfur atom or a nitrogen atom. From the viewpoint of improving sensitivity, a sulfur atom is more preferable, and from the viewpoint of suppressing non-specific adsorption, a nitrogen atom is more preferable. Since a sulfide bond has a structure showing a weak hydrophobic tendency, it can be expected that the water-binding force of the highly hydrophilic side chain is moderately weakened to suppress osmotic flocculation that may occur by mixing with the sample at a high concentration. Thus, the sulfide bond can contribute to improving sensitivity. On the other hand, an amino group has a structure showing a hydrophilic tendency and can contribute to reducing non-specific adsorption.

[0051] When the glycidyl group derived from glycidyl (meth)acrylate reacts with 3-mercapto-1,2-propanediol, in formula (5), a structural unit A is formed in which L 1 、L 2 and L 3 each represents a methylene group and X represents a sulfur atom, which is further more preferable from the viewpoint of improving the sensitivity as described above.

[0052] In addition, when the glycidyl group derived from glycidyl (meth)acrylate reacts with 3-amino-1,2-propanediol, in formula (5), a structural unit A is formed in which L 1 、L 2 and L 3 each represents a methylene group and X represents a sulfur atom, which is further more preferable from the viewpoint of reducing the non-specific adsorption as described above.

[0053] The side chain of structural unit B has a carboxyl group for chemically bonding with a ligand. In particular, when there are two or more carboxyl groups, the reaction efficiency with the ligand is improved.

[0054] In the test particles of the present invention, preferably, structural unit B contains a structure represented by the following formula (6):

[0055]

[0056] Wherein, R 3 represents a methyl group or a hydrogen atom, L 4represents 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, and X represents S or NH.

[0057] When carboxyl groups are present very close to each other as in formula (6), the interaction between them inhibits their dissociation from each other, making the dispersibility of the particles moderately unstable. Therefore, using such particles in the immunolatex agglutination measurement method is advantageous for detecting the target substance with high sensitivity.

[0058] L in formula (6) 4 is preferably a linear or branched alkylene group such as methylene, ethylene, n-propylene, isopropyl, n-butylene, and isobutylene. L in formula (6) 5 is preferably a single bond or a linear or branched alkylene group such as methylene, ethylene, n-propylene, and isopropyl. L in formula (6) 4 and L 5 is more preferably methylene. For groups having too many carbon atoms, there is a risk of promoting non-specific adsorption to the particles due to increased hydrophobicity.

[0059] X in formula (6) can be either a sulfur atom or a nitrogen atom, and is more preferably a sulfur atom. Since the thioether bond has a structure showing a weak hydrophobic tendency, it is desirable to moderately weaken the water-binding force of the side chain, thereby suppressing osmotic flocculation that may occur by mixing with the sample at a high concentration.

[0060] When the glycidyl group derived from glycidyl (meth)acrylate reacts with mercaptosuccinic acid, a structural unit B is formed in which L 4 and L 5 are each methylene and X is a sulfur atom, which is more preferable as described above.

[0061] Preferably, [structural unit A] / [structural unit B] is 0.2 or more and 20 or less (mole fraction). When the mole fraction is less than 0.2, the proportion of carboxyl groups contained in structural unit B is too large, which may impair the dispersion stability of the particles, and when chemically bonded to the ligand, the unreacted carboxyl groups may interact with the ligand to denature the ligand. Or, when the mole fraction is greater than 20, due to the small proportion of carboxyl groups, the reaction efficiency of chemical bonding with the ligand may decrease, or the electrostatic repulsion that sometimes contributes to the dispersion stability of the particles may decrease.

[0062] From the viewpoint of the mechanical strength of the particles, it is preferable that the particles of the present invention have a structural unit C represented by the following formula (3) or formula (4), and in the total amount of the structural unit A, the structural unit B, and the structural unit C, the total amount of the structural unit A and the structural unit B is preferably 43 mol% or less, and more preferably 5 mol% or more and 43 mol% or less:

[0063]

[0064] Wherein, in formula (3), R 5 represents a methyl group or a hydrogen atom, R 6 represents a linear or branched alkyl group having 1 or more and 9 or less carbon atoms or a hydrogen atom, and n represents an integer of 1 or more and 5 or less; and wherein, in formula (4), R 7 represents a methyl group or a hydrogen atom, and R 8 represents a linear or branched alkyl group having 1 or more and 12 or less carbon atoms.

[0065] Due to the inclusion of the structural unit C, the particles of the present invention are physically strengthened, so that even when operations such as centrifugation are repeatedly performed during the refining process of the particles, damage to the particles such as cracks and debris can be suppressed. In addition, from the viewpoint of reducing the specific gravity of the particles, it is preferable to include the structural unit C.

[0066] Examples of the compounds from which the components represented by formula (3) are 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.

[0067] Examples of the compounds from which the components represented by formula (4) are derived include (meth)acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, n-pentyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate, cyclohexyl acrylate, methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, n-pentyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, and n-nonyl methacrylate.

[0068] In any case, as long as the object of the present invention can be achieved, the compounds are not limited thereto. Alternatively, two or more components can be used in combination.

[0069] The compound from which structural unit C is derived is preferably a styrene represented by formula (3), more preferably styrene. Additionally, the structural unit C of the particles of the present invention is preferably at least one selected from the group consisting of styrenes, and more preferably styrene. The monomers of styrenes have a small specific gravity and excellent refractive index, and are preferred from the viewpoints of reducing the specific gravity of the particles and improving the refractive index of the particles to improve sensitivity.

[0070] In the particles of the present invention, in the total amount of structural unit A, structural unit B, and structural unit C, the amount of structural unit C is preferably 57 mol% or more and 95 mol% or less, and more preferably 68 mol% or more and 93 mol% or less. (In other words, as described above, in the total amount of structural unit A, structural unit B, and structural unit C, the total amount of structural unit A and structural unit B is preferably 5 mol% or more and 43 mol% or less.)

[0071] <Specific Gravity of the Particles of the Present Invention>

[0072] The specific gravity of the particles of the present invention is in the range of 1.00 or more and 1.10 or less. When the specific gravity is 1.10 or less, excellent dispersion stability can be maintained even when the dispersion of the test particles as a test reagent is stored statically. Since the sedimentation of the particles is reduced, the service life of the test reagent can be extended. Specifically, the dispensing accuracy can be maintained even after storing for a specific time, thereby preventing a decrease in detection sensitivity.

[0073] The main factors determining the specific gravity of the particles include the constituent components of the particles. In addition, in the case of particles having a crosslinked property, a crosslinking degree above a certain level also affects the specific gravity.

[0074] The constituent components of the particles of the present invention are mainly components derived from the reactive monomers used to form the above-mentioned mother particles. Specifically, since the main components are the glycidyl group-containing monomers from which structural units A and B are derived (in the present invention, more preferably glycidyl (meth)acrylate) and the monomers from which structural unit C is derived (in the present invention, more preferably styrene), the specific gravity of the particles of the present invention is roughly determined by the content ratio of these monomers used to form the mother particles.

[0075] In other words, the side chain components and ligands added to the surface after forming the mother particles are only present near the particle surface and exist in a very small proportion in the constituent components of the particles, such that the contribution to the specific gravity of the particles of the present invention is very small.

[0076] The specific gravity of the particles increases in proportion to the content of glycidyl group-containing monomers having a specific gravity greater than 1 in the monomers used for forming the mother particles (in the present invention, glycidyl (meth)acrylate is more preferred). Therefore, in order to maintain the dispersion stability while reducing sedimentation, it is preferable to reduce the content of epoxy group-containing monomers in the formation of the mother particles. Specifically, in the total amount of structural unit A, structural unit B, and structural unit C, the total amount of structural unit A and structural unit B is preferably 43 mol% or less, and more preferably 32 mol% or less.

[0077] On the other hand, the structures of structural units A and B are necessary for excellent non-specific adsorption and chemical binding of ligands to the particle surface, and if the amount is too small, it may lead to deteriorated non-specific adsorption and binding with an insufficient amount of ligands. Therefore, in the total amount of structural unit A, structural unit B, and structural unit C, the total amount of structural unit A and structural unit B is preferably 20 mol% or more.

[0078] <Composition ratio quantified by XPS measurement of the particles of the present invention>

[0079] As described above, although it is preferable to use glycidyl (meth)acrylate as a component of the particles to reduce non-specific adsorption and enable chemical binding of ligands to the particle surface, it is speculated that the content of glycidyl (meth)acrylate needs to be reduced in the formation of the particles to maintain the dispersion stability while reducing sedimentation. However, simply reducing the blending amount not only reduces the amount over the entire area of the particles but also reduces the amount in the "surface layer" of the particles. In this case, since the amount of carboxyl groups and hydroxyl groups imparted in the post-treatment is reduced, the amount of ligands that can be bound may be reduced, or the non-specific adsorption property may be significantly deteriorated.

[0080] Preferably, the particles of the present invention are particles in which the amount ratio in the "surface layer" of the particles is appropriately controlled even when the amount ratio of the components in the entire area of the particles, that is, the formulation ratio, is changed. (A production method of particles with appropriately controlled is described below.)

[0081] The particles of the present invention are particles in which the composition ratio of element C to element O quantified by XPS measurement is controlled within a range of 2.1 or more and 3.3 or less. In XPS measurement, photoelectrons generated in the region from the outermost surface to about 10 nm are detected, whereby the components in the "surface layer" of the particles can be analyzed. When the composition ratio is less than 2.1, element O in the "surface layer" of the particles is excessive, so a sufficient amount of hydroxyl groups and carboxyl groups are added. On the other hand, due to the very high hydrophilicity, it is difficult for the particles to agglomerate, so the detection sensitivity based on the principle of latex agglutination method may decrease. Conversely, when the composition ratio is greater than 3.3, element O in the "surface layer" of the particles is too little, so the hydrophilicity is insufficient, which not only results in poor dispersion stability of the particles during long-term storage, but also results in deteriorated non-specific adsorption property. As described above, by optimizing the amount of components in the "surface layer" of the particles, particles with excellent detection sensitivity and non-specific adsorption property can be provided.

[0082] <DSC Curve of the Particles of the Present Invention>

[0083] In the DSC curve obtained during the second heating in the differential scanning calorimetry (DSC) measurement of the particles of the present invention, a straight line passing through the point on the DSC curve at a temperature of 80 °C and the point on the DSC curve at a temperature of 100 °C is drawn. The intersection point between this straight line and the DSC curve in the temperature region of 105 °C or more and 140 °C or less is defined as intersection point A. Preferably, the DSC curve has an endothermic peak in the temperature region between the temperature At of intersection point A and 100 °C.

[0084] The endothermic peak originates from the property of styrene-based (structural unit C of the present invention). The observed endothermic peak indicates that polystyrene aggregates at a high concentration in the particles, and structural units A and B are compositionally separated from polystyrene (structural unit C) in the particles. In the particles of the present invention, since structural units A and B are mainly arranged on the surface layer to control the "surface layer", on the other hand, it is speculated that polystyrene (structural unit C) is restricted inside the particles. Since the presence of polystyrene, which is a hydrophobic material, in the surface layer of the particles can be a main factor deteriorating the non-specific adsorption property, by firmly restricting polystyrene inside the particles, particles with excellent non-specific adsorption property can be provided.

[0085] When the structural unit C of the present invention contains a poly(methyl)acrylate-based structure represented by the formula (4), on the DSC curve obtained during the second heating in the DSC measurement, a straight line passing through the point at a temperature of 80 °C and the point at a temperature of 100 °C is drawn. Preferably, the straight line and the DSC curve have intersection point A in the region of 95 °C or more and 140 °C or less, and have an endothermic peak in the temperature region between the temperature At of intersection point A and 90 °C.

[0086] <Zeta potential of the particles of the present invention>

[0087] Preferably, the zeta potential of the particles of the present invention is -50 mV or more and -10 mV or less. A zeta potential less than -50 mV indicates that the surface layer of the particles has many carboxyl groups. When the particles are combined with a ligand to form test particles, the reactivity of the ligand may be reduced due to the electrostatic interaction between the ligand and the particle surface, or the charge shift on the surface of the test particles caused by the zeta potential difference between the ligand and the particle surface may cause heteroagglutination. To reduce the possibility, the zeta potential is preferably -50 mV or more. A zeta potential greater than -10 mV indicates that the surface layer of the particles has a small amount of carboxyl groups. To allow a sufficient amount of ligand to bind to the particles to achieve excellent detection sensitivity, the zeta potential is preferably -10 mV or less.

[0088] <Particle diameter of the particles of the present invention>

[0089] The volume average particle diameter of the particles of the present invention in the aqueous dispersion is preferably 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.

[0090] When the particle diameter is 280 nm or more, the change in absorbance or the like detected by the latex agglutination method increases, so that the detection sensitivity is improved particularly when the target substance is present at a low concentration. Generally, when particles having a size of 280 nm or more and containing a component derived from glycidyl (meth)acrylate are used as a test reagent, problems in actual use easily 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. Even when the particle size is 280 nm or more, by using the particles of the present invention with the specific gravity controlled, the dispersion stability of the test reagent and particle sedimentation during long-term storage can be improved. From the same viewpoint, the particle diameter is more preferably 400 nm or less, and even more preferably 350 nm or less.

[0091] [Second Embodiment]

[0092] The second embodiment relates to a method for producing particles.

[0093] As described above, although it is preferable to use glycidyl (meth)acrylate as a component of the particles, it is presumed that the content of glycidyl (meth)acrylate needs to be reduced during particle formation. However, simply reducing the blending amount not only results in a decrease in the amount in the entire region of the particles but also a decrease in the amount in the "surface layer" of the particles.

[0094] Preferably, the particles of the present invention are particles in which the quantitative ratio in the "surface layer" is appropriately controlled even when the quantitative ratio of the components, i.e., the formulation ratio, is changed throughout the particles. To meet the above requirements, the production method of the particles of the present invention includes the following steps.

[0095] Step 1: Mix a compound represented by the following formula (7), a compound represented by the following formula (8), water, and a radical polymerization initiator to initiate polymerization;

[0096] Step 2: After Step 1, further add a compound represented by formula (7) to the reaction system;

[0097] wherein, relative to the sum of the mass of the compound represented by formula (7) and the mass of the compound represented by formula (8) mixed in Step 1, the mass ratio of the compound represented by formula (7) added in Step 2 is 0.16 or more and 0.30 or less:

[0098]

[0099] wherein, in formula (7), R 9 represents a methyl group or a hydrogen atom, and L 6 represents an alkylene group having 1 or more and 4 or less carbon atoms; and wherein, in formula (8), R 10 represents a methyl group or a hydrogen atom, and R 11 represents a linear or branched alkyl group having 1 or more and 9 or less carbon atoms or a hydrogen atom.

[0100] L in formula (7) 6 is preferably a linear or branched alkylene group such as methylene, ethylene, n-propylene, isopropyl, n-butylene, and isobutylene. R in formula (8) 11 is preferably methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, and n-nonyl.

[0101] The compound represented by formula (7) added in Step 2, i.e., the glycidyl group-containing monomer, immediately dissolves and adheres to the surface layer of the particles after being added to the water in the reaction system for the polymerization reaction.

[0102] When the amount of the compound represented by the formula (7) added in Step 2, i.e., the glycidyl group-containing monomer, is controlled to be 16% by mass or more, even when the total 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. Therefore, a sufficient amount of hydroxyl groups and carboxyl groups can be added to the surface layer of the particles in the post-treatment. The composition ratio of element C to element O becomes 3.3 or less, enabling the provision of particles with high hydrophilicity and excellent non-specific adsorption properties. In addition, when the amount of the glycidyl group-containing monomer added in Step 2 is controlled to be 30% by mass or less, the glycidyl groups in the surface layer of the mother particles do not increase excessively, thereby preventing an excessive amount of hydroxyl groups and carboxyl groups from being added to the surface layer of the particles. The composition ratio of element C to element O quantified by XPS measurement of the particles becomes 2.1 or more, providing appropriate hydrophobicity, ensuring the aggregability required for the latex aggregation method, and enabling the provision of particles with excellent detection sensitivity.

[0103] In the method for producing the particles of the present invention, preferably, the addition of the compound represented by the formula (7), i.e., the glycidyl group-containing monomer, is carried out in several batches in Step 2. In order to more precisely control the amount of glycidyl groups in the surface layer of the mother particles, it is necessary to consider the amount of the monomer remaining in the reaction system during the addition in Step 2. When the monomer is added in several batches in Step 2, the monomer remaining in the reaction system (especially styrene-based monomers with low solubility in water) can be efficiently dissolved in water, thereby promoting the polymerization reaction during the first addition. Therefore, in the addition in Step 2, the amount of the monomer remaining in the reaction system can be reduced, and thus the dissolution of the remaining monomer during the last addition can be reduced. Therefore, the ratio of glycidyl groups finally present in the "surface layer" of the particles can be effectively increased. "Adding the monomer in several batches" means that the time interval between the first addition and the last addition is more than 1 minute. As a specific method, it can be added in batches at intervals, or continuous dropping can be carried out without intervals.

[0104] The method for producing the particles of the present invention preferably includes the following Step 3 after Step 2.

[0105] Step 3: Mix the aqueous dispersion of the obtained granular copolymer, 3-mercapto-1,2-propanediol, and mercaptosuccinic acid to prepare a mixed solution, thereby reacting the epoxy groups derived from the compound represented by the formula (7) with the thiol groups derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid.

[0106] By reacting the epoxy groups of the obtained granular copolymer (masterbatch) with 3-mercapto-1,2-propanediol and mercaptosuccinic acid, side chains represented by structural unit A of formula (5) and structural unit B of formula (6) can be imparted to the surface layer of the particles. The reasons for more preferably using 3-mercapto-1,2-propanediol and mercaptosuccinic acid are as described above.

[0107] In the method for producing the particles of the present invention, other typical production methods are as described below. However, the method for producing the particles of the present invention is not limited thereto as long as the object of the present invention can be achieved.

[0108] The radical polymerization initiator in Step 1 is preferably a water-soluble polymerization initiator.

[0109] Although the water-soluble polymerization initiator is not particularly limited, water-soluble azo compounds and water-soluble peroxides are preferably used.

[0110] 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], and 2,2′-azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate.

[0111] The water-soluble peroxide is preferably any one of potassium persulfate, ammonium persulfate, sodium persulfate, tert-butyl hydroperoxide, cumene hydroperoxide, p-menthane hydroperoxide, and diisopropylbenzene hydroperoxide.

[0112] In Step 1, a radically polymerizable monomer having crosslinkability may further be used. Examples of the radically polymerizable monomer having crosslinkability 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, pentaerythritol tetraacrylate, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butanediol 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, pentaerythritol tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether, although not limited thereto as long as the object of the present invention can be achieved. Further, two or more kinds of radically polymerizable monomers having crosslinkability may be used in combination.

[0113] The amount of the radically polymerizable monomer having crosslinkability is preferably 0.1% by mass to 5% by mass of the total amount of the monomers used in the polymerization. When the amount is greater than 5% by mass, the particle density increases, and thus the specific gravity of the particles may increase.

[0114] In the case of forming a granular copolymer (master particle) by radical polymerization, emulsion polymerization, soap-free emulsion polymerization, and suspension polymerization are preferably used, and emulsion polymerization or soap-free emulsion polymerization is more preferably used. Even more preferably, soap-free emulsion polymerization is used. Generally, the granular polymer obtained by emulsion polymerization or soap-free emulsion polymerization has a narrower particle size distribution than the granular polymer obtained by suspension polymerization. In the case of using particles bound to a ligand as test particles, there is a concern that an anionic surfactant or a cationic surfactant usually used in emulsion polymerization present as a residue may denature the ligand. Therefore, in the case of forming a granular polymer (master particle) by emulsion polymerization, a nonionic surfactant is preferably used.

[0115] Step 3 includes adjusting the pH to an alkaline region with an organic base free of primary amines. In Step 3, 3-mercapto-1,2-propanediol and a thiol group derived from mercaptosuccinic acid are used to cause a reaction of an epoxy group possessed by the granular copolymer (master particle) in the depth direction from the surface of the granular copolymer (master particle).

[0116] To cause a sufficient reaction in the depth direction, it is preferable to select triethylamine, which has permeability to the granular copolymer (master particle), as the organic base.

[0117] Preferably, the method for producing the particles of the present invention includes the following step 3' after step 2.

[0118] Step 3': Mix the aqueous dispersion of the obtained granular copolymer, 3-amino-1,2-propanediol, and mercaptosuccinic acid to prepare a mixed solution, so that the epoxy groups derived from the compound represented by formula (7) react with the amino groups derived from 3-amino-1,2-propanediol and the thiol groups derived from mercaptosuccinic acid.

[0119] [Application Example]

[0120] Examples of the application examples of the present invention include test particles, reagents, kits, and detection methods. Each item is described as follows.

[0121] <Test Particles>

[0122] The test particles of the present invention preferably include the above-mentioned particles chemically bound to a ligand, and more preferably include the above-mentioned particles bound to a ligand having an affinity for a target substance. Preferably, the test particles of the present invention are used to detect a target substance in a sample by an agglutination method.

[0123] A ligand refers to a compound that specifically binds to a receptor of a specific target substance. The region where the ligand binds to the target substance is specific and has a selective or specific high affinity. 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., although it is not limited thereto within the scope where the object of the present invention can be achieved. Specific examples of ligands include antigens, antibodies, fragments that bind to antigens (such as Fab, F(ab′)2, F(ab′), Fv, and scFv, etc.), nucleic acids of natural origin, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, and coenzymes, etc. The ligand of the test particles of the present invention is preferably an antibody or an antigen.

[0124] In the present invention, as a chemical reaction method for binding the carboxyl group or carboxylate salt derived from structural unit B to a ligand, within the scope where the object of the present invention can be achieved, conventionally known methods can be applied. Preferred examples of the chemical reaction include, for example, carbodiimide-mediated reactions and NHS ester activation reactions. Alternatively, the carboxyl group bound to avidin can be bound to a ligand modified with biotin. However, the chemical reaction method for binding the carboxyl group or carboxylic acid derived from structural unit B to a ligand is not limited thereto within the scope where the object of the present invention can be achieved.

[0125] As described above, with respect to the particles of the present invention, it can be determined that the retention specific gravity is the same between the particles before binding to the ligand and the test particles having the chemically bound ligand. This is because the ratio of the ligand relative to the particles is sufficiently small.

[0126] <Reagent>

[0127] Preferably, the reagent of the present invention is a reagent for detecting a target substance in a sample by in vitro diagnosis, and comprises the above-mentioned test particles and a dispersion medium.

[0128] In the present invention, the case of using an antibody (antigen) as a ligand and an antigen (antibody) as a target substance as a method for detecting a target substance in a sample in in vitro diagnosis can be extremely preferably applied to the latex agglutination method in immunological tests widely used in fields such as clinical tests and biochemical research. In the case of using commonly used particles as the particles in the latex agglutination method, there is a concern that the antigen (antibody) as the target substance and foreign substances in serum or plasma are non-specifically adsorbed to the particle surface, thereby causing unintentional agglutination between the particles, which impairs the accuracy of the immunological test.

[0129] Preferably, the reagent of the present invention is for detecting a target substance in a sample by an agglutination method and comprises 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 in the range of 0.001% by mass to 20% by mass, more preferably in the range of 0.01% by mass to 10% by mass. The reagent of the present invention may contain a third substance other than the particles for the latex agglutination method, such as a solvent and a blocking agent, within the range capable of achieving the object of the present invention. Examples of the solvent used in the present invention include various aqueous buffer solutions, such as phosphate buffer solution, glycine buffer solution, Good's buffer solution, Tris buffer solution, HEPES buffer solution, MES buffer solution, and ammonia buffer solution, although the solvent contained in the reagent of the present invention is not limited thereto.

[0130] <Kit>

[0131] The kit of the present invention is a kit for detecting a target substance in a specimen through in vitro diagnosis, and preferably includes at least the above-mentioned reagent. In addition to the reagent of the present invention (hereinafter referred to as Reagent 1), the kit of the present invention preferably further includes a reaction buffer solution containing albumin (hereinafter referred to as Reagent 2). Examples of albumin include serum albumin, etc., and may include albumin treated with protease. The estimated amount of albumin contained in Reagent 2 is in the range of 0.001% by mass to 5% by mass, although the kit of the present invention is not limited thereto. A sensitizer for latex agglutination measurement may be contained in both Reagent 1 and Reagent 2, or either of them. Examples of the sensitizer for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, and alginic acid, although the kit of the present invention is not limited thereto. In addition, in addition to Reagent 1 and Reagent 2, the kit of the present invention may further include a positive control, a negative control, a serum diluent, etc. The culture medium for the positive control or the negative control may include a solvent in addition to serum and physiological saline that do not contain a measurable target substance. The kit of the present invention can be used in the detection method of the target substance of the present invention in the same manner as a kit for detecting a target substance in a specimen through ordinary in vitro diagnosis. In addition, this kit can also be used to measure the concentration of the target substance by a conventionally known method, and is particularly suitable for detecting the target substance in a specimen by the latex agglutination method.

[0132] <Detection method>

[0133] The method for detecting a target substance in a specimen through in vitro diagnosis preferably includes mixing the above-mentioned test particles with a specimen that may contain the target substance, and more preferably is a method for detecting the target substance in the specimen through an agglutination reaction. In addition, preferably, the mixing of the test particles of the present invention with the specimen is carried out within the range of pH 3.0 to pH 11.0. The mixing temperature is within the range of 20°C to 50°C, and the mixing time is within the range of 1 minute to 20 minutes. In this detection method, it is preferable to use a solvent. In the detection method of the present invention, the concentration of the test particles of the present invention in the reaction system is preferably 0.001% by mass to 5% by mass, and more preferably 0.01% by mass to 1% by mass. The detection method of the present invention is characterized in that the agglutination between the particles generated by mixing the test particles of the present invention with the specimen is optically detected. By optically detecting the agglutination between the particles, the target substance in the specimen is detected, and in addition, the concentration of the target substance can also be measured. In order to optically detect the agglutination reaction, changes in scattered light intensity, transmitted light intensity, absorbance, etc. can be measured with an optical device capable of detecting them.

[0134] [Method for measuring the specific gravity of particles]

[0135] A method for measuring the specific gravity of the particles of the present invention will be described. The particles are dispersed in ion-exchanged water at 0.5 mass%, and the sedimentation rate of the particles is measured under a specified centrifugal force. The specific gravity of the particles is calculated based on the measured sedimentation value, the specific gravity and viscosity of the dispersion medium, and the volume-average particle size of the particles. The conductivity of the ion-exchanged water used is 10 μS / cm or less. Specifically, a LUMISizer (manufactured by MS Scientific Co., Ltd.) is used to measure the sedimentation rate determined by the change in transmitted light when a centrifugal force of 4000 rpm is applied to the particle dispersion. Using the specific gravity and viscosity of the ion-exchanged water as the dispersion medium, and the volume-average particle size of the particles measured by the method described below, the specific gravity of the particles is calculated from the Stokes formula shown below. The measurement is performed three times, and the average value of the three measurement values is taken as the specific gravity.

[0136] (Stokes formula)

[0137] Sedimentation rate Vs = D ρ 2 (ρ p - ρ f )g / 18η

[0138] where Vs represents the sedimentation rate (m / s), Dρ represents the particle size (m), ρ p represents the specific gravity of the particles (kg / m 3 ), ρ f represents the specific gravity of the dispersion medium (kg / m 3 ), g represents the acceleration due to gravity (m / s 2 ), and η represents the viscosity of the dispersion medium (Pa·s).

[0139] [Method for measuring the C / O ratio of particles]

[0140] A method for quantifying the composition ratio measured by XPS of the particles of the present invention will be described. In the XPS measurement of the composition ratio of the particles of the present invention, the freeze-dried particles are fixed to indium foil. The measurement equipment and measurement conditions are as follows.

[0141] · Measurement equipment: X-ray photoelectron spectroscopy Quantum 2000 (trade name, manufactured by ULVAC-PHI Inc.)

[0142] · X-ray source: Monochromatic AlKα

[0143] · X-ray setting: 100 μmφ (25W (15KV))

[0144] · Photoelectron emission angle: 45 degrees

[0145] · Neutralization condition: The neutralization gun and the ion gun are used in combination

[0146] · Analysis area: 300 μm × 200 μm

[0147] · Pass energy: 58.70 eV

[0148] · Step size: 0.125 eV

[0149] · Analysis software: MultiPak (PHI, Inc.)

[0150] In the measurement, the cumulative number C1 s and O1 s is 15, and N1 s is 30. From the quantitative values of the obtained elements (composition ratio (atomic %) of element C and composition ratio (atomic %) of element O with respect to the total amount of the three elements), the composition ratio of element C with respect to element O is obtained.

[0151] [Method for obtaining DSC curve of particles]

[0152] Describe the method for obtaining the DSC curve of the particles of the present invention. The measurement of the sample of the freeze-dried particles is carried out using a differential scanning calorimeter (DSC) (trade name “Discovery DSC2500”, manufactured by TA Instruments). The sample sealed in an aluminum pan is measured using the temperature program described below. According to the temperature program, initially, after the first temperature increase from 20 °C to 180 °C at a heating rate of 10 °C / min, the temperature is maintained at 180 °C for 10 minutes, then cooled to 10 °C at a cooling rate of 10 °C / min, then maintained at 10 °C for 10 minutes, and subsequently, the second temperature increase to 180 °C is performed again at a heating rate of 10 °C / min. The DSC curve during the second temperature increase is obtained.

[0153] [Method for measuring ζ potential of particles]

[0154] Describe the method for measuring the ζ potential of the particles in the present invention. In the present invention, the ζ potential is measured in a state where the particles are dispersed at 0.001 mass % in a 0.01 N potassium aqueous solution at pH 7.8. The 0.01 N potassium aqueous solution with a pH of 7.8 used is obtained by appropriately mixing a 0.01 N potassium chloride aqueous solution, a 0.01 N potassium hydroxide aqueous solution, and a 0.01 N hydrochloric acid aqueous solution prepared using ion-exchanged water with a conductivity of 10 μS / cm or less. The measurement device used is a Zetasizer (Nano-ZS, manufactured by Malvern Panalytical), and the measurement is carried out at 25 °C. The selection of the analysis parameters includes latex as the refractive index of the particles and purified water as the dispersion medium. The measurement is carried out 10 times, and the average value of the 10 measurements is taken as the ζ potential.

[0155] [Method for Measuring Volume-Average Particle Size of Particles in Aqueous Dispersion]

[0156] A method for measuring the volume-average particle size of particles in the aqueous dispersion of the present invention is described. In the present invention, the volume-average particle size of particles in the aqueous dispersion is measured under the condition that the particles are dispersed at 0.001% by mass in ion-exchanged water having a conductivity of 10 μS / cm or less. For the measurement of the particle size in water, the dynamic light scattering method is used. Specifically, Zetasizer (Nano-ZS, manufactured by Spectris) is used for measurement at 25°C. The selection of analysis parameters includes latex as the refractive index of particles and purified water as the dispersion medium. The measurement is carried out 10 times, and the average value of the 10 measurements is taken as the particle size in water.

[0157] [Method for Measuring Antibody Sensitization Rate of Test Particles]

[0158] A method for measuring the antibody sensitization rate of test particles prepared from the particles of the present invention is described. The antibody sensitization rate (%) of the test particles is determined by protein quantification. The antibody sensitization rate (%) is the ratio of the amount of antibody bound to the particles to the amount of antibody used for the reaction (the amount of antibody prepared).

[0159] First, 7 mL of Solution A and 140 μL of Solution B of a protein assay BCA kit (manufactured by Wako Pure Chemical) are mixed to prepare AB solution. Then, 200 μL of the AB solution is added to 25 μL (particle amount: 25 μg) of the dispersion (0.1% solution) of the test particles, and the mixture is incubated at 60°C for 30 minutes. The solution is centrifuged at 4°C and 15,000 rpm (20,400 g) for 5 minutes, and 200 μL of the supernatant is pipetted into a 96-well microplate. The absorbance of the sample and standard samples (several samples of antibodies in the range of 0 to 200 μg / mL in 10 mM HEPES) is measured at 562 nm using a microplate reader, 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 mass of particles (μg / mg)) is obtained by dividing the calculated amount of antibody by the particle mass (in this case, 0.025 mg). Finally, the sensitization rate is calculated. When the amount of antibody prepared is 25 μg / 1 mg of particles and the amount of antibody sensitized is 12.5 μg / mg, the sensitization rate is 50%.

[0160] [Examples]

[0161] The present invention will be described in more detail with reference to the following examples, although the present invention is not limited to these examples.

[0162] [Example 1-1]

[0163] (Synthesis of Granular Copolymer (Master Particle) 1)

[0164] In a 2 L four-neck separable flask, 27.8 g of styrene (St, manufactured by Kishida Chemical Co., Ltd.), 17.3 g of glycidyl methacrylate (GMA, manufactured by Kishida Chemical Co., Ltd.), 0.70 g of divinylbenzene (DVB, manufactured by Kishida Chemical Co., Ltd.), and 1305.5 g of ion-exchanged water were weighed out to prepare a mixed solution. While stirring the mixed solution at 200 rpm at 70 °C, nitrogen was flowed at a rate of 200 ml / min to purge the oxygen in the four-neck separable flask. Then, a separately prepared solution containing 0.68 g of V-50 (manufactured by FUJIFILM Wako Pure Chemical Corporation) dissolved in 18 g of ion-exchanged water was added to the mixed solution to initiate soap-free emulsion polymerization.

[0165] Two hours after the initiation of polymerization, as a step of adding additional GMA, 5.2 g of GMA was added to the four-neck separable flask, and after 30 minutes, 5.2 g of GMA was further added (5.2 g of GMA was added additionally, twice). While stirring at 200 rpm for 22 hours, the temperature was maintained at 70 °C to obtain an aqueous dispersion containing granular copolymer 1. After the dispersion was slowly cooled to room temperature, a part of the dispersion was collected to evaluate the polymerization conversion rate by proton NMR and gas chromatography. As a result, it was confirmed that the polymerization conversion rate was approximately 100%. In other words, the molar ratio of GMA to the total amount of St and GMA in granular copolymer 1 was 42.2 mol%. In addition, the mass ratio of the amount of additional GMA to the amount of monomers at the start of polymerization was 0.23. The volume average particle diameter of granular copolymer 1 was 240 nm. Granular copolymer 1 was stored under light-shielded conditions at 4 °C.

[0166] [Example 1-2]

[0167] (Synthesis of Granular Copolymer (Master Particle) 2)

[0168] Aqueous dispersion of granular copolymer 2 was obtained in the same manner as in Example 1-1, except that the amount of St 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 added in two steps was changed from 5.2 g to 6.2 g in two steps. After the dispersion was slowly cooled to room temperature, a portion of the dispersion was collected and the polymerization conversion was evaluated in the same manner as in Example 1-1. As a result, the polymerization conversion was confirmed to be approximately 100%. In other words, the molar ratio of GMA to the total amount of St and GMA in granular copolymer 2 was 32.7 mol%. In addition, the mass ratio of the amount of additional GMA to the amount of monomers at the start of polymerization was 0.27. The volume average particle diameter of granular copolymer 2 was 245 nm.

[0169] [Example 1-3]

[0170] (Synthesis of granular copolymer (master particle) 3)

[0171] Aqueous dispersion of granular copolymer 3 was obtained in the same manner as in Example 1-1, except that the amount of GMA was changed from 17.3 g to 19.3 g and the amount of additional GMA added in two steps was changed from 5.2 g to 4.1 g in two steps. After the dispersion was slowly cooled to room temperature, a portion of the dispersion was collected and the polymerization conversion was evaluated in the same manner as in Example 1-1. As a result, the polymerization conversion was confirmed to be approximately 100%.

[0172] In other words, the molar ratio of GMA to the total amount of St and GMA in granular copolymer 3 was 41.9 mol%. In addition, the mass ratio of the amount of additional GMA to the amount of monomers at the start of polymerization was 0.17. The volume average particle diameter of granular copolymer 3 was 234 nm.

[0173] [Example 1-4]

[0174] (Synthesis of granular copolymer (master particle) 4)

[0175] Aqueous dispersion of granular copolymer 4 was obtained in the same manner as in Example 1-1, except that the amount of ion-exchanged water was changed from 1305.5 g to 1958.3 g and the amount of V-50 was changed from 0.68 g to 1.02 g. After the dispersion was slowly cooled to room temperature, a portion of the dispersion was collected and the polymerization conversion was evaluated in the same manner as in Example 1-1. As a result, the polymerization conversion was confirmed to be approximately 100%.

[0176] In other words, similar to Example 1-1, the molar ratio of GMA to the total amount of St and GMA in the granular copolymer 4, and the mass ratio of the amount of GMA added relative to the monomer amount at the start of polymerization are 42.2 mol% and 0.23, respectively. The volume average particle diameter of the granular copolymer 4 is 212 nm.

[0177] [Example 1-5]

[0178] (Synthesis of granular copolymer (master particle) 5)

[0179] Except for changing St in Example 1-1 to methyl methacrylate (MMA, manufactured by Tokyo Chemical Industry Co., Ltd.), an aqueous dispersion of the granular copolymer 5 was obtained in the same manner as in Example 1-1. After slowly cooling the dispersion to room temperature, a part of the dispersion 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 polymerization conversion rate was approximately 100%. In other words, in the granular copolymer 5, the molar ratio of GMA to the total amount of methyl methacrylate and GMA is 41.2 mol%. The mass ratio of the added GMA relative to the monomer amount at the start of polymerization is 0.23. The volume average particle diameter of the granular copolymer 5 is 262 nm.

[0180] [Example 1-6]

[0181] (Synthesis of granular copolymer (master particle) 6)

[0182] Except for changing the amount of GMA in Example 1-1 to 6.0 g, changing the amount of added GMA from 5.2 g twice to 4.2 g twice, and adding the added GMA 6 hours after the start of polymerization, an aqueous dispersion of the granular copolymer 6 was obtained in the same manner as in Example 1-1. After slowly cooling the dispersion to room temperature, a part of the dispersion 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 polymerization conversion rate was approximately 100%. In other words, in the granular copolymer 6, the molar ratio of GMA to the total amount of St and GMA is 27.5 mol%. The mass ratio of the added GMA relative to the monomer amount at the start of polymerization is 0.27. The volume average particle diameter of the granular copolymer 2 is 232 nm.

[0183] [Example 2-1]

[0184] (Synthesis of particle 1)

[0185] In 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, manufactured by FUJIFILM Wako Pure Chemical Corporation), and 230 mg (2.12 mmol) of 3-mercapto-1,2-propanediol (MPD, manufactured by FUJIFILM Wako Pure Chemical Corporation) were weighed out, and triethylamine (manufactured by Kishida Chemical Co., Ltd.) was added thereto to adjust the pH to 11.3. While stirring the mixture in the round-bottom flask at 200 rpm, the temperature was raised to 70 °C and then maintained in this state for 18 hours. Thus, a dispersion of Particle 1 was obtained. Particle 1 was separated from the dispersion using a centrifuge. Further, the operation of redispersing Particle 1 in ion-exchanged water was repeated 8 times to purify Particle 1. Finally, an aqueous dispersion containing 1.0 wt% of Particle 1 was prepared and stored.

[0186] Storage was carried out at 4 °C under light-shielded conditions. In Table 1-1, the physical properties of the particles are shown.

[0187] [Example 2-2]

[0188] (Synthesis of Particle 2)

[0189] An aqueous 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. In Table 1-1, the physical properties of the particles are shown.

[0190] [Example 2-3]

[0191] (Synthesis of Particle 3)

[0192] An aqueous dispersion of Particle 3 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 3. In Table 1-1, the physical properties of the particles are shown.

[0193] [Example 2-4]

[0194] (Synthesis of Particle 4)

[0195] 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. In Table 1-1, the physical properties of the particles are shown.

[0196] [Example 2-5]

[0197] (Synthesis of Particle 5)

[0198] Aqueous dispersions of Particle 5 were 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 this particle are shown in Table 1-1.

[0199] [Example 2-6]

[0200] (Synthesis of Particle 6)

[0201] Aqueous dispersions of Particle 6 were 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 of 2-aminopentanedioic acid (APA: L-glutamic acid, manufactured by FUJIFILM Wako Pure Chemical). The physical properties of this particle are shown in Table 1-2.

[0202] [Example 2-7]

[0203] (Synthesis of Particle 7)

[0204] Aqueous dispersions of Particle 7 were 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, manufactured by Tokyo Chemical Industry Co., Ltd.). The physical properties of this particle are shown in Table 1-2.

[0205] [Example 2-8]

[0206] (Synthesis of Particle 8)

[0207] Aqueous dispersions of Particle 8 were 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 this particle are shown in Table 1-2.

[0208] [Example 2-9]

[0209] (Synthesis of Particle 9)

[0210] Aqueous dispersions of Particle 9 were 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 mmol). The physical properties are shown in Table 1-2.

[0211] [Example 2-10]

[0212] (Synthesis of Particle 10)

[0213] 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, manufactured by Tokyo Chemical Industry Co., Ltd.). Physical properties are shown in Table 1-2.

[0214] [Comparative Example 1-1]

[0215] (Synthesis of Comparative Granular Copolymer (Mother Particle) 1)

[0216] 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, and the amount of additional GMA was changed to 5.8 g and added only once 2 hours after the start of polymerization. After the dispersion was slowly cooled to room temperature, a part of the dispersion 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 polymerization conversion rate was approximately 100%. In other words, the molar ratio of GMA was 53.0 mol% with respect to the total amount of St and GMA in Comparative Granular Copolymer 1. In addition, the mass ratio of the amount of additional GMA with respect to the amount of monomers at the start of polymerization was 0.11. The volume average particle diameter of Comparative Granular Copolymer 1 was 238 nm.

[0217] [Comparative Example 1-2]

[0218] (Synthesis of Comparative Granular Copolymer (Mother Particle) 2)

[0219] Except that the amount of GMA in Example 1-1 was changed from 17.3 g to 21.8 g, the amount of additional GMA was changed to 5.8 g, and the addition timing was changed to only once, 2 hours after the start of polymerization, an aqueous 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 the dispersion 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 polymerization conversion rate was approximately 100%. In other words, although the molar ratio of GMA to the total amount of St and GMA in Comparative Granular Copolymer 2 was 42.2 mol% as in Example 1-1, the mass ratio of the amount of additional GMA to the monomer amount at the start of polymerization was 0.12. The volume average particle diameter of Comparative Granular Copolymer 2 was 239 nm.

[0220] [Comparative Example 1-3]

[0221] (Synthesis of Comparative Granular Copolymer (Mother Particle) 3)

[0222] Except that the amount of GMA in Example 1-1 was changed from 17.3 g to 13.4 g, the amount of additional GMA was changed to 4.8 g each time, and the addition timing was changed to three times, i.e., 2 hours after the start of polymerization, 30 minutes after the first addition, and 30 minutes after the second addition, an aqueous dispersion of Comparative Granular Copolymer 3 was obtained in the same manner as in Example 1-1. After the dispersion was slowly cooled to room temperature, a part of the dispersion 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 polymerization conversion rate was approximately 100%. In other words, although the molar ratio of GMA to the total amount of St and GMA in Comparative Granular Copolymer 3 was 42.2 mol% as in Example 1-1, the mass ratio of the amount of additional GMA to the monomer amount at the start of polymerization was 0.34. The volume average particle diameter of Comparative Granular Copolymer 3 was 243 nm.

[0223] [Comparative Example 2-1]

[0224] (Synthesis of Comparative Particle 1)

[0225] Except that Granular Copolymer 1 in Example 2-1 was changed to Comparative Granular Copolymer 1, an aqueous dispersion of Comparative Particle 1 was obtained in the same manner as in Example 2-1. The physical properties of this particle are shown in Table 2.

[0226] [Comparative Example 2-2]

[0227] (Synthesis of Comparative Particle 2)

[0228] Aqueous dispersions of Comparative Particle 2 were 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 2. The physical properties of the particles are shown in Table 2.

[0229] [Comparative Example 2-3]

[0230] (Synthesis of Comparative Particle 3)

[0231] Aqueous dispersions of Comparative Particle 3 were 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 mmol). The physical properties of the particles are shown in Table 2.

[0232] [Comparative Example 2-4]

[0233] (Synthesis of Comparative Particle 4)

[0234] Aqueous dispersions of Comparative Particle 4 were 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 3. The physical properties of the particles are shown in Table 2.

[0235] [Evaluation 1] Evaluation of Non-Specific Adsorptivity of Particles

[0236] In a phosphate buffer solution (containing 0.01% Tween 20), Particles 1 to 10 and Comparative Particles 1 to 4 were each dispersed to a content of 0.1 wt% to prepare a dispersion (Liquid P). Then, 55 μL of a sample diluent (Liquid Q) containing a normal human sample (serum sample, 5 μL) and phosphate buffer (50 μL) was added to each 50 μL of the dispersion and stirred to prepare a mixed solution, and the absorbance at a wavelength of 572 nm was immediately measured. In the absorbance measurement, a spectrophotometer "Biospectrometer" manufactured by Eppendorf SE was used. Then, each mixed solution was allowed to stand at 37 °C for 5 minutes, and then the absorbance at a wavelength of 572 nm was measured again to calculate the absorbance change value ΔABS×10000. The results are summarized in Tables 1-1, 1-2, and 2. It is speculated that the occurrence of non-specific adsorption increases proportionally to this value. When using particles in the latex agglutination method in sample tests, there is a concern that normal samples may be judged as false positives. In addition, considering the noise risk when detecting low-concentration target substances by the latex agglutination method, the obtained change value ΔABS×10000 was rated based on the following criteria.

[0237] A: Less than ±50

[0238] B: 50 or more and less than 100

[0239] C: 100 or more and less than 500

[0240] D: 500 or more

[0241] As can be seen from Table 1-1 and Table 1-2, Particles 1 to 10 in which the composition ratio of Element C to Element O in the particle surface layer is 2.1 or more and 3.3 or less are excellent in the ability to suppress non-specific adsorption.

[0242] On the other hand, it can be seen that the non-specific adsorption of Comparative Particles 2 and 3 in which the composition ratio of Element C to Element O is greater than 3.3 is large. Although, as in the examples, in order to achieve long-term storage, the GMA ratio in the mother particles of Comparative Particles 2 and 3 is lower than a certain level, the ratio of the additional amount of GMA used in Comparative Particles 2 and 3 is less than 16% by mass in the synthesized mother particles. As a result, compared with the particles of the present invention, the relatively small amount of GMA in the surface layer of the mother particles cannot be compensated, and thus a high styrene ratio in the surface layer of the mother particles is expected. Even in the particles after adding hydroxyl groups and carboxyl groups, styrene in the surface layer of the mother particles remains in the surface layer unchanged. As a result, the composition ratio of Element C to Element O in the particle surface layer is high, making it expected that Comparative Particles 2 and 3 have greater non-specific adsorption.

[0243] In addition, based on the fact that no specific endothermic peak (derived from polystyrene) is detected in the DSC curve, it is expected that, compared with the particles of the present invention, Comparative Particles 2 and 3 do not have a high-concentration region of polystyrene in the particles, and polystyrene is not firmly enclosed in the particles.

[0244] On the other hand, no specific endothermic peak is detected in the DSC curve of Comparative Particle 1 either, and it has good non-specific adsorption properties. It is speculated that since Comparative Particle 1 has a GMA ratio higher than the scope of the present invention, when the amount of polystyrene in the original particle composition is small, the behavior derived from polystyrene is hardly observed.

[0245] [Evaluation 2] Preparation of test particles by antibody sensitization of particles and evaluation of the antibody sensitization rate.

[0246] (Preparation of test particles)

[0247] For each of Particles 1 to 10 and Comparative Particles 1 to 4, 180 μL of a 1.7 wt% aqueous suspension was provided in 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 sodium N-hydroxysulfosuccinimide were added thereto to prepare a mixed solution. The mixed solution was stirred at room temperature for 30 minutes to obtain a dispersion of particles having activated carboxyl groups (activated particle dispersion).

[0248] After centrifugal washing, 270 μL of a phosphate buffer-saline solution having a pH of 7.2 (hereinafter referred to as PBS) was added, and the particles with activated carboxyl groups were dispersed by ultrasonic waves.

[0249] 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 (manufactured by Funakoshi Co., Ltd.) was added thereto, and the mixture was stirred at room temperature for 3 hours to obtain test particles by sensitizing the particles with the antibody. Each test particle was centrifugally washed, and then 1 mL of PBS was added thereto to form a mixture, and the mixture was stored in a dispersed state.

[0250] As a result of evaluating the specific gravity of the test particles of Particle 1, the particle specific gravity was 1.079, and the particle specific gravity before antibody sensitization was 1.082. From the above, it was determined that the actual particle specific gravity of the test particles as a component of the test reagent was comparable to the particle specific gravity before sensitization.

[0251] (Evaluation of Antibody Sensitization Rate of Test Particles)

[0252] Next, according to the method for measuring the antibody sensitization rate of the above test particles, the antibody sensitization rate (%) was determined.

[0253] The results are summarized in Table 1-1, Table 1-2, and Table 2.

[0254] It can be seen from Table 1-1 and Table 1-2 that any of Particles 1 to 10 in which the composition ratio of Element C to Element O in the particle surface layer was 2.1 or more and 3.3 or less exhibited a sufficient antibody sensitization rate. It was speculated that especially when the ζ potential was lower than -10 mV (large absolute value), it was expected that there were many carboxyl groups on the particle surface layer, resulting in a particularly high antibody sensitization rate.

[0255] Specifically, among the comparison particles, the comparison particle 3 with a composition ratio of element C to element O greater than 3.3 and a ζ potential greater than -10 mV (small absolute value) has a poor antibody sensitization rate. The comparison particle 3 is a particle made from a mother particle synthesized at a ratio of an additional GMA amount of less than 16% by mass as described in [Evaluation 1], and the amount of GMA in the surface layer of the mother particle is small, making it inherently difficult to impart a sufficient amount of carboxyl groups. In addition, the comparison particle 3 uses a relatively small amount of mercapto succinic acid and has a relatively small amount of carboxyl groups as predicted from the small absolute value of the ζ potential, thus presumably failing to achieve binding with a sufficient amount of antibodies.

[0256] [Evaluation 3] Evaluation of the latex agglutination sensitivity and non-specific adsorption of test particles

[0257] (Evaluation of standard serum)

[0258] Dilute the standard serum of CRP with PBS to a concentration of 0.15 mg / dL to prepare a CRP sample solution. Mix 1 μL of the CRP sample solution and 50 μL of a buffer solution (PBS containing 0.01% Tween 20) to prepare a mixed solution (hereinafter referred to as R1+), and incubate at 37°C. As a control, mix 1 μL of a physiological saline solution and 50 μL of a buffer solution (PBS containing 0.01% Tween 20) to prepare a mixed solution (hereinafter referred to as R1-), which is also incubated at 37°C.

[0259] Next, mix 50 μL each of the dispersion liquid of the test particles (particle concentration 0.1 wt%, denoted as R2) that has been ultrasonically dispersed again immediately before use with R1+ or R1-. Measure the absorbance of the mixed solution (volume: 101 μL) immediately after stirring at a wavelength of 572 nm. The absorbance is measured using a spectrophotometer "Biospectrometer" manufactured by Eppendorf SE. Let the mixed solution stand at 37°C for 5 minutes, and then measure the absorbance at a wavelength of 572 nm again to calculate the change value of absorbance ΔABS×10000. The results are summarized in Table 1-1, Table 1-2, and Table 2.

[0260] A large value of R- (reactivity with physiological saline solution) means that agglutination caused by non-specific adsorption or osmotic flocculation has occurred in the test particles. In this case, there is a concern that using latex agglutination particles in the sample test may cause false positives in normal samples due to noise. However, it has been confirmed that no such agglutination has occurred in all the particles evaluated in this example.

[0261] The larger the R+ value (reactivity with 0.15 mg / dl human CRP) of the test particles using the latex agglutination method in the sample test, the more sensitive the detection of the target substance is expected to be.

[0262] Rate the obtained R+ values according to the following criteria.

[0263] A: Above 1500

[0264] B: Above 1200 and less than 1500

[0265] C: Above 1000 and less than 1200

[0266] D: Less than 1000

[0267] As can be seen from Table 1-1 and Table 1-2, test particles 1 to 10 with a composition ratio of element C to element O of 2.1 or more and 3.3 or less can detect human CRP at a concentration lower than the standard value (0.15 mg / dl) with high sensitivity by latex agglutination method. Especially when the volume average particle diameter is greater than 280 nm, when the ζ potential with sufficient antibody sensitization rate is lower than -10 mV (large absolute value), and when using a granular copolymer of styrene and glycidyl methacrylate as the mother particle, the detection sensitivity is excellent.

[0268] On the other hand, comparative particle 4 with a composition ratio of element C to element O less than 2.1 has poor sensitivity in the latex agglutination method. Comparative particle 4 is a particle made from a mother particle synthesized at a ratio of an additional GMA amount of more than 30% by mass. As a result, the amount of GMA in the surface layer of the mother particle is large, enabling sufficient hydroxyl and carboxyl groups to be obtained with a high antibody sensitization rate. On the other hand, due to extremely high hydrophilicity, almost no agglutination of particles occurs, resulting in reduced detection sensitivity based on the principle of the latex agglutination method. It is also expected that the low amount of antibody sensitization of comparative particle 3 described in [Evaluation 2] leads to insufficient detection sensitivity.

[0269] [Evaluation 4]

[0270] (Evaluation of latex agglutination sensitivity after static storage of the test particle dispersion)

[0271] Disperse the test particles prepared in [Evaluation 2] in a 10 mM HEPES solution with pH 7.9 at a particle concentration of 0.1 wt%, using 0.01% Tween 20. Divide 1 mL of the dispersion into 1.5 mL microtubes respectively. Store the dispersion statically at 4 °C for 1 week, and then visually test the sedimentation of the particles. At this time, carefully collect 50 μL of the dispersion from a position 3 mm deep from the liquid surface of the dispersion. Using this liquid, evaluate the latex agglutination sensitivity to human CRP (measurement of R+) in the same manner as in [Evaluation 3]. The results are summarized in Table 1-1, Table 1-2 and Table 2.

[0272] The change rate (%) of the R+ value is calculated by subtracting the value ΔABS×10000 (Δ(1)) obtained in [Evaluation 3] from the value ΔABS×10000 (Δ(2)) obtained in [Evaluation 4], and then dividing by (Δ(1)). The change rate of the obtained R+ value is rated according to the following criteria.

[0273] A: -5% or more

[0274] B: More than -10% and less than -5%

[0275] C: More than -15% and less than -10%

[0276] D: Less than -15%

[0277] As shown in Table 1-1 and Table 1-2, for the test particles 1 to 10 with a particle specific gravity of 1.10 or less and a composition ratio of element C to element O on the particle surface of 2.1 or more and 3.3 or less, even after standing for 1 week, no particle sedimentation determined by visual inspection occurred. In addition, for the dispersion liquid collected from the upper part of the dispersion liquid after static storage, in [Evaluation 3] which is an evaluation immediately after ultrasonic dispersion, no significant difference in latex agglutination sensitivity was caused. In particular, it is speculated that particles with a volume average particle size of 350 nm or less are particularly excellent in dispersion stability because, due to the particle size, the natural sedimentation rate is slower compared to particles with a size of 350 nm or more.

[0278] On the other hand, for the dispersion liquid of the comparative particle 1 with a particle specific gravity greater than 1.10, a transparent liquid phase was identified near the liquid surface, so particle sedimentation was expected to progress. In addition, the dispersion liquid collected from the upper part of the dispersion liquid after static storage had a significantly reduced latex agglutination sensitivity. Among the comparative particles with a particle specific gravity less than 1.10, the comparative particles 2 and 3 with a composition ratio of element C to element O of 3.3 or more had a small amount of transparent liquid phase identified by visual inspection. Due to the insufficient hydrophilicity of the particle surface layer, it is speculated that sedimentation was accelerated to a certain extent. Therefore, the latex agglutination sensitivity of the dispersion liquid collected after static storage was greatly reduced.

[0279] [Table 1-1]

[0280]

[0281] [Table 1-2]

[0282]

[0283] [Table 2]

[0284]

[0285] According to the present invention, there can be provided particles used in a latex agglutination method having reactive functional groups chemically bonded to a ligand, low non-specific adsorption, and excellent stability during static storage, and a method for producing the same. Further, there can be provided test particles having a chemically bonded ligand, an in vitro diagnostic reagent and a kit containing the particles, and a method for detecting a target substance.

[0286] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. The scope of the appended claims should be given the broadest interpretation so as to cover all such modifications as well as equivalent structures and functions.

Claims

1. A test particle comprising: ligand, and A particle chemically bonded to the ligand, wherein the particle has a polymer comprising a structural unit A represented by the following formula (1) and a structural unit B represented by the following formula (2), The specific gravity of the particles is within a range of 1.00 or more and 1.10 or less, and The composition ratio of the element C to the element O of the particles, which is quantitatively determined by XPS measurement, is within a range of 2.1 to 3.

3. in, In the formula (1), R 1 represents a methyl group or a hydrogen atom, L 1 represents an alkylene group having 1 to 4 carbon atoms, and R 2 is a group containing a thioether group or a secondary amine and a hydroxyl group; and wherein, in the formula (2), R 3 represents a methyl group or a hydrogen atom, L 4 represents an alkylene group having 1 to 4 carbon atoms, and R 4 It is a group containing a thioether group or a secondary amine and a carboxyl group.

2. The test particle according to claim 1, wherein The particles further comprise a structural unit C represented by the following formula (3) or the following formula (4), Among the total amount of the structural unit A, the structural unit B and the structural unit C, the total amount of the structural unit A and the structural unit B is 5 mol% or more and 43 mol% or less: In the formula (3), R 5 represents a methyl group or a hydrogen atom, R 6 represents a linear or branched alkyl group or a hydrogen atom having 1 to 9 carbon atoms, and n represents an integer of 1 to 5; wherein, in the formula (4), R 7 represents a methyl group or a hydrogen atom, R 8 It represents a linear or branched alkyl group having 1 to 12 carbon atoms. The test particle according to claim 1 , wherein the zeta potential of the particle is greater than or equal to −50 mV and less than or equal to −10 mV. The test particle according to claim 2 , wherein the structural unit C is at least one selected from the group consisting of styrenes. 5 . The test particle according to claim 2 , wherein in the total amount of the structural unit A, the structural unit B, and the structural unit C, the amount of the structural unit C is 57 mol % or more and 95 mol % or less.

6. The test particle according to claim 1, wherein when a straight line passing through a point at which the temperature is 80°C and a point at which the temperature is 100°C on the DSC curve is drawn on the DSC curve obtained in the second temperature increase of differential scanning calorimetry (DSC) measurement of the particle, and the intersection between the straight line and the DSC curve in the temperature region of 105°C to 140°C is defined as an intersection A, the DSC curve has an endothermic peak in the temperature region between the temperature At of the intersection A and 100°C.

7. The test particle according to claim 1, wherein the structural unit A comprises a structure represented by the following formula (5): in, R 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 represents any one of a single bond and an alkylene group having 1 to 3 carbon atoms, and X represents S or NH.

8. The test particle according to claim 1, wherein the structural unit B comprises a structure represented by the following formula (6): in, R 3 represents a methyl group or a hydrogen atom, L 4 represents an alkylene group having 1 to 4 carbon atoms, L 5 represents any of a single bond and an alkylene group having 1 to 3 carbon atoms, and X represents S or NH. 9 . The test particle according to claim 2 , wherein the structural unit C is styrene. 10 . The test particle according to claim 1 , wherein a volume average particle diameter of the particle in the aqueous dispersion is 280 nm or more and 400 nm or less. The test particle of claim 1 , wherein the ligand is an antibody or an antigen. 12 . A reagent for detecting a target substance in a test sample by in vitro diagnosis, the reagent comprising the test particle according to claim 1 and a dispersion medium.

13. The reagent according to claim 12, which is used for detecting a target substance in a sample by an agglutination method. 14 . A kit for detecting a target substance in a sample by in vitro diagnosis, the kit comprising at least the reagent according to claim 12 or 13.

15. A method for detecting a target substance in a sample by agglutination reaction, comprising: The test particles according to any one of claims 1 to 11 are mixed with a sample which may contain a target substance.

16. A method for producing particles, comprising the following steps: Step 1: mixing a compound represented by the following formula (7), a compound represented by the following formula (8), water and a radical polymerization initiator to initiate polymerization; Step 2: After step 1, further adding a compound represented by formula (7) to the reaction system; The ratio of the mass of the compound represented by the formula (7) added in the step 2 to the sum of the mass of the compound represented by the formula (7) and the mass of the compound represented by the formula (8) mixed in the step 1 is 0.16 or more and 0.30 or less: Wherein, in the formula (7), R 9 represents a methyl group or a hydrogen atom, and L 6 represents an alkylene group having 1 or more and 4 or less carbon atoms; and wherein, in the formula (8), R 10 represents a methyl group or a hydrogen atom, and R 11 It represents a linear or branched alkyl group having 1 to 9 carbon atoms or a hydrogen atom.

17. The method for producing particles according to claim 16, further comprising the following step 3 after step 2: Step 3: The obtained aqueous dispersion of the granular copolymer, 3-mercapto-1,2-propanediol and mercaptosuccinic acid are mixed to prepare a mixed solution, thereby reacting the epoxy group derived from the compound represented by the formula (7) with the thiol group derived from 3-mercapto-1,2-propanediol and mercaptosuccinic acid.

18. The method for producing particles according to claim 16, further comprising the following step 3' after step 2: Step 3': The obtained aqueous dispersion of the granular copolymer, 3-amino-1,2-propanediol and mercaptosuccinic acid are mixed to prepare a mixed solution, thereby reacting the epoxy group derived from the compound represented by the formula (7) with the amino group derived from 3-amino-1,2-propanediol and the thiol group derived from mercaptosuccinic acid.

19. The method for producing particles according to any one of claims 16 to 18, wherein: In the step 2, the compound represented by the formula (7) is added in several batches.

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