Particles and their manufacturing method, affinity particles, test reagent, test kit, and detection method

Abstract

To provide a particle with which it is possible to suppress nonspecific adsorption without using BSA, a particle that adsorbs and sediments impurities that cause a decrease in measurement sensitivity, and a manufacturing method for these.SOLUTION: Provided is a polymer particle including polystyrene, the polymer particle being composed of a random copolymer that includes a unit having a pyrrolidone ring and a unit having a functional group that can bind a ligand. Also provided is a particle including an inorganic matter, the surface of the particle being composed of a random copolymer that includes a unit having a pyrrolidone ring and a unit having a functional group that can bind a ligand, or composed of a first layer of a polymer having a pyrrolidone ring and a second layer of a polymer having a carboxyl group in the same molecule and a silicon alkoxide group or a silanol group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to particles and methods for producing the same, affinity particles, test reagents, test kits, and detection methods. [Background technology]

[0002] Polymer particles are becoming increasingly important in the fields of basic biology and medicine, for example in the application of in vitro diagnostics using latex agglutination.

[0003] Furthermore, the importance of nanoparticles is increasing not only in fundamental research but also in applied fields such as medicine and material engineering. As an example of an application in the medical field, nanoparticles are used in the latex agglutination method. The principle of the latex agglutination method is as follows: Nanoparticles with antibodies (or antigens) bound to their surface agglutinate through antibody-antigen interactions depending on the amount of antigen (or antibody) present. In other words, we can know the amount of antigen (or antibody) present by measuring the degree of agglutination spectroscopically.

[0004] In principle, the sensitivity of latex agglutination reactions can be increased if the desired antigen or antibody can be adsorbed onto the particle surface. However, many impurities exist in samples such as serum, and these impurities also adsorb (non-specifically adsorb) onto the particle surface, preventing the desired antigen or antibody from adsorbing onto the particle surface.

[0005] Furthermore, if particles aggregate via impurities adsorbed to the particles, the measurement accuracy will decrease. Impurities are substances that are not the antigens to be detected but are contained in the reaction system. In the latex agglutination method, particle aggregation via impurities that are not the antigens to be detected is undesirable because it causes a decrease in measurement sensitivity. Therefore, there is a method in which the desired antibodies are adsorbed onto the prepared particles, and then the particle surfaces are coated with BSA (bovine serum albumin) to suppress the adsorption of impurities to the particle surfaces (non-specific adsorption) (Patent Document 1).

[0006] In addition, there is a method for suppressing non-specific adsorption by coating the particle surfaces with a compound having a hydroxyl group (Patent Document 2). Another method is to make some particles magnetic, that is, to attract particles aggregated by latex agglutination in the direction of a magnetic field, thereby relatively reducing nonspecific adsorption and increasing sensitivity (Patent Document 3). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2004-151000 A [Patent Document 2] Japanese Patent Application Publication No. 4-218772 [Patent Document 3] Patent No. 2603843 Summary of the Invention [Problem to be solved by the invention]

[0008] However, when the particle surface was coated with BSA, the adsorption of impurities was sometimes sufficiently suppressed and sometimes not. Therefore, when BSA-coated particles were used in the latex agglutination method, there was a problem of reduced reproducibility. This is presumably because BSA is a natural product, and the degree of hydrophilicity and hydrophobicity varies from lot to lot.

[0009] The BSA-coated particles disclosed in Patent Document 1 did not have sufficient performance in suppressing nonspecific adsorption. One of the reasons for this is presumably that BSA is a natural product that has been separated and purified. In other words, it is considered that the performance of BSA varies depending on the individual and production lot, and therefore the performance in suppressing nonspecific adsorption is insufficient. Furthermore, in the method disclosed in Patent Document 2, coating the surface with hydrophilic functional groups requires complicated operations involving multiple reaction steps. Furthermore, in the method using magnetic particles disclosed in Patent Document 3, it is necessary to incorporate a mechanism for applying a magnetic field, which may result in a complicated device configuration.

[0010] Therefore, the present invention aims to provide particles that can suppress non-specific adsorption without using BSA, particles that adsorb and precipitate impurities that cause a decrease in measurement sensitivity, and methods for producing them. [Means for solving the problem]

[0011] A particle according to one embodiment of the present invention is a polymer particle containing polystyrene, characterized in that the polymer particle is composed of a random copolymer including a unit having a pyrrolidone ring and a unit having a functional group capable of binding to a ligand.

[0012] A method for producing particles according to one embodiment of the present invention is characterized by comprising a first step of mixing a radically polymerizable monomer, a radical polymerization initiator, and a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding to a ligand with an aqueous medium to prepare a suspension, and a second step of stirring the suspension to polymerize the monomer.

[0013] A particle according to another embodiment of the present invention is an inorganic particle, characterized in that the surface of the particle contains a random copolymer including a unit having a pyrrolidone ring and a unit having a functional group capable of binding to a ligand, or the particle contains a first layer of a polymer having a pyrrolidone ring and a second layer of a polymer having a carboxy group and a silicon alkoxide group or a silanol group in the same molecule. Effect of the Invention

[0014] According to one aspect of the present invention, it is possible to provide particles capable of suppressing non-specific adsorption without using BSA, and a method for producing the same.

[0015] According to one aspect of the present invention, it is possible to provide particles capable of suppressing non-specific adsorption, which can be synthesized in a single polymerization reaction without requiring complicated steps, and a method for producing the same.

[0016] According to another aspect of the present invention, it is possible to provide particles that contain an inorganic substance and therefore have a high specific gravity and a high sedimentation rate, and a method for producing such particles.

[0017] Furthermore, according to another aspect of the present invention, the particles are coated with a copolymer having a pyrrolidone ring or a copolymer having a pyrrolidone ring, a carboxy group, and a silicon alkoxide group or a silanol group in the same molecule, thereby suppressing non-specific adsorption, and as a result, it is possible to provide particles and a method for producing particles that have the effect of adsorbing and precipitating impurities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, the embodiments of the present invention will be described in detail, but the present invention is not limited to these. First Embodiment (Particles and method for producing particles) The method for producing particles according to the present embodiment includes a step (first step) of preparing an emulsion by mixing at least a radical polymerizable monomer, a radical polymerization initiator, and a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand with an aqueous solution (aqueous medium). The method further includes a step (second step) of heating the obtained emulsion to polymerize the radical polymerizable monomer. The particles produced by the production method according to the present embodiment may be referred to as polymer fine particles below. The particles produced by the production method for particles according to the present embodiment have a high hydrophilicity on the particle surface due to the presence of an organic compound having a pyrrolidone ring, so that non-specific adsorption can be suppressed without using BSA. In addition, since natural products such as BSA are not used, the difference in the ability to suppress non-specific adsorption for each particle is small. In other words, the reproducibility of the ability to suppress non-specific adsorption is high in producing particles. Furthermore, since a copolymer is used as the organic compound having a pyrrolidone ring, many active sites are present on the particle surface, and the proportion of ligands bound to the copolymer is also high. As a result, the detection sensitivity of antigen-antibody reactions by agglutination methods, such as latex agglutination methods, is high. Here, the term "unit" refers to a unit structure corresponding to one monomer.

[0019] (An example of particle manufacturing method) Next, an example of a method for producing particles according to the present embodiment will be described. The polymer microparticles according to this embodiment are prepared by mixing at least a radical polymerizable monomer, a radical polymerization initiator, and a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand with an aqueous medium to prepare an emulsion (first step). Here, the functional group capable of binding a ligand can be any of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, and a silicon alkoxide group. Further, the method includes a step (second step) of heating the emulsion to polymerize the monomers. The polymer particles according to this embodiment are polymer particles obtained by copolymerizing a compound having a radically polymerizable double bond.

[0020] (Radical polymerizable monomer) The radical polymerizable monomer is characterized by including at least a styrene-based monomer. In addition, a monomer selected from the group consisting of an acrylate-based monomer and a methacrylate-based monomer may be included. For example, butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, etc. can be mentioned. In addition to the styrene-based monomer, at least one selected from the above-mentioned monomer group can be used. That is, among these monomers, one or more types can be used in combination. Also, a monomer having two or more double bonds in one molecule, such as divinylbenzene, can be used as a crosslinking agent.

[0021] Also, a radical polymerizable monomer containing an organic silane may be used. Examples of the organic silane compound include vinyl trimethoxysilane, vinyl triethoxysilane, p-styryl trimethoxysilane, 3-methacryloxypropyl methyl dimethoxysilane, 3-methacryloxypropyl trimethoxysilane, 3-methacryloxypropyl methyl diethoxysilane, 3-methacryloxypropyl triethoxysilane, and 3-acryloxypropyl trimethoxysilane. At least one selected from the group of monomers can be used. That is, among these organic silane compounds, one may be used alone or in combination. The radical polymerizable monomer containing an organic silane forms a skeleton of an inorganic oxide in the synthesized polymer microparticles, and plays a role in improving the physical and chemical stability of the polymer microparticles. Furthermore, it plays a role in increasing the affinity between the random copolymer containing a unit having a pyrrolidone ring on the surface of the polymer microparticle and a unit having a functional group capable of binding a ligand, and the skeleton material of the polymer microparticles mainly composed of styrene.

[0022] Furthermore, by using a radical polymerizable monomer containing an organic silane, it is possible to add silanol groups to the surface of the polymer particles. The PVP copolymer is more firmly adsorbed to the surface of the polymer particles due to hydrogen bonding between the silanol groups and the PVP copolymer.

[0023] (Radical polymerization initiator) As the radical polymerization initiator, a wide variety of azo compounds, organic peroxides, etc. can be used. Specifically, 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionate) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), etc. can be mentioned.

[0024] (Copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding to a ligand) The copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand is contained in the synthesized polymer microparticles and is mainly present on the particle surface. Hereinafter, the copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand is abbreviated as PVP copolymer. By adding the above PVP copolymer from the time of synthesis of the polymer microparticles, it is possible to simultaneously impart non-specific adsorption suppression ability and ligand binding ability to the polymer microparticles. Since the PVP copolymer added during synthesis is more hydrophilic than the radical polymerizable monomer, it is present at the interface between the solvent and the polymer microparticles being produced during synthesis. The polymer microparticles adsorb the PVP copolymer on the surface of the particles by engulfing a part of the polymer chain of the PVP copolymer during synthesis, or by physical and chemical adsorption such as the interaction between the pyrrolidone ring and styrene (radical polymerizable monomer). The copolymer is preferably a copolymer of vinylpyrrolidone and acrylic acid, and the molar ratio of vinylpyrrolidone to acrylic acid is preferably 2:8 to 9:1. The polymer microparticles may also contain a silicon compound, and the ratio of the silicon compound to the styrene monomer is preferably 40% by weight or less.

[0025] The molecular weight of the PVP copolymer is preferably 10,000 to 100,000, more preferably 40,000 to 70,000. If the molecular weight is less than 10,000, the hydrophilicity of the polymer particle surface is weak, and non-specific adsorption is likely to occur. If the molecular weight is more than 100,000, the hydrophilic layer on the surface of the polymer particle becomes too thick, and the polymer particle gels, making it difficult to handle.

[0026] In the PVP copolymer, the molar ratio of the unit having a pyrrolidone ring to the unit having a functional group capable of binding to a ligand is preferably about 2:8 to 9:1. If the number of functional groups capable of binding to a ligand such as an antibody is too small, the efficiency of the latex agglutination reaction decreases, and if the number is too large, the nonspecific adsorption ability decreases.

[0027] The functional group in the PVP copolymer capable of binding a ligand is not particularly limited as long as it is a functional group capable of binding an antibody, an antigen, an enzyme, etc., and examples of the functional group that can be used include a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, and a silicon alkoxide group. The PVP copolymer is a random polymer (hereinafter, also referred to as "PVP random copolymer"). In addition to the PVP copolymer, a hydrophilic polymer may be added as a protective colloid during the synthesis of the polymer particles. The PVP random copolymer of the present embodiment has a high molecular weight and exhibits an appropriate hydrophilicity that can provide a good balance between the steric repulsion effect for particle dispersion and the adsorption to the polystyrene particle surface. The appropriate hydrophilicity without a significant bias in the degree of hydrophilicity within the molecule is believed to contribute to achieving both good particle dispersibility and ligand binding. On the other hand, since the block copolymer has a clearly separated high hydrophilicity (PVP) and low hydrophilicity (units capable of binding ligands) within the molecule, it is difficult to uniformly adjust the degree of hydrophilicity throughout the molecule. For example, the hydrophobic portion of the block copolymer self-associates in water to form a self-assembled body such as a polymer micelle. In that case, as shown in the manufacturing method of the present embodiment, when a PVP copolymer is added to an aqueous medium in particle synthesis, the block copolymer is likely to become micelles, the amount of adsorption to the polystyrene particle surface decreases, and the resulting microparticles are considered to have poor dispersibility in water.

[0028] Furthermore, the above particles have an absorption spectrum difference of 0.1 or less at a wavelength of 572 nm in an optical path of 10 mm before and after adding 30 μL of a 0.1 wt % particle dispersion to 60 μL of a buffer solution mixed with 16 μL of serum and leaving the mixture for 5 minutes at 37° C. Particles with such an absorption spectrum difference are preferred because they have little nonspecific adsorption of impurities in serum.

[0029] (aqueous medium) The aqueous solution (aqueous medium) preferably contains 80% or more and 100% or less of water in the medium. Examples of the aqueous solution include water and a solution obtained by mixing water with a water-soluble organic solvent, such as methanol, ethanol, isopropyl alcohol, or acetone. If the aqueous solution contains more than 20% of an organic solvent other than water, there is a risk that the polymerizable monomer will dissolve during the production of polymer microparticles.

[0030] In addition, when a radical polymerizable monomer containing an organic silane is used, the aqueous solution (aqueous medium) is preferably adjusted in advance to a pH of 6 or more and 9 or less. If the pH is less than 6 or more than 9, the alkoxide group and silanol group in the organic silane compound may undergo condensation polymerization or react with other functional groups before the formation of polymer fine particles, which may cause the resulting particles to aggregate. In this embodiment, condensation polymerization of alkoxide is not intentionally performed before the formation of polymer fine particles.

[0031] The pH is preferably adjusted using a pH buffer, but may also be adjusted using an acid or a base. In addition, surfactants, antifoaming agents, salts, thickeners, etc. may be added in proportions of 10% or less relative to the aqueous medium.

[0032] In the method for producing polymer microparticles according to the present embodiment, first, a PVP copolymer is dissolved in an aqueous medium adjusted to a pH of 6 to 9. The PVP copolymer is 0.01 to 10% by weight, preferably 0.03 to 5% by weight, based on the aqueous medium. If it is less than 0.01% by weight, the amount of the PVP copolymer adsorbed to the polymer microparticles is small and the effect is not achieved. If it is more than 10% by weight, the viscosity of the aqueous medium increases, and there is a possibility that stirring cannot be performed sufficiently.

[0033] Next, the radical polymerizable monomer (A) is added to the aqueous medium to form an emulsion. At this time, in addition to (A), the radical polymerizable monomer (B) containing an organic silane may also be added to the aqueous medium to form an emulsion. The weight ratio of (A) to (B) is 6:4 to 10:0.

[0034] If (A) is 6 or less, the specific gravity of the particles as a whole increases, and there is a risk of the particles sedimenting significantly. Also, (B) is not necessary, but in order to increase the adhesion between the PVP copolymer and the polymer particles, it is more preferable to add (B).

[0035] The weight ratio of the aqueous medium to the total amount of (A) and (B) is preferably 5:5 to 9.5:0.5. If the ratio of the aqueous medium is 5 or less, the generated polymer particles may be significantly aggregated. If the ratio is 9.5 or more, there is no problem in generating the polymer particles, but the amount of the generated particles may be small.

[0036] The radical polymerization initiator is used by dissolving it in water, a buffer, etc. The amount of the radical polymerization initiator to be used relative to the total weight of (A) and (B) is between 0.5% by weight and 10% by weight.

[0037] The step of heating the emulsion does not matter as long as the entire emulsion is heated uniformly. The heating temperature can be set to any value between 50°C and 90°C, and the heating time can be set to any value between 2 hours and 24 hours. By heating the emulsion, the monomers are polymerized.

[0038] The particle diameter of the polymer microparticles is preferably 50 nm or more and 400 nm or less, more preferably 100 nm or more and 400 nm or less. The coefficient of variation (CV value) of the particle size distribution of the polymer microparticles is preferably 5 or less. The main measurement methods for the average particle diameter are dynamic light scattering (DLS), laser diffraction (LD), and observation with an electron microscope, but in this embodiment, dynamic light scattering is preferably used. For example, a dispersion of polymer microparticles is measured by dynamic light scattering, the obtained light intensity is converted into a number distribution, and the average value is taken as the particle diameter. If the average particle diameter is less than 50 nm, the washing process during synthesis may take a long time. If the average particle diameter is greater than 400 nm, aggregation due to sedimentation during storage may become significant.

[0039] The coefficient of variation (CV value) can be determined by taking images of polymer microparticles with an electron microscope, determining the diameters of, for example, 100 or more particles, and performing statistical processing to determine the coefficient of variation (CV value). If the coefficient of variation (CV value) is 5 or more (the distribution width of particle sizes is wide), there is a risk that the reaction between the particles for specimen testing and the antigen will be unstable.

[0040] The particle size of the polymer particles can be controlled by the ratio of the monomer to the aqueous medium during synthesis, the amount of the water-soluble polymer added, the reaction temperature, and the reaction time.

[0041] The dispersion of polymer particles thus produced is purified by classification, such as by filtration, decantation by centrifugation, or ultrafiltration, and by removing unreacted materials, aggregates, and the like.

[0042] <Particles for specimen testing> The polymer microparticles thus prepared are precursor particles for specimen testing, and can be used as specimen testing particles by immobilizing antibodies on the precursor particles.

[0043] The immobilization method utilizes the functional groups of the polymer particles and involves immobilization by chemical bonding, physical adsorption, etc. The present invention is not limited to this method.

[0044] When an antigen is present in a sample, the particles for specimen testing, which have antibodies immobilized on polymer microparticles, will agglutinate with each other via the antigen, and the antigen can be detected by observing this agglutination. If the target antigen is not present in the sample, agglutination is unlikely to occur.

[0045] (Ligand affinity particles) In this embodiment, an affinity particle having a particle according to this embodiment and a ligand bound to a functional group capable of binding the ligand can be provided. In this embodiment, the ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site at which the ligand binds to the target substance is determined, and the ligand has a selective or specific high affinity. For example, an antigen and an antibody, an enzyme protein and its substrate, a signal substance such as a hormone or a neurotransmitter and its receptor, a nucleic acid, etc. are exemplified, but the ligand in this embodiment is not limited to these. An example of the nucleic acid is deoxyribonucleic acid, etc. The affinity particle in this embodiment has a selective or specific high affinity (affinity) for the target substance. For example, an antibody, an antigen, an antigen-binding fragment (for example, Fab, F(ab')2, F(ab'), Fv, scFv, etc.), a naturally occurring nucleic acid, an artificial nucleic acid, an aptamer, a peptide aptamer, an oligopeptide, an enzyme, or a coenzyme, etc. are exemplified. It is preferable that the ligand in this embodiment is any one of an antibody, an antigen, a protein, and a nucleic acid.

[0046] In this embodiment, the method of chemical reaction for chemically bonding the reactive functional group of the particle according to this embodiment with the ligand can be a conventionally known method within the scope of the present invention. In addition, when bonding the ligand with an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used.

[0047] When the affinity particles of this embodiment use an antibody (antigen) as the ligand and an antigen (antibody) as the target substance, they can be preferably applied to an immuno-latex agglutination assay that is widely used in the fields of clinical testing, biochemical research, and the like.

[0048] (In vitro diagnostic test reagents) The test reagent for in vitro diagnosis in this embodiment, that is, the test reagent for use in detecting a target substance in a specimen by in vitro diagnosis, has the affinity particles according to this embodiment and a dispersion medium for dispersing the affinity particles. The amount of the affinity particles according to this embodiment contained in the reagent in this embodiment is preferably 0.001% by mass to 20% by mass, more preferably 0.01% by mass to 10% by mass. The reagent according to this embodiment may contain a third substance such as a solvent or a blocking agent in addition to the affinity particles according to this embodiment within a range that can achieve the object of the present invention. The third substance such as a solvent or a blocking agent may be contained in combination of two or more types. Examples of the solvent used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvent contained in the reagent in this embodiment is not limited to these.

[0049] When used to detect an antigen or antibody in a sample by latex agglutination, the ligand can be an antibody or an antigen.

[0050] (Test kit) The test kit for use in detecting a target substance in a specimen by in vitro diagnosis in this embodiment has the above-mentioned reagent and a housing containing the above-mentioned reagent. The kit according to this embodiment may contain a sensitizer for latex agglutination measurement. Examples of sensitizers for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, etc., but the present invention is not limited thereto. The kit according to this embodiment may also include a positive control, a negative control, a serum dilution solution, etc. As a medium for the positive control and the negative control, a solvent may be used in addition to serum and physiological saline that do not contain a measurable target substance. The kit according to this embodiment can be used in the detection method of a target substance according to this embodiment in the same manner as the kit for use in detecting a target substance in a specimen by normal in vitro diagnosis. In addition, the concentration of the target substance can also be measured by a conventionally known method, and it is particularly suitable for use in detecting a target substance in a specimen by the latex agglutination method.

[0051] (Detection method) The method for detecting a target substance in a specimen by in vitro diagnosis in this embodiment includes a step of mixing the affinity particles according to this embodiment with a specimen that may contain the target substance. The affinity particles according to this embodiment are preferably mixed with the specimen at a pH range of 3.0 to 11.0. The mixing temperature is in the range of 20° C. to 50° C., and the mixing time is in the range of 1 to 20 minutes. The present detection method preferably uses a solvent. The concentration of the affinity particles in the detection method according to this embodiment is preferably 0.001% by mass to 5% by mass, more preferably 0.01% by mass to 1% by mass, in the reaction system. The detection method according to this embodiment preferably detects the target substance in the specimen by optically detecting the agglutination reaction that occurs as a result of mixing the affinity particles with the specimen, that is, by the latex agglutination method. Specifically, the method includes a step of mixing the specimen with the test reagent to obtain a mixed solution, a step of irradiating the mixed solution with light, and a step of detecting at least one of the transmitted light and the scattered light of the light irradiated to the mixed solution. By optically detecting the agglutination reaction occurring in the mixed solution, the target substance in the specimen can be detected and the concentration of the target substance can also be measured. The method for optically detecting the agglutination reaction may involve measuring the changes in scattered light intensity, transmitted light intensity, absorbance, etc., using an optical device capable of detecting these values.

[0052] Second Embodiment The second embodiment of the present invention will be described in detail below, but the present invention is not limited thereto.

[0053] (particle) The first embodiment of the particle according to the present invention is a particle containing an inorganic substance, the surface of which is coated with a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand. The second embodiment of the particle according to the present invention includes an inorganic substance, a first layer of a polymer having a pyrrolidone ring, and a second layer of a polymer having a carboxy group, a silicon alkoxide group, or a silanol group in the same molecule. In the first embodiment of the particle according to the present invention, the copolymer that coats the surface of the particle is a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand, so that, as described in the first embodiment, both good particle dispersibility and ligand binding can be achieved.

[0054] The particles according to the present embodiment have a high hydrophilicity on the particle surface due to the presence of pyrrolidone rings, and therefore can suppress non-specific adsorption without using BSA. Since BSA, whose performance varies depending on individual particles and manufacturing lots, is not used, the particles according to the present embodiment can suppress non-specific adsorption without changes between manufacturing lots. Furthermore, since a copolymer having a functional group capable of binding a ligand is used, many functional groups are present on the particle surface, and the proportion of ligands bound is also high. As a result, when the ligand is an antibody (or antigen), the antigen-antibody reaction becomes highly efficient. It is preferable that a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand coats particles containing inorganic matter. Here, the coating may be in any state of binding as long as the particles are stably dispersed. In other words, it is not necessary to cover the entire particle, but it may be partial, and the coating layer may not be uniform.

[0055] Furthermore, the particles according to this embodiment are composed of two layers as described above. The first layer can suppress non-specific adsorption due to the presence of pyrrolidone rings. Furthermore, the second layer has a high ratio of ligand binding due to the presence of carboxyl groups. In addition, the second layer has silicon alkoxide groups or silanol groups, which interact with the pyrrolidone rings of the first layer to stabilize the coating structure.

[0056] (Inorganic) Inorganic substances constituting the particles according to the present invention include metals, metal oxides, metal nitrides, alloys, and combinations thereof. Metals include silver, gold, copper, tin, iron, nickel, palladium, molybdenum, platinum, zinc, cobalt, silicon, and the like. Metal oxides include oxides of the compounds listed above as metals, as well as silica, zirconium oxide, aluminum oxide, titanium oxide, yttrium oxide, cerium oxide, indium tin oxide, bismuth oxide, and the like. Nitrides include nitrides of the compounds listed above as metals, as well as titanium carbonitride and aluminum nitride. Alloys include alloys of the compounds listed above as metals (e.g., iron-nickel alloys, etc.). The inorganic substance in this embodiment may be a silicon compound, and the ratio of the silicon compound to the particles is preferably 40% by weight or more.

[0057] These inorganic substances are preferred because the sedimentation rate increases with increasing specific gravity. Furthermore, it is preferred that these inorganic substances are obtained as stable particles. For the above reasons, silica is preferred among these inorganic substances. Silica can also be synthesized using a radical polymerizable monomer (or oligomer) containing an organic silane. Examples of the organic silane compound include vinyl trimethoxysilane, vinyl triethoxysilane, p-styryl trimethoxysilane, 3-methacryloxypropyl methyl dimethoxysilane, 3-methacryloxypropyl trimethoxysilane, 3-methacryloxypropyl methyl diethoxysilane, 3-methacryloxypropyl triethoxysilane, and 3-acryloxypropyl trimethoxysilane. At least one selected from the group of these monomers can be used. That is, one or more of these may be used alone or in combination. The radical polymerizable monomer containing an organic silane plays a role in forming a skeleton of an inorganic oxide within the synthesized particles and improving the physical and chemical stability of the particles.

[0058] (Covering material) The copolymer comprising a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand, which constitutes the particles of the first embodiment of the present invention, is present on the surface of the synthesized particles. Hereinafter, the copolymer comprising a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand is abbreviated as PVP copolymer. The molecular weight of the PVP copolymer is preferably 10,000 or more and 100,000 or less, more preferably 40,000 or more and 70,000 or less. If the molecular weight is small, the hydrophilicity of the particle surface is weak, and non-specific adsorption is likely to occur. If the molecular weight is large, the hydrophilic layer on the surface of the particle becomes too thick, causing the particle to gel and become difficult to handle. The copolymer is preferably a copolymer of vinylpyrrolidone and acrylic acid, and the molar ratio of vinylpyrrolidone to acrylic acid is preferably 2:8 to 9:1.

[0059] In the PVP copolymer, the molar ratio of the unit having a pyrrolidone ring to the unit having a functional group capable of binding to a ligand is preferably about 2: 8 to 9: 1. If the number of functional groups capable of binding to a ligand such as an antibody is too small, the efficiency of the antigen-antibody reaction decreases, and if the number is too large, the performance of suppressing nonspecific adsorption decreases.

[0060] The functional group in the PVP copolymer capable of binding a ligand is not particularly limited as long as it is a functional group capable of binding an antibody, an antigen, an enzyme, etc., and examples thereof include a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, and a silicon alkoxide group.

[0061] The PVP copolymer is a random polymer. In addition to the PVP copolymer, a hydrophilic polymer or a surfactant may be added as a protective colloid during particle synthesis.

[0062] By adding the PVP copolymer from the time of particle synthesis, it is possible to simultaneously impart non-specific adsorption suppression ability and ligand binding ability to the particles. Furthermore, when an inorganic substance is formed using a radical polymerizable monomer (or oligomer) containing an organic silane, silanol groups can be imparted to the surface of the silica particles. Due to hydrogen bonds between the silanol groups and the PVP copolymer, the PVP copolymer is more firmly adsorbed to the particle surface.

[0063] An example of the polymer having a pyrrolidone ring in the first layer constituting the particles of the second embodiment according to the present invention is polyvinylpyrrolidone. For the same reason as the PVP copolymer, the molecular weight of polyvinylpyrrolidone is preferably 10,000 to 100,000, more preferably 40,000 to 70,000. Next, the polymer having a carboxy group and a silicon alkoxide group or a silanol group in the same molecule of the second layer constituting the particles of the second embodiment according to the present invention can be synthesized using a radical polymerizable monomer (or oligomer) containing an organic silane. Examples of the radical polymerizable monomer (or oligomer) containing an organic silane include 3-trimethoxysilylpropylsuccinic anhydride, silane-PEG-carboxylic acid (manufactured by Nanocs), diethoxysilane-PEG-carboxylic acid (manufactured by Nanocs), and monoethoxysilane-PEG-carboxylic acid (manufactured by Nanocs). The molecular weight of the PEG can be selected from 600 to 70,000.

[0064] (particle size and specific gravity) The particle size and specific gravity of the particles according to this embodiment can be determined by the settling velocity required to remove impurities. The relationship between the settling velocity, particle size, and specific gravity in a certain medium can be expressed by the following formula (1) using Stokes' equation.

number

[0065] In formula (1), υ s is the (terminal) settling velocity, D p is the particle size, ρ pis the specific gravity (density) of the particle, ρ f where ρ is the specific gravity (density) of the medium, g is the acceleration of gravity, and η is the viscosity of the medium. From formula (1), it can be seen that the larger the particle size and the larger the specific gravity, the faster the settling velocity. However, as the particle size increases, the surface area per unit volume of the particle decreases, and the amount of ligand that can be bound decreases, which is not necessarily preferable. Therefore, the average particle size of the particles according to this embodiment is preferably 50 nm or more and 100 μm or less, and more preferably 100 nm or more and 10 μm or less. In addition, if the natural settling velocity in water is 2.2×10 -5 It is preferable that the speed is cm / sec or more.

[0066] The coefficient of variation (CV value) of the particle size distribution of the particles is preferably 5 or less. The main methods for measuring the average particle size include dynamic light scattering (DLS), laser diffraction (LD), and observation with an electron microscope, but in this embodiment, dynamic light scattering is preferably used. For example, a dispersion of particles is measured by dynamic light scattering, the light intensity obtained is converted into a number distribution, and the average value is taken as the particle size.

[0067] The coefficient of variation (CV value) can be determined by taking images of particles with an electron microscope, determining the diameters of, for example, 100 or more particles, and then performing statistical processing to determine the coefficient of variation (CV value). If the coefficient of variation (CV value) is 5 or more (the distribution width of particle sizes is wide), there is a risk of large inter-measurement errors.

[0068] The particle size can be controlled by the ratio of the monomer to the aqueous medium during synthesis, the amount of the water-soluble polymer added, the reaction temperature and the reaction time.

[0069] The specific gravity of the particles according to this embodiment can be changed by the composition and content of the inorganic substance. The specific gravity of the particles can be calculated or measured. When calculated, it can be calculated from the specific gravity of each single component and the composition ratio. When measuring the specific gravity, it can be measured by a known method such as the Gay-Lussac type pycnometer method or the Le Chatelier pycnometer method.

[0070] (Manufacturing method) An example of a method for producing particles according to the present embodiment will be described. When a metal oxide is used as the inorganic substance, the method includes a step of mixing a radical polymerizable monomer such as a metal alkoxide, a radical polymerization initiator, and a random copolymer containing a unit having a pyrrolidone ring and a unit having a functional group capable of binding a ligand with an alcohol-based solvent to prepare an emulsion. The radical polymerization initiator is not necessary, but ion-exchanged water or the like can be added. As the alcohol-based solvent, methanol, ethanol, isopropyl alcohol, or the like can be used. When silica is used as the inorganic substance, the particles can be synthesized according to the Stöber method.

[0071] Another example includes a step of preparing an emulsion by mixing a radical polymerizable monomer such as a metal alkoxide, a radical polymerization initiator, and a polymer having a pyrrolidone ring with an alcohol-based solvent. After purification, the second step includes a step of mixing the emulsion prepared in the first step and a radical polymerizable monomer containing an organic silane with an aqueous solvent to react. When using the radical polymerizable monomer containing an organic silane, it is preferable that the pH of the aqueous solution (aqueous medium) during the reaction is adjusted in advance to 6 to 9. If the pH is less than 6 or more than 9, the alkoxide group and silanol group in the organic silane compound may undergo condensation polymerization or react with other functional groups before reacting with the particles, and the resulting particles may aggregate. In this embodiment, the alkoxide is not intentionally condensed before the formation of the particles. The pH is preferably adjusted using a pH buffer, but may be adjusted using an acid or base. In addition, a surfactant, an antifoaming agent, a salt, a thickener, etc. may be added to the aqueous medium at a ratio of 10% or less. It is also possible to produce particles by carrying out only the second step using commercially available water-dispersed inorganic particles.

[0072] The particle dispersion thus produced can be purified by classification and removal of unreacted materials, aggregates, etc., through filtration, decantation by centrifugation, ultrafiltration, etc.

[0073] <Particles for specimen testing> The microparticles thus prepared can be used as precursor particles for specimen testing, and can be used as impurity separation particles by binding a ligand to the precursor particles.

[0074] The method for binding the ligand utilizes the functional groups of the fine particles and can be by chemical bonding, physical adsorption, etc. The present invention is not limited to this method.

[0075] Impurity separation particles, which have ligands bound to them, bind to the particles when impurities to be separated are present in a specimen, and precipitate, thereby suppressing non-specific reactions in the specimen.

[0076] (Ligand / impurity separation particles) In this embodiment, it is possible to provide impurity separation particles having a ligand bound to a functional group capable of binding the ligand. The impurity separation particles in this embodiment have a high affinity for the impurities to be separated. In this embodiment, the ligand is a compound that specifically binds to a specific substance. Examples of combinations of a ligand and a compound that binds to the ligand include an antibody and an antigen, a nucleic acid and a nucleic acid having a complementary sequence, avidin (or streptavidin, or neutravidin) and biotin, various immunoglobulins and protein A (or protein G), a chelating agent coordinated with a histidine tag peptide and a metal ion, various proteins (including tag peptides) and their interacting substances, an enzyme protein and its substrate, a signal substance such as a hormone or a neurotransmitter and its receptor, and the like, but the ligand in this embodiment is not limited to these. For example, if the impurities to be separated can be removed collectively, a ligand with low specificity can be used. Examples of ligands include antibodies, antigens, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, or coenzymes.

[0077] The ligand in this embodiment is preferably any one of an antibody, an antigen, a protein, and a nucleic acid.

[0078] In this embodiment, the method of chemical reaction for chemically bonding the functional group of the particle with the ligand can be a conventionally known method within the scope of the present invention. For example, when the particle according to this embodiment has a carboxy group, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used to bond the ligand with an amide bond.

[0079] When the impurity separation particles in this embodiment use an antibody (or antigen) as the ligand and an antigen (antibody) as the target substance, they can be preferably used as impurity separation particles for immunoturbidimetric assays and Enzyme-Linked ImmunoSorbent Assays (ELISA), which are widely used in the fields of clinical testing, biochemical research, and the like.

[0080] (In vitro diagnostic test reagents) The in vitro diagnostic test reagent in this embodiment, that is, the test reagent for use in detecting a target substance in a specimen by in vitro diagnosis, has a test reagent capable of detecting a target substance, impurity separation particles, and a dispersion medium for dispersing them. The amount of impurity separation particles contained in the reagent in this embodiment is preferably 0.001% by mass to 20% by mass, more preferably 0.01% by mass to 10% by mass. The reagent according to this embodiment may contain a third substance such as a solvent or a blocking agent in addition to the impurity separation particles within a range that can achieve the object of the present invention. The third substance such as a solvent or a blocking agent may be contained in combination of two or more types. Examples of the third substance include polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, etc., which are sensitizers for latex agglutination measurement, but the present invention is not limited to these. Examples of the solvent used in this embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, Hepes buffer, and ammonia buffer, but the solvent contained in the reagent in this embodiment is not limited to these.

[0081] When used to remove impurities from a specimen by latex agglutination, the ligand of the particles according to this embodiment can be an antibody against the impurities to be separated.

[0082] (Test kit) The test kit for use in detecting a target substance in a specimen (test subject) by in vitro diagnosis in this embodiment has the above test reagent and a housing containing the above reagent. The kit according to this embodiment may contain a sensitizer for latex agglutination measurement. Examples of sensitizers for latex agglutination measurement include polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, etc., but the present invention is not limited thereto. The kit according to this embodiment may also include a positive control, a negative control, a serum dilution solution, etc. As a medium for the positive control and the negative control, serum not containing a measurable target substance, physiological saline, or a solvent may be used. The kit according to this embodiment can be suitably used for detecting a target substance in a specimen by the latex agglutination method, in particular, by suppressing non-specific agglutination by removing impurities in the specimen.

[0083] (Detection method) The method for detecting a target substance in a specimen by in vitro diagnosis in this embodiment includes a step of mixing the impurity separation particles with a specimen that may contain the impurities to be separated. The mixing of the impurity separation particles and the specimen according to this embodiment is preferably performed at a pH range of 3.0 to 11.0. The mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 1 to 20 minutes. The detection method preferably uses a solvent. The concentration of the impurity separation particles in the detection method according to this embodiment is preferably 0.001% by mass to 5% by mass, more preferably 0.01% by mass to 1% by mass in the reaction system.

[0084] After mixing the impurity separation particles with a specimen, if the impurities to be separated are present in the specimen, the particles bind to the impurities and precipitate, thereby suppressing non-specific reactions in the specimen. This precipitation may occur by natural precipitation, or may be efficiently caused by applying acceleration in one direction to the mixed solution. For example, centrifugation can be performed to apply acceleration from the center of a circle toward the outside, thereby causing precipitation.

[0085] After removing the impurities, the target substance in the specimen can be detected by optically detecting the agglutination reaction that occurs as a result of mixing the specimen with a test reagent capable of detecting the target substance, for example, by the latex agglutination method. Specifically, the method includes a step of mixing the test reagent with the specimen to obtain a mixed liquid, a step of irradiating the mixed liquid with light, and a step of detecting at least one of the transmitted light and the scattered light of the light irradiated to the mixed liquid. By optically detecting the agglutination reaction that occurs in the mixed liquid, the target substance in the specimen can be detected, and the concentration of the target substance can also be measured. As a method for optically detecting the agglutination reaction, the amount of change in these values ​​can be measured using an optical device that can detect scattered light intensity, transmitted light intensity, absorbance, degree of polarization, particle size change, etc. It is to be noted that the removal of impurities and the detection of the target substance by the latex agglutination method can be performed simultaneously. EXAMPLES

[0086] The present invention will now be described more specifically with reference to examples and comparative examples. However, the present invention is not limited to the following examples.

[0087] Example 1 157 g of phosphate buffer pH 7.4 (Kishida Chemical Co., Ltd.) was added to a 300 ml flask, and Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)) (Polymer Source Co., Ltd., molecular weight 60,000, molecular weight of pyrrolidone ring moiety) was added. number : Molecules with carboxyl groups numberIn the flask, 1.3 g of styrene (ratio = 4:6) was dissolved. Next, 4.0 g of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) and 13.0 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and the mixture was stirred for 15 minutes while blowing nitrogen into the mixture at room temperature. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 10 ml of phosphate buffer solution pH 7.4 (manufactured by Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70°C. After stirring for 7 hours at 70°C, the mixture was returned to room temperature to obtain a dispersion of polymer microparticles.

[0088] Furthermore, this dispersion was centrifuged to recover the polymer microparticles, and the supernatant was discarded. The recovered polymer microparticles were redispersed in ion-exchanged water and then centrifuged again. The recovery of the polymer microparticles using the centrifuge and redispersion in ion-exchanged water were repeated three times. The concentration of the polymer microparticle dispersion thus obtained was adjusted to 1.0 wt%.

[0089] Example 2 157 g of phosphate buffer pH 7.4 (Kishida Chemical Co., Ltd.) was added to a 300 ml flask, and Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)) (Polymer Source Co., Ltd., molecular weight 60,000, molecular weight of pyrrolidone ring moiety) was added. number : Molecules with carboxyl groups number0.9g of styrene (Kishida Chemical Co., Ltd., molecular weight 40000) and 0.4g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) were dissolved in the flask. Next, 4.0g of 3-methacryloxypropyltrimethoxysilane (Shin-Etsu Chemical Co., Ltd., LS-3380) and 13.0g of styrene (Kishida Chemical Co., Ltd.) were added, and the mixture was stirred for 15 minutes at room temperature while blowing in nitrogen. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 10ml of phosphate buffer pH 7.4 (Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70°C. After stirring for 7 hours at 70°C, the mixture was returned to room temperature to obtain a dispersion of polymer fine particles.

[0090] Furthermore, this dispersion was centrifuged to recover the polymer microparticles, and the supernatant was discarded. The recovered polymer microparticles were redispersed in ion-exchanged water and then centrifuged again. The recovery of the polymer microparticles using the centrifuge and redispersion in ion-exchanged water were repeated three times. The concentration of the polymer microparticle dispersion thus obtained was adjusted to 1.0 wt%.

[0091] Example 3 157 g of phosphate buffer pH 7.4 (Kishida Chemical Co., Ltd.) was added to a 300 ml flask, and Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)) (Polymer Source Co., Ltd., molecular weight 60,000, molecular weight of pyrrolidone ring moiety) was added. number : Molecules with carboxyl groups number0.9g of styrene (Kishida Chemical Co., Ltd. ratio = 4:6) and 0.4g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) were dissolved in the flask. 17.0g of styrene (Kishida Chemical Co., Ltd.) was added and stirred for 15 minutes at room temperature while blowing in nitrogen. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 10ml of phosphate buffer pH 7.4 (Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70°C. After stirring for 7 hours at 70°C, the temperature was returned to room temperature to obtain a dispersion of polymer microparticles.

[0092] Furthermore, this dispersion was centrifuged to recover the polymer microparticles, and the supernatant was discarded. The recovered polymer microparticles were redispersed in ion-exchanged water and then centrifuged again. The recovery of the polymer microparticles using the centrifuge and redispersion in ion-exchanged water were repeated three times. The concentration of the polymer microparticle dispersion thus obtained was adjusted to 1.0 wt%.

[0093] Comparative Example 1 157g of phosphate buffer solution pH 7.4 (Kishida Chemical Co., Ltd.) was added to a 300ml flask, and 1.3g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved in it. 13.0g of styrene (Kishida Chemical Co., Ltd.) was then added, followed by 4.0g of 3-methacryloxypropyltrimethoxysilane (LS-3380, Shin-Etsu Chemical Co., Ltd.), and the mixture was stirred for 15 minutes at room temperature while blowing in nitrogen. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 10ml of phosphate buffer solution pH 7.4 (Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70°C. After stirring for 7 hours at 70°C, the mixture was returned to room temperature to obtain a dispersion of polymer microparticles.

[0094] Furthermore, this dispersion was centrifuged to recover the polymer microparticles, and the supernatant was discarded. The recovered polymer microparticles were redispersed in ion-exchanged water and then centrifuged again. Recovery of the polymer microparticles by centrifugation and redispersion in ion-exchanged water were repeated three times. The concentration of the polymer microparticle dispersion thus obtained was adjusted to 10.0% by weight.

[0095] The 10.0 wt% polymer particle dispersion prepared was diluted to 1.0 wt%, and 10.0 wt% ammonia water and 0.25 mmol of trimethoxypropylsuccinic anhydride were added. The total volume of the liquid was 10 mL. The mixed liquid was stirred at room temperature for 14 hours, and the surface of the polymer particles was modified with dicarboxyl groups.

[0096] Comparative Example 2 157 g of phosphate buffer solution pH 7.4 (Kishida Chemical Co., Ltd.) was added to a 300 ml flask, and 1.3 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40,000) was dissolved in it. 17.0 g of styrene (Kishida Chemical Co., Ltd.) was added, and stirring was performed for 15 minutes while blowing nitrogen into the solution at room temperature. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 10 ml of phosphate buffer solution pH 7.4 (Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70°C. After stirring for 7 hours at 70°C, the solution was returned to room temperature to obtain a dispersion of polymer microparticles.

[0097] Furthermore, this dispersion was centrifuged to recover the polymer microparticles, and the supernatant was discarded. The recovered polymer microparticles were redispersed in ion-exchanged water and then centrifuged again. Recovery of the polymer microparticles by centrifugation and redispersion in ion-exchanged water were repeated three times. The concentration of the polymer microparticle dispersion thus obtained was adjusted to 10.0% by weight.

[0098] The 10.0 wt% polymer microparticle dispersion prepared was added at 1.0 wt% and a dimethyl sulfoxide solution of trimethoxypropylsuccinic anhydride was added at 0.4 wt% to the solution, and the mixture was stirred at room temperature for 14 hours to modify the surface of the polymer microparticles with dicarboxyl groups.

[0099] Comparative Example 3 157 g of phosphate buffer solution pH 7.4 (Kishida Chemical Co., Ltd.) was added to a 300 ml flask, and 1.3 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40,000) was dissolved in it. 13.0 g of styrene (Kishida Chemical Co., Ltd.) and 2.0 mL of light ester HO-MS (Kyoeisha Chemical Co., Ltd.) were added, and the mixture was stirred for 15 minutes while blowing nitrogen into the mixture at room temperature. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) dissolved in 10 ml of phosphate buffer solution pH 7.4 (Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70°C. After stirring for 7 hours at 70°C, the mixture was returned to room temperature to obtain a dispersion of polymer microparticles.

[0100] Furthermore, this dispersion was centrifuged to recover the polymer microparticles, and the supernatant was discarded. The recovered polymer microparticles were redispersed in ion-exchanged water and then centrifuged again. Recovery of the polymer microparticles by centrifugation and redispersion in ion-exchanged water were repeated three times. The concentration of the polymer microparticle dispersion thus obtained was adjusted to 10.0% by weight.

[0101] Comparative Example 4 IMMUTEX immunodiagnostic polymer particles P0113 (polymer particles made by JSR Corporation, in which a carboxy group is bonded to polystyrene particles having a particle size of 187 nm) were prepared to a particle concentration of 1.0% by weight.

[0102] <Antibody binding> To each of the polymer particle dispersions prepared in the Examples and Comparative Examples other than Comparative Example 4, an anti-PSA antibody was bound as follows.

[0103] First, each polymer microparticle dispersion was centrifuged at 15000 rpm (20400 g) for 15 minutes to precipitate the microparticles. After removing the supernatant, the microparticle pellet was redispersed in a buffer solution, to which water-soluble carbodiimide WSC was added. N-hydroxysuccinimide was further added. The microparticle dispersion was stirred at room temperature for 30 minutes, and the particles were collected by centrifugation. The microparticles were washed with a buffer solution. The microparticles were redispersed in a buffer solution, and anti-PSA antibody was added to a final antibody concentration of 2 mg / mL. After stirring at room temperature for 60 minutes, the particles were collected by centrifugation. The particles were thoroughly washed with a buffer solution to obtain microparticles to which the anti-PSA antibody was bound. The binding of the antibody was confirmed by measuring the decrease in antibody concentration in the buffer solution to which the antibody was added by a BCA assay.

[0104] <Non-specific agglutination inhibition evaluation> 60 μl of human serum solution diluted 15-fold with a buffer solution was added to 30 μl of each polymer microparticle dispersion prepared in Examples 1 to 3 and Comparative Examples 1 to 4, and the mixture was incubated at 37° C. for 5 minutes. The absorbance at 527 nm was measured before and after incubation, and the change in absorbance before and after incubation was measured three times. Table 1 shows the average value of the three measurements. A change in the absorbance×10,000 value of less than 1,000 was considered to indicate that nonspecific aggregation had been inhibited, and a change of 1,000 or more was considered to indicate that nonspecific aggregation had occurred. The results are shown in Table 1.

[0105] [Table 1]

[0106] In Comparative Examples 3 and 4, an agglutination reaction (non-specific agglutination) occurred in reaction with human serum. In the Examples, it is believed that the hydrophilic PVP copolymer present on the surface of the polymer particles acts to suppress non-specific adsorption.

[0107] <Latex agglutination test evaluation> A dispersion of each polymer particle bound with the anti-PSA antibody prepared by antibody binding was prepared to a concentration of 0.1% by weight, and a latex agglutination reaction was carried out.

[0108] A human serum-derived PSA standard solution was mixed with the polymer microparticle dispersion prepared in antibody binding 1, and the absorbance at a wavelength of 572 nm was measured before and after mixing. If the anti-PSA antibody on the microparticles can capture the antigen PSA, an agglutination reaction between the microparticles occurs via PSA. At this time, a decrease in transmittance occurs, and an increase in absorbance is observed. The change in absorbance (absorbance x 10,000) was measured three times before and after treatment at a reaction temperature of 37°C and a reaction time of 5 minutes, both when PSA was added to a solution containing antibody-bound microparticles and when PSA was not added. Table 1 shows the average absorbance change rate for the three times. From Table 1, it can be seen that an agglutination reaction occurs due to the antigen-antibody reaction when PSA is added, and a clear difference can be confirmed compared to the increase rate when PSA is not added.

[0109] As can be seen from Table 1, it was confirmed by evaluation of the latex agglutination reaction that the polymer microparticles of the examples of the present invention to which antibodies were bound recognized PSA and caused the particles to aggregate with each other. On the other hand, the amount of change was small in Comparative Examples 1 and 2. Taking into consideration the results of non-specific adsorption overall, it is possible for the comparative examples to achieve a sample-to-noise ratio of at least 3. On the other hand, it can be seen that the sample-to-noise ratios of Comparative Examples 1 and 2, which had low detection sensitivity, were much smaller in order of magnitude than those of the examples.

[0110] Therefore, it was found that the microparticles according to this embodiment can bind antibodies without using BSA, and the bound antibodies have sufficient antigen recognition ability. The microparticles according to this embodiment can be suitably used as test particles that utilize immune reactions, for example, antibody-sensitized latex used in latex agglutination methods.

[0111] In addition, the synthesis can be performed in only one emulsion polymerization reaction, making it easier to prepare than with conventional methods.

[0112] (Measurement of physical properties of fine particles) The solid / particle concentration was determined by evaporating the moisture of a known weight of sample using a heat-drying moisture meter (MX-50, A&D Co., Ltd.). The particle size was measured by dynamic light scattering using a Zetasizer (Nano-ZS, manufactured by Malvern Instruments). The zeta potential was measured using a Zetasizer with a dedicated cell. The solvent was pure water. The spontaneous sedimentation rate was measured using a LUMISizer (registered trademark) (manufactured by LUM Japan Co., Ltd.) based on the light transmittance profile during centrifugation.

[0113] Example 4 In a 110 ml sample bottle, 50 mL of isopropyl alcohol (Kishida Chemical Co., Ltd.) and 0.0555 ml of ion-exchanged water were added, and 6.2 ml of tetraethyl orthosilicate (Tokyo Chemical Industry Co., Ltd.) and 0.5 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) were dissolved. Next, while stirring the mixed solution in the sample bottle prepared above, 6.5 ml of 28% ammonia water (Kishida Chemical Co., Ltd.) was slowly added dropwise. After stirring overnight at room temperature, the mixed solution was centrifuged to collect the fine particles, and the supernatant was discarded. The fine particles were redispersed in ion-exchanged water and then centrifuged again. The collection of the fine particles by the centrifuge and the redispersion by ion-exchanged water were repeated three times. The dispersion of the fine particles thus obtained was adjusted to a concentration of 10.0% by weight.

[0114] Next, a dispersion of the fine particles was prepared with a phosphate buffer solution (Kishida Chemical Co., Ltd., pH 7.4) to a concentration of 0.25% by weight and 10 ml. Next, Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)) (Polymer Source Co., Ltd., molecular weight 60000, ratio of N-vinyl pyrrolidone molecular number:acrylic acid molecular number = 4:6) was dissolved in the phosphate buffer solution to a concentration of 0.033 g / ml and 1.5 ml, and added to the dispersion of the fine particles. After stirring overnight at room temperature, the mixed solution was centrifuged to collect the fine particles, and the supernatant was discarded. The fine particles were redispersed in ion-exchanged water and then centrifuged again. The collection of the fine particles by the centrifuge and the redispersion by ion-exchanged water were repeated three times. The dispersion of the fine particles thus obtained was adjusted to a concentration of 1% by weight.

[0115] The particle size, zeta potential, and natural sedimentation rate of the prepared microparticles are summarized in Table 2. By adding Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)), the particle size became larger and the zeta potential became negative. This is thought to be because the surface of the microparticles is coated with the polymer.

[0116] Example 5 Spherical silica particles Silbol-S 240 nm (manufactured by Fuji Chemical Co., Ltd.) were prepared with a phosphate buffer solution to a concentration of 0.25% by weight and 10 ml. As in Example 4, a phosphate buffer solution in which Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)) was dissolved was added to the dispersion of the particles. The same operations as in Example 4 were then carried out to prepare a dispersion of the particles.

[0117] The particle size, zeta potential, and natural sedimentation rate of the prepared microparticles are summarized in Table 2. By adding Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)) as in Example 4, the particle size increased and the zeta potential became negative. This is considered to be due to the surface of the microparticles being coated with the polymer.

[0118] [Table 2]

[0119] <Antibody binding> To each of the microparticle dispersions prepared in Examples 4 and 5 after adding Poly(N-vinyl pyrrolidone-co-acrylic acid), random (Poly(N-vinyl pyrrolidone-ran-acrylic acid)), an anti-CRP antibody was bound as follows.

[0120] First, each microparticle dispersion was centrifuged to precipitate the microparticles. After removing the supernatant, the microparticle pellet was redispersed in a buffer solution, to which water-soluble carbodiimide (Tokyo Chemical Industry Co., Ltd.) was added. N-hydroxysuccinimide (Tokyo Chemical Industry Co., Ltd.) was further added. The microparticle dispersion was stirred at room temperature for 30 minutes, and then the particles were collected by centrifugation. The microparticles were washed with a buffer solution. The microparticles were redispersed in a buffer solution, and anti-C-reactive protein (hereinafter sometimes abbreviated as CRP) antibody was added so that the antibody was 200 μg per 1 mg of particles. After stirring at room temperature for 60 minutes, the particles were collected by centrifugation. The particles were thoroughly washed with a buffer solution to obtain microparticles to which anti-CRP antibodies were bound. Protein was quantified by a bicinchoninic acid (hereinafter sometimes abbreviated as BCA) assay to confirm that the antibodies were bound to the microparticles (Table 3).

[0121] [Table 3]

[0122] <Evaluation of impurity removal performance> CRP was selected as a model of impurities. Human serum-derived CRP (Sigma-Aldrich) was diluted with a buffer solution to prepare an absorbance of about 0.5 at a wavelength of 280 nm with a path length of 1 cm. The diluted CRP solution was mixed with each of the microparticle dispersions to which the anti-CRP antibody was bound, which had been prepared to a concentration of 0.2% by weight, at a volume ratio of 1:1. For comparison, the diluted CRP solution was mixed with a solution not containing microparticles of the same volume as the microparticle dispersion. After stirring at room temperature for 1 hour, the solution was centrifuged to recover the supernatant. The absorbance of the supernatant at a wavelength of 280 nm was measured, and the rate of change in absorbance was measured. The rate of change in absorbance was calculated using the following formula. {(Absorbance of the supernatant to which each microparticle was added) - (Absorbance of the supernatant to which a solution not containing microparticles was added)} ÷ (Absorbance of the supernatant to which a solution not containing microparticles was added) x 100

[0123] As a result of the absorbance measurement, the change in absorbance was largely negative for both the microparticles of Examples 4 and 5. This indicates that the amount of CRP in the supernatant was reduced. In other words, it was confirmed that the microparticles had the effect of capturing the CRP in the solution and causing it to precipitate.

[0124] From the above, it was demonstrated that by binding an anti-CRP antibody to the microparticles prepared in Examples 4 and 5, CRP, which is a model of impurities, can be precipitated and removed.

[0125] (Example 6) Synthesis and evaluation of silica particles A A dispersion of polymer fine particles was obtained in the same manner as in Example 1, except that the amount of styrene monomer (used without distillation) was changed from 13.0 g to 12.8 g and that the amount of potassium peroxodisulfate was changed from 0.3 g to 0.5 g in Example 1. The polymer fine particles are referred to as silica particles A. The particle size of the silica particles A in water (diameter, measured by dynamic light scattering) was 235 nm, and the polydispersity index (PDI) was 0.027. As a result of measuring the electrical conductivity of the dispersion solution of the silica particles A, the amount of carboxy groups per unit weight of the silica particles A was 87 nmol / mg.

[0126] (Example 7) Evaluation of the inhibition of non-specific aggregation of albumin by silica particles A To evaluate the inhibition of nonspecific aggregation of silica particles A, the nonspecific aggregation property of silica particles A with respect to albumin was confirmed. First, 50 μL of an aqueous dispersion of silica particles A was mixed with 50 μL of a 0.05% or 0.5% bovine serum albumin solution (bovine serum albumin dissolved in phosphate buffered saline), and the mixture was incubated at 37° C. for 5 minutes. The absorbance at 572 nm was measured before and after incubation, and the amount of change in absorbance before and after incubation was measured. If nonspecific aggregation with respect to albumin occurs, the value of the amount of change in absorbance x 10,000 will be 1,000 or more. The values ​​calculated by multiplying the change in absorbance by 10,000 are shown below. 0.05% albumin solution: 40 0.5% albumin solution: 130

[0127] From the above results, it was revealed that silica particles A do not aggregate with albumin.

[0128] (Example 8) Evaluation of the inhibition of non-specific agglutination of silica particles A against human serum globulin fractions To evaluate the inhibition of nonspecific aggregation of silica particles A, the nonspecific aggregation property of silica particles A against human serum globulin fractions was confirmed. First, 16 μL of globulin solution with a globulin concentration of 0.48 mg / mL or 48 mg / mL was added to 60 μL of phosphate buffer and incubated at 37°C for 5 minutes. Then, 30 μL of dispersion of silica particles A was added to this and incubated at 37°C for 5 minutes. The absorbance at 572 nm was measured before and after incubation, and the change in absorbance before and after incubation was measured. When nonspecific aggregation occurs against human serum globulin fractions, the value of the change in absorbance x 10,000 will be 1,000 or more. The values ​​calculated by multiplying the change in absorbance by 10,000 are shown below. Globulin concentration 0.48mg / mL:-20 Globulin concentration 48mg / mL:100

[0129] These results demonstrate that silica particles A do not aggregate with human serum globulin fractions.

[0130] (Example 9) Evaluation of the inhibition of non-specific aggregation of human serum fibrin by silica particles A To evaluate the inhibition of nonspecific aggregation of silica particles A, the nonspecific aggregation property of silica particles A against human serum fibrin was confirmed. First, 50 μL of an aqueous dispersion of silica particles A was mixed with 50 μL of a human serum fibrin solution (prepared before use) and incubated at 37° C. for 5 minutes. After incubation, particle aggregation was visually observed, and no particle aggregation was observed.

[0131] (Example 10) Antibody sensitization to silica particles A and evaluation (preparation of affinity particles) An anti-CRP antibody was bound to the silica particles A prepared in Example 6 as follows. First, the dispersion of silica particles A was centrifuged to precipitate the fine particles. After removing the supernatant, the pellet of silica particles A was redispersed in a buffer solution, to which water-soluble carbodiimide (manufactured by Tokyo Chemical Industry Co., Ltd.) was added. N-hydroxysuccinimide (manufactured by Tokyo Chemical Industry Co., Ltd.) was further added. The dispersion of silica particles A was stirred at room temperature for 30 minutes, and then the silica particles A were collected by centrifugation. The silica particles A were washed with a buffer solution. The silica particles A were redispersed in a buffer solution, and an anti-CRP antibody was added so that the antibody was 200 μg per 1 mg of particles. After stirring at room temperature for 60 minutes, the silica particles A were collected by centrifugation. The silica particles A were thoroughly washed with a buffer solution to obtain silica particles A to which the anti-CRP antibody was bound. The protein was quantified by a BCA assay, and it was confirmed that the antibody was bound to the silica particles A. Next, the latex agglutination reaction was evaluated. A human serum-derived CRP standard solution was mixed with a dispersion of antibody-bound silica particles A, and the absorbance at a wavelength of 572 nm was measured before and after mixing, and an increase in absorbance was observed. From the above results, the antigen-antibody reactivity of antibody-bound silica particles A with CRP was confirmed. Silica particles A can be suitably used as test particles that utilize immune reactions, for example, antibody-sensitized latex used in latex agglutination methods.

Claims

1. Particles comprising polystyrene and further comprising a random copolymer of poly(N-vinylpyrrolidone-ran-acrylic acid) in which the molar ratio of the vinylpyrrolidone units to the acrylic acid units is from 2:8 to 9:

1.

2. 2. The particle according to claim 1, wherein the molecular weight of the random copolymer is 10,000 or more and 100,000 or less.

3. 3. The particle according to claim 1 or 2, characterized in that it comprises a silicon compound.

4. A particle described in any one of claims 1 to 3, characterized in that the difference in absorption spectrum at a wavelength of 572 nm at an optical path of 10 mm before and after adding 30 μL of a 0.1 wt % dispersion of the particle to 60 μL of buffer solution mixed with 16 μL of serum and leaving it at 37°C for 5 minutes is 0.1 or less.

5. a first step of preparing an emulsion by mixing at least a radical polymerizable monomer containing styrene, a radical polymerization initiator, and a random copolymer of poly(N-vinylpyrrolidone-ran-acrylic acid) having a molar ratio of the vinylpyrrolidone unit to the acrylic acid unit of 2:8 to 9:1 with an aqueous medium; a second step of stirring the emulsion and polymerizing the radical polymerizable monomer.

6. A method for producing a test reagent, comprising the step of dispersing the particles according to claim 1 in a dispersion medium.

7. The method for producing a test reagent according to claim 6 , wherein the average particle diameter of the particles in the test reagent is 100 nm or more and 400 nm or less.

8. 5. An affinity particle comprising the particle according to claim 1 and a ligand bound to a carboxy group contained in the acrylic acid unit.

9. 9. The affinity particle according to claim 8, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid.

10. 10. An in vitro diagnostic test reagent comprising the affinity particles according to claim 8 or 9 and a dispersion medium for dispersing the affinity particles.

11. The test reagent according to claim 10 , wherein the ligand is an antibody or an antigen, and the test reagent is used for detecting an antigen or an antibody in a specimen by an agglutination method.

12. 12. An in vitro diagnostic test kit comprising: the test reagent according to claim 10 or 11; and a housing that contains the test reagent.

13. A method for detecting a target substance in a specimen, comprising: A detection method comprising the step of mixing a specimen with the test reagent according to claim 10 or 11.

14. A method for detecting a target substance in a specimen by an agglutination method, comprising the steps of: A step of mixing a specimen with the test reagent according to claim 10 or 11 to obtain a mixed solution; irradiating the mixture with light; detecting at least one of transmitted light and scattered light of the light irradiated onto the mixed liquid; A detection method comprising the steps of:

15. The particles contain an inorganic substance, and the surface of the particles contains a random copolymer in which a unit having a pyrrolidone ring and a unit having any functional group selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group are randomly copolymerized with each other, and the molar ratio of the unit having the pyrrolidone ring to the unit having the functional group is 2:8 to 9:

1.

16. 16. The particle according to claim 15, wherein the random copolymer is a copolymer of vinylpyrrolidone and acrylic acid.

17. 17. The particle according to claim 16, wherein the molar ratio of the vinylpyrrolidone unit to the acrylic acid unit in the random copolymer is from 2:8 to 9:

1.

18. The particle according to any one of claims 15 to 17, characterized in that the molecular weight of the random copolymer is 10,000 or more and 100,000 or less.

19. 19. The particle according to claim 15, wherein the inorganic substance is a silicon compound.

20. 20. The particle according to claim 19, characterized in that the content of the silicon compound is 40% by weight or more.

21. A particle described in any one of claims 15 to 20, characterized in that a ligand is bound to the functional group.

22. 22. The particle of claim 21, wherein the ligand is an antibody, an antigen, a protein, or a nucleic acid.

23. The natural settling velocity of the particles in water is 2.2 × 10 －５ 23. The particle according to claim 15, characterized in that the particle has a viscosity of at least cm / sec.

24. a first step of preparing an emulsion by mixing, with a solvent, a random copolymer comprising at least a metal alkoxide, a unit having a pyrrolidone ring, and a unit having any functional group selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group, the molar ratio of the unit having the pyrrolidone ring to the unit having the functional group being 2:8 to 9:1; and a second step of stirring the emulsion and polymerizing the monomer.

25. 23. An in vitro diagnostic test reagent comprising at least the particle according to claim 22 and a dispersion medium for dispersing the particle.

26. The test reagent according to claim 25, characterized in that the test reagent is used for separating impurities.

27. 27. An in vitro diagnostic test kit comprising: the test reagent according to claim 25 or 26; the test reagent according to claim 10 or 11; and a housing that contains the test reagent.

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