Test reagent containing thiourea compound and resin particle containing sulfur element, and method for detecting target substance in specimen by in vitro diagnosis using test reagent

Abstract

To provide a test reagent having small nonspecific adsorption and capable of detecting a low-concentration target substance, and a detection method using the test reagent.SOLUTION: A test reagent for in vitro diagnosis, comprising a thiourea compound and a resin particle, wherein the resin particle contains a sulfur element.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a test reagent containing a thiourea compound and resin particles having elemental sulfur, and a detection method. [Background technology]

[0002] In in vitro diagnostic pharmaceuticals used for diagnosing diseases, a method using resin particles is known as a method for detecting target substances. In this method, a ligand is immobilized on the resin particles, and the target substance is reacted with the ligand by contacting the resin particles with a sample. In the above-mentioned method, when the resin particles come into contact with the sample, the target substance or impurities in the sample may be nonspecifically adsorbed to the surface of the resin particles rather than to the ligand, which may cause noise in the measurement. Therefore, in the above-mentioned method using resin particles, it is desirable that the resin particles have a property of adsorbing substances other than the target substance, i.e., have little nonspecific adsorption.

[0003] One method to reduce nonspecific adsorption is to coat the resin particle surface in a post-process using biologically derived polymers such as albumin, casein, and gelatin. However, the physical properties of these biologically derived substances can vary from production lot to production lot. Therefore, when coating the resin particle surface in a subsequent process, a method using a highly biocompatible hydrophilic polymer instead of a biopolymer is also effective in reducing nonspecific adsorption. However, if the adsorption of the polymer to the resin particle is due to physical adsorption, the polymer may be released by dilution, and nonspecific adsorption may not be sufficiently suppressed. A known method for preventing nonspecific adsorption on the surface of resin particles is to introduce sulfur element near the surface of resin particles with a polystyrene main chain. It is believed that the inclusion of sulfur element improves the hydrophilicity of the resin particles and prevents nonspecific adsorption by electrostatic repulsion between the particles. Patent Documents 1 and 2 disclose reagents that use resin particles with side chains containing sulfonic acid groups or sulfinyl groups on the surface. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2012 / 133771 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-153140 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have conducted an investigation using the latex agglutination method, which is one of the methods for detecting a target substance. As a result, it was confirmed that, in a system for measuring a target substance at a low concentration in a sample, resin particles containing elemental sulfur, such as those described in Patent Documents 1 and 2, cause nonspecific adsorption, resulting in a false positive, in which even a negative sample is judged as positive.

[0006] The present invention has been made in view of the above background art and problems. An object of the present invention is to provide a test reagent containing a thiourea compound and resin particles containing elemental sulfur, which has low nonspecific adsorption and is capable of detecting a target substance at low concentrations, and a detection method using the test reagent. [Means for solving the problem]

[0007] The present invention relates to an in vitro diagnostic test reagent containing a thiourea compound and resin particles, the resin particles containing elemental sulfur.

[0008] The present invention also relates to a method for detecting a target substance in a specimen by in vitro diagnosis, which comprises a step of mixing the above-mentioned test reagent with a specimen that may contain the target substance. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide an in vitro diagnostic test reagent that significantly reduces nonspecific adsorption and is capable of detecting a target substance at low concentrations, and further to provide a detection method using the same. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail, but the technical scope of the present invention is not limited to these embodiments. In this specification, the term "(meth)acrylic" means "acrylic or methacrylic". For example, "(meth)acrylate" means "acrylate or methacrylate".

[0011] The test reagent of the present invention is an in vitro diagnostic test reagent containing a thiourea compound and resin particles, the resin particles containing elemental sulfur. The inventors conducted a study using latex agglutination, a method for detecting target substances. As a result, it was confirmed that conventional resin particles containing elemental sulfur cause nonspecific adsorption in a system for measuring low concentrations of target substances in a sample, resulting in a false positive, in which even negative samples are judged as positive. In particular, it was found that it is necessary to suppress nonspecific adsorption at the side chain moiety containing elemental sulfur in the resin particles. The present inventors have intensively investigated solutions based on the mechanism by which specific causative substances in a specimen are nonspecifically adsorbed to resin particles, and as a result have come up with the invention of a test reagent that uses a test reagent containing both a thiourea compound and resin particles containing elemental sulfur, which has low nonspecific adsorption and is capable of detecting low concentrations of target substances.

[0012] <Resin particles> The resin particles of the present invention are characterized by containing elemental sulfur. The presence of elemental sulfur is believed to contribute to the suppression of nonspecific adsorption between resin particles and the detection of low concentrations of target substances. The reason for this is believed to be as follows: By including a structure containing elemental sulfur in the resin particles, the particles have a certain degree of polarity and are moderately hydrophilic, improving the dispersibility of the resin particles while also preventing the particles from agglomerating during target substance detection, which is believed to contribute to the suppression of nonspecific adsorption between resin particles and the detection of low concentrations of target substances.

[0013] Although there are no particular limitations on the structure containing sulfur elements, from the viewpoint of the aforementioned moderate hydrophilicity, it is preferable that the resin particles have a polymer having at least one of a sulfide group and a thiol group. By having a sulfide group or a thiol group, it is easier to suppress agglutination inhibition during target substance detection due to excessive hydrophilicity. Other structures containing sulfur elements include a sulfo group and a thioketone group.

[0014] In the resin particles of the present invention, the amount of elemental sulfur as determined by X-ray photoelectron spectroscopy (ESCA) is preferably 0.1 atm% to 8.0 atm%, more preferably 0.5 atm% to 6.0 atm%. As will be described in detail later, it is preferable that the resin particles contain a polymer having a carboxy group or a structure derived from a carboxy group. In this case, the amount of elemental sulfur in the resin particles as determined by ESCA is more preferably 1.5 atm% to 2.0 atm%, from the viewpoint of detecting low concentrations of target substances. When the resin particles do not contain a carboxy group or a structure derived from a carboxy group, it is more preferable that the amount of elemental sulfur as determined by ESCA is 3.1 atm% to 6.0 atm%, from the viewpoint of suppressing nonspecific adsorption.

[0015] By containing a certain amount of elemental sulfur, the resin particles have sufficient dispersibility to suppress nonspecific adsorption between themselves, which is thought to suppress agglutination inhibition during target substance detection, resulting in excellent results. As will be described in detail later, it is thought that the effect of suppressing nonspecific adsorption of resin particles containing elemental sulfur with substances in a specific sample can also be easily achieved when mixed with a thiourea compound in a test reagent. To control the amount of elemental sulfur in the resin particles, as quantified by ESCA, within the most preferred range, for example, if the resin particles contain sulfide groups as structures containing elemental sulfur, one method is to replace some of them with amine groups.

[0016] As long as the object of the present invention can be achieved, the chemical structure of the resin particles is not particularly limited except that it contains sulfur element, but it is preferable that the resin particles have a polymer having a structure derived from at least one of styrenes and (meth)acrylates in the main chain. It is also preferable that the resin particles have a polymer having a hydroxy group. It is also preferable that the resin particles have a polymer having at least one of a carboxy group and a structure derived from a carboxy group.

[0017] Having at least one of a structure derived from styrenes and a structure derived from (meth)acrylates is preferred because the glass transition temperature is high and the resin particles have sufficient strength. Furthermore, styrenes are highly hydrophobic and tend to aggregate when detecting a target substance in a sample, which is preferable because it also has the effect of increasing detection sensitivity. Below, examples of styrenes and (meth)acrylates that can be used in this embodiment are listed, but are not limited to these. Furthermore, two or more types of oil-based radical polymerizable monomers may be used.

[0018] Styrenes: styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, and the like.

[0019] (Meth)acrylates: glycidyl (meth)acrylate, methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, n-amyl acrylate, n-hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, n-nonyl acrylate, cyclohexyl acrylate, benzyl acrylate, dimethyl phosphate ethyl acrylate, diethyl phosphate ethyl acrylate, dibutyl phosphate ethyl acrylate, 2-benzoyloxyethyl acrylate, methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, n-amyl methacrylate, n-hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, n-nonyl methacrylate, diethyl phosphate ethyl methacrylate, and dibutyl phosphate ethyl methacrylate.

[0020] The reason why resin particles preferably contain hydroxyl groups is thought to be as follows. For example, the styrene-derived structure, which was exemplified as a preferred structure for the polymer main chain of resin particles, is highly hydrophobic. Therefore, the inclusion of hydrophilic hydroxyl groups is thought to suppress nonspecific aggregation of resin particles due to hydrophobic interactions. Conventionally, bio-derived hydrophilic additives such as BSA have been used to suppress aggregation, but there are concerns about performance variations between lots and the nonspecific adsorption of BSA to substances in the sample. In contrast, the inclusion of hydroxyl groups in resin particles suppresses nonspecific adsorption of resin particles without the use of additives such as BSA, making it easier to design assays that are less likely to produce false positives or false negatives.

[0021] The reason why resin particles preferably have carboxy groups or polymers with structures derived from carboxy groups is believed to be as follows: For example, during the process of adsorbing ligands such as antibodies or antigens onto resin particles, carboxy groups act as reactive functional groups, which are thought to facilitate both high reactivity and the suppression of interparticle aggregation due to charge repulsion between resin particles. This facilitates uniform adsorption of ligands such as antibodies and antigens onto resin particles, resulting in improved low-concentration sensitivity. Examples of other reactive functional groups include maleimide groups, amide groups, tosyl groups, amino groups, epoxy groups, and thiol groups. A carboxy group-derived structure possessed by a polymer in resin particles refers to a ligand such as an antigen or antibody bound via a carboxy group. In other words, when resin particles have ligands such as antigens or antibodies, the antigens or antibodies can be bound to the resin particles via carboxy groups.

[0022] One method for producing polymers containing hydroxyl or carboxyl groups according to the intended design is to incorporate glycidyl (meth)acrylate into resin particles, open the epoxy moiety, and bond it. For example, by reacting mercaptopropanediol or aminopropanediol, a structure containing hydroxyl groups can be obtained. Similarly, by reacting glycidyl (meth)acrylate with mercaptosuccinic acid, a structure containing carboxyl groups can be obtained.

[0023] Furthermore, the resin particles containing sulfur element can contain a structure derived from a group of radically polymerizable monomers having crosslinkability. Examples of the group of radically polymerizable monomers 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, tetramethylolmethane tetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, and triethylene glycol dimethacrylate. Examples of suitable crosslinkable monomers include, but are not limited to, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, 1,6-hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis(4-(methacryloxydiethoxy)phenyl)propane, 2,2'-bis(4-(methacryloxypolyethoxy)phenyl)propane, trimethylolpropane trimethacrylate, tetramethylolmethane tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether, provided that the object of the present invention can be achieved. In addition, two or more types of radically polymerizable monomers having crosslinkability may be used in combination.

[0024] A radical polymerization initiator may be used during the production of the resin particles. Examples of the polymerization initiator include 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]tetrahydrate, 2,2'-azobis(2-methylpropionamidine)dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, potassium peroxodisulfate, ammonium peroxodisulfate, and sodium peroxodisulfate, but are not limited to these as long as the object of the present invention can be achieved.

[0025] The particle size of the resin particles is preferably 0.05 μm to 1.00 μm, more preferably 0.10 μm to 0.60 μm, and even more preferably 0.10 μm to 0.40 μm, in terms of number average particle size. When the resin particles are dispersed in an aqueous medium and used, centrifugation for purification is possible, making handling easier, and particle sedimentation is less likely to occur when the resin particles are stored in the form of a resin particle dispersion.

[0026] <Ligand> In the present invention, a ligand refers to 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 fixed, and the ligand has a selective or specific high affinity. The ligand is not particularly limited, but it is preferable to use an antigen or an antibody. Other examples include enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, and nucleic acids.

[0027] <Ligand-bound resin particles> The test reagent of the present invention may contain resin particles before ligand binding, or may contain ligand-bound resin particles. Ligand-bound resin particles are formed by binding the aforementioned ligand to reactive functional groups present on resin particles before ligand binding. In other words, the resin particles of the present invention may have a ligand (such as an antigen or antibody) bound to their surface (reactive functional group). The binding mode between the reactive functional group and the ligand is not particularly limited. However, in terms of selective binding to the ligand, the reactive functional group is preferably selected from carboxyl groups, amino groups, thiol groups, and maleimide groups. Furthermore, carboxyl groups are most preferably selected in terms of ease of reaction. Conventional methods can be used for chemical reaction to chemically bond the carboxyl group to the ligand, provided that the objectives of the present invention can be achieved. For example, carbodiimide-mediated reactions and NHS ester activation reactions are commonly used chemical reaction methods, but the chemical reaction method for chemically bonding the carboxyl group to the ligand is not limited to these.

[0028] <Activated resin particles> When the substrate of a ligand-bound carrier is particulate, the specific surface area of ​​the carrier surface is maximized, allowing the carrier to exhibit its effects most effectively. Ligand-bound resin particles, whose substrate is particulate, are also called activated resin particles because they have a high affinity (activation) for target substances selectively or specifically. Ligand-bound carriers whose substrate is particulate, i.e., activated resin particles, are highly suitable for use in in vitro diagnostics to detect target substances in samples, as an example of which is the application of particles for latex agglutination. When general particles are used as particles for latex agglutination, target substances such as antigens (antibodies) and foreign substances in serum or plasma may nonspecifically adsorb to the particle surface, which can cause unintended inter-particle aggregation and potentially impair the accuracy of immunoassays. The use of the activated resin particles of the present invention reduces unintended inter-particle aggregation due to nonspecific adsorption and enables the detection of low concentrations of target substances.

[0029] <Test reagents for use in in vitro diagnostics to detect target substances in specimens> The test reagent for use in in vitro diagnostic detection of a target substance in a specimen according to the present invention is characterized by containing a thiourea compound and resin particles containing elemental sulfur. This allows for detection of low concentrations of the target substance while suppressing nonspecific adsorption. The reason for this is believed to be as follows: The thiourea compound can assume a form having an appropriate amount of thiol groups through an equilibrium reaction, trapping substances contained in a specific specimen that are prone to nonspecific adsorption with the resin particles containing elemental sulfur. Therefore, by combining a thiourea compound with resin particles containing elemental sulfur, nonspecific adsorption between substances in the specimen and the resin particles, and between the resin particles themselves, is thought to be less likely to occur, and low concentrations of the target substance can also be detected. Furthermore, as mentioned above, controlling the amount of elemental sulfur in the resin particles is thought to enable both suppression of nonspecific adsorption and detection of low concentrations of the target substance at a superior level.

[0030] The inventors believe that it is important to use thiourea compounds to ensure that the appropriate amount of thiol is present in the test reagent. By not using too much, the reactivity of antibodies, antigens, etc. in the test reagent is fully maintained, while by not using too little, sufficient amounts of substances that tend to nonspecifically adsorb to resin particles contained in specific samples can be trapped. For example, compounds that actively supply thiol groups, such as 2-mercaptoethanol, which is known as a reducing agent, or compounds that do not have a thiol group, such as urea, a chaotropic agent, were not sufficiently effective in the present invention when added to the test reagent.

[0031] Assuming that the test reagent is aqueous, and taking into consideration the solubility in water, the thiourea compound preferably contains a compound represented by the following formula (1): [ka] (In formula (1), R 1 ~R 4 each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may be branched, an allyl group, an acetyl group, or a group represented by the following formula (2): [ka] (In formula (2), * indicates the bonding position in the compound represented by formula (1).)

[0032] Examples of the compound represented by formula (1) include thiourea, 1,3-diethyl-2-thiourea, 2,5-dithiobiurea, 1-methyl-2-thiourea, 1-ethyl-2-thiourea, 1-allyl-2-thiourea, 1-tert-butyl-2-thiourea, N,N'-dimethylthiourea, 1,3-dibutyl-2-thiourea, 1,3-di-tert-butyl-2-thiourea, 1-acetylthiourea, and trimethylthiourea, with thiourea being more preferred.

[0033] The content of the thiourea compound in the test reagent is preferably 0.0005 M or more and 2 M or less, more preferably 0.005 M or more and 2 M or less, from the viewpoint of suppressing nonspecific adsorption. Here, "test reagent" refers to the total amount of the test reagent. For example, when the test reagent is composed of two liquids, a buffer solution and a resin particle dispersion liquid, as described below, it refers to the total amount of the buffer solution and the resin particle dispersion liquid (total amount of the test reagent), and the content of the thiourea compound in the test reagent refers to the content relative to the total amount of the test reagent.

[0034] The test reagent according to the present invention may be a test reagent composed of two or more liquids, including a buffer solution and a resin particle dispersion. For example, when a buffer solution is used as the first reagent (specimen dilution liquid) and a resin particle dispersion is used as the second reagent, the resin particles of the present invention can be contained in the second reagent (resin particle dispersion). In this case, the first reagent (buffer solution) does not contain resin particles, but can contain a thiourea compound. When the content of the thiourea compound in the test reagent according to the present invention is W(A) and the content of the resin particles containing elemental sulfur in the test reagent is W(B), the mass ratio W(A) / W(B) can be 0.2 or more and 450 or less. The amount of activated resin particles contained in the test reagent according to the present invention is preferably 0.001% by mass to 20% by mass, more preferably 0.01% by mass to 10% by mass.

[0035] The pH of the buffer solution in the present invention is preferably 6.0 to 9.0, more preferably 6.5 to 8.5. By adjusting the pH within the above range, the thiourea compound can be used in an equilibrium reaction to allow the thiol groups to be present in the test reagent in just the right amount. The pH adjuster is not particularly limited, but examples of acids include hydrochloric acid, acetic acid, and oxalic acid, and examples of bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

[0036] Furthermore, the test reagent according to the present invention may contain a third substance in addition to the above-mentioned resin particles, etc., within the scope that allows the object of the present invention to be achieved. The test reagent of the present invention preferably contains a buffer. While the type of buffer is not particularly limited, it is preferable to use at least one buffer selected from the group consisting of MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPSO, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, and CAPS, as this facilitates maintaining the pH at which the thiourea compound has an appropriate amount of thiol groups through an equilibrium reaction. Other buffers include Tris-HCl (e.g., PBS, Tris-HCl), boric acid, phosphoric acid, acetic acid, citric acid, succinic acid, phthalic acid, glutaric acid, maleic acid, glycine, and salts thereof.

[0037] Furthermore, the test reagent of the present invention preferably contains a chelating agent. The chelating agent adsorbs metal ions, such as calcium ions, contained in the test reagent. Therefore, it is thought that the chelating agent inhibits the thiourea compound from adsorbing metal ions, making it easier to more selectively adsorb nonspecific causative substances in the sample. Examples of chelating agents include EDTA, EDTA-2Na, EDTA-3Na, EDTA-4Na, EDTA-2K, EDTA-3K, EDTA-4K, calcium disodium edetate, and calcium dipotassium edetate.

[0038] In order to increase the sensitivity of the reaction, the reagent may contain water-soluble additives such as polyethylene glycol, carboxymethylcellulose, methylcellulose, dextran, polyvinylpyrrolidone, polyglycosylethyl methacrylate, pullulan, dextran, elsinan, polyacrylic acid, and other water-soluble polymers. Furthermore, for the purpose of improving the specificity and the stability of the test reagent, proteins such as casein and gelatin, or their decomposition products or denatured products, quaternary ammonium salts such as choline chloride, polyanions, chaotropic ions (Cl - ,I - ,SCN - etc.), amino acids, surfactants, sugars, etc.

[0039] <Detection method> The method for detecting a target substance in a specimen by in vitro diagnosis according to the present invention is characterized by comprising a step of mixing the test reagent according to the present invention with a specimen that may contain the target substance (a step of obtaining a reaction solution). When the test reagent is a test reagent composed of two or more liquids including a buffer solution and a resin particle dispersion, the step of obtaining the reaction solution may comprise a step of mixing the buffer solution with the specimen to obtain a mixture, and a step of mixing the mixture with the resin particle dispersion. In this case, the buffer solution may not contain resin particles but may contain a thiourea compound, and the resin particle dispersion may contain resin particles. Incidentally, the content of the thiourea compound in the reaction solution can be set to be 0.0005 M or more and 2 M or less. The mixing of the activated resin particles and the specimen is preferably carried out within the range of pH 3.0 to pH 11.0. Further, the mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 1 minute to 20 minutes. Also, in this detection method, it is preferable to use a solvent. Further, the concentration of the activated resin particles in the detection method according to the present invention is preferably 0.001% by mass to 5% by mass, and preferably 0.01% by mass to 1% by mass in the reaction system. The detection method according to the present invention is characterized by optically detecting the inter-particle aggregation resulting from the mixing of the resin particles and the specimen in the present invention. By optically detecting this inter-particle aggregation, the target substance in the specimen can be detected, and further, the concentration of the target substance can also be measured. As a method for optically detecting the above aggregation reaction, the amount of change in these values may be measured using an optical instrument capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc.

[0040] <Measurement of molecular weight by GPC> The polymer (resin particles) having at least one of the structures derived from at least one of styrenes and (meth)acrylates, the structure having a hydroxy group, and the structure having a carboxy group in the present invention can be measured for its molecular weight by GPC. The method is as follows. Put high-performance liquid chromatography methanol and the sample into a vial and dissolve them. After confirming that the sample has dissolved, filter it using a disposable disk filter (trade name: Micron Disc, aperture 0.5 μm) manufactured by Tosoh Corporation, and the passed material is used as the GPC sample. Incidentally, the sample solution is adjusted so that the concentration is about 1.0% by mass. Using this sample solution, measurement is carried out under the following conditions. Apparatus: Waters APC system (Waters Japan Co., Ltd.) Detector: RI, PDA Column: ACQUITY APC XT900 (150 mm), XT200 (75 mm), XT-125 (75 mm), XT45 (150 mm) Temperature: 40.0℃ Solvent: High-performance liquid chromatographic methanol Flow rate: 0.8mL / min Injection volume: 10μL

[0041] [Method for measuring the amount of sulfur (S) near the surface of resin particles] The method for quantifying the atomic weight ratio of resin particles by ESCA measurement in the present invention will be described. The atomic weight ratio of resin particles by ESCA measurement in the present invention is measured using freeze-dried resin particles fixed on indium foil. The measurement device and measurement conditions are as follows. Measurement equipment: X-ray photoelectron spectrometer: Quantum2000 (product name, manufactured by ULVAC-PHI, Inc.) X-ray source: Monochrome Al Kα ·Xray Setting:100μmφ(25W(15KV)) Photoelectron take-off angle: 45 degrees Neutralization Condition: Neutralization Gun and Ion Gun Used Together ·Analysis area: 300×200μm Pass Energy: 58.70 eV Step size: 0.125eV Analysis software: Maltipak (PHI) The cumulative number of measurements was 30 for S2p. The quantitative value of S2p obtained was taken as the amount of elemental sulfur near the surface of the resin particles. From the viewpoint of detection accuracy, the lower detection limit was set at 0.05 atm%, and values ​​below 0.05 atm were considered undetected.

[0042] [Method for measuring number average particle size of resin particles in aqueous dispersion] The method for measuring the number-average particle size of resin particles containing elemental sulfur in an aqueous dispersion of the present invention will be described. The number-average particle size in an aqueous dispersion of the present invention is measured in a state where the resin particles are dispersed in ion-exchanged water to a concentration of 0.001% by mass. The ion-exchanged water used has an electrical conductivity of 10 μS / cm or less. The number-average particle size of resin particles in an aqueous dispersion is measured using a dynamic light scattering method. Specifically, a Zetasizer (Nano-ZS, Spectris Inc.) is used and the measurement is performed at 25°C. The analytical parameters are latex (n≈1.59) as the refractive index of the particles and pure water as the dispersion medium. The measurement is performed 10 times, and the average value of the 10 measurements is used as the number-average particle size of the resin particles in the aqueous dispersion.

[0043] [Method for measuring antibody (antigen) sensitization rate of activated resin particles] The method for measuring the antibody (antigen) sensitization rate of activated resin particles in the present invention, which are obtained by sensitizing resin particles with antibodies or antigens and activating them, is described below. The antibody sensitization rate (%) of activated resin particles was determined by protein quantification. Here, the antibody sensitization rate (%) refers to the ratio of the amount of antibody bound to the resin particles to the amount of antibody used in the reaction (antibody charge amount).

[0044] First, 7 mL of Solution A and 140 μL of Solution B from the Protein Assay BCA Kit (Wako Pure Chemical Industries) were mixed to prepare a solution called Solution AB. Next, 200 μL of Solution AB was added to 25 μL of a 0.1% dispersion of activated resin particles (25 μg of resin particles) and incubated at 60°C for 30 minutes. The solution was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and 200 μL of the supernatant was pipetted into a 96-well microwell. The absorbance at 562 nm was measured using a microplate reader along with standard samples (several antibody concentrations ranging from 0 to 200 μg / mL in 10 mM HEPES), and the antibody amount was calculated from the standard curve. The amount of antibody sensitized to the resin particles (antibody binding amount per 1 mg of resin particles (μg / mg)) was calculated by dividing the calculated antibody amount by the mass of the resin particles (0.025 mg in this case). Finally, the sensitization rate was calculated. When the amount of antibody charged is 25 μg per 1 mg of resin particles, and the antibody sensitization amount is 12.5 μg / mg, the sensitization rate is 50%. [Example]

[0045] The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. The synthesis examples of resin base particles are described below. The amount of sulfur element (S amount) near the surface of each resin base particle measured by ESCA is shown in Table 1.

[0046] [Resin mother particle synthesis example] (Resin base particle 1) A 200 ml flask was charged with 1.2 g of styrene (Kishida Chemical Co., Ltd.), 1.8 g of glycidyl methacrylate (Kishida Chemical Co., Ltd.), 0.04 g of divinylbenzene (Kishida Chemical Co., Ltd.), and 100 g of ion-exchanged water to obtain a mixed solution. The mixed solution was then stirred at 200 rpm while being maintained at 70°C, and nitrogen bubbling was performed for 30 minutes. The nitrogen bubbling was then switched to nitrogen flow.

[0047] Radical polymerization was initiated by adding a separately prepared solution of 0.06 g of V-50 (Fujifilm Wako Pure Chemical Corporation) dissolved in 3 g of purified water to the above mixture. Two hours after the initiation of polymerization, 0.3 g of glycidyl methacrylate was added to the radical polymerization reaction field. The mixture was then maintained at 70 °C with stirring at 200 rpm for an additional 8 hours, after which it was gradually cooled to room temperature. At this point, the contents of the 200 ml flask were sampled, and the radical polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was essentially 100%. The polymerization conversion was calculated from the amount of monomer charged in the polymerization process. Specifically, the polymerization conversion was determined by analyzing the solution after the polymerization reaction using proton NMR or other methods to quantify the remaining monomer, and then calculating the degree to which the monomer had been polymerized by the polymerization reaction (polymerization conversion rate) from the remaining amount of monomer.

[0048] A previously prepared aqueous solution of 3-amino-1,2-propanediol (FUJIFILM Wako Pure Chemical Corporation) and mercaptosuccinic acid (FUJIFILM Wako Pure Chemical Corporation) (adjusted to pH 7 using triethylamine (Kishida Chemical Co., Ltd.) and 2N hydrochloric acid) was added. The solution was then stirred at 200 rpm for 3 hours at room temperature. The aqueous solution had a molar ratio of 3-amino-1,2-propanediol to mercaptosuccinic acid of 8.5:1.5. The total number of moles of 3-amino-1,2-propanediol and mercaptosuccinic acid was equal to the number of moles of glycidyl methacrylate. The pH of the contents of the 200 ml flask was then adjusted to 10 using triethylamine. The temperature was then raised to 70°C and the solution was further stirred at 200 rpm for 3 hours. This resulted in a chemical reaction between the epoxy groups derived from glycidyl methacrylate, the amino groups derived from 3-amino-1,2-propanediol, and the thiol groups derived from mercaptosuccinic acid, yielding resin base particles 1. No agglomerates were formed during the chemical reaction. Resin base particles 1 were purified by centrifugation, and the dispersion medium was replaced with a phosphate buffer solution before storage (the dispersion medium replacement was also performed by centrifugation). Resin base particles 1 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was found to be 238 nm.

[0049] (Resin base particle 2) Resin base particles 2 were prepared in the same manner as resin base particles 1, except that the molar ratio of 3-amino-1,2-propanediol to mercaptosuccinic acid was set to 7:3. Resin base particles 2 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was found to be 235 nm.

[0050] (Resin base particle 3) Resin base particles 3 were prepared in the same manner as resin base particles 1, except that 3-mercapto-1,2-propanediol was used instead of 3-amino-1,2-propanediol and the molar fraction of 3-mercapto-1,2-propanediol to mercaptosuccinic acid was set to 9:1. Resin base particles 3 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 236 nm.

[0051] (Resin base particle 4) Resin base particles 4 were produced in the same manner as resin base particles 1, except that 3-mercapto-1,2-propanediol was used instead of 3-amino-1,2-propanediol, aminosuccinic acid was used instead of mercaptosuccinic acid, and the molar fraction of 3-mercapto-1,2-propanediol to aminosuccinic acid was set to 1.5:8.5. Resin base particles 4 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.), and the number average particle size was found to be 233 nm.

[0052] (Resin base particle 5) A 200 ml flask was charged with 1.2 g of styrene (Kishida Chemical Co., Ltd.), 0.02 g of acrylic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.04 g of divinylbenzene (Kishida Chemical Co., Ltd.), and 100 g of ion-exchanged water to obtain a mixed solution. The mixed solution was then stirred at 200 rpm while being maintained at 70°C, and nitrogen bubbling was performed for 30 minutes. The nitrogen bubbling was then switched to nitrogen flow. Radical polymerization was initiated by adding a separately prepared solution of 0.06 g of potassium peroxodisulfate (Fujifilm Wako Pure Chemical Corporation) dissolved in 3 g of pure water to the above mixture. The mixture was stirred at 200 rpm for 6 hours at 85°C and then slowly cooled to room temperature. At this point, the contents of the 200 ml flask were sampled and the radical polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography. The conversion rate was confirmed to be essentially 100%, yielding resin base particles 5. No agglomerates were formed during the chemical reaction. Resin base particles 5 were purified by centrifugation, and the dispersion medium was replaced with phosphate buffer solution and stored (the dispersion medium replacement was also performed by centrifugation). Dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) of resin base particles 5 revealed a number-average particle size of 218 nm.

[0053] (Resin base particle 6) Resin base particles 6 were produced in the same manner as resin base particles 5, except that the amount of potassium peroxodisulfate was changed to 0.03 g. Resin base particles 6 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 227 nm.

[0054] (Resin base particle 7) A 200 ml flask was charged with 1.2 g of styrene (Kishida Chemical Co., Ltd.), 1.8 g of glycidyl methacrylate (Kishida Chemical Co., Ltd.), 0.04 g of divinylbenzene (Kishida Chemical Co., Ltd.), 0.05 g of sodium styrene sulfonate, and 100 g of ion-exchanged water to obtain a mixed solution. The mixed solution was then stirred at 200 rpm while being maintained at 70°C, and nitrogen bubbling was performed for 30 minutes. The nitrogen bubbling was then switched to nitrogen flow.

[0055] Radical polymerization was initiated by adding a separately prepared solution of 0.02 g of potassium peroxodisulfate (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in 3 g of purified water to the mixture. Two hours after the start of polymerization, 0.3 g of glycidyl methacrylate was added to the radical polymerization reaction field. The mixture was then stirred at 200 rpm for an additional 8 hours at 70°C, after which it was slowly cooled to room temperature. At this point, the contents of the 200 ml flask were sampled, and the radical polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was essentially 100%.

[0056] A pre-prepared aqueous solution of 3-amino-1,2-propanediol (adjusted to pH 7 using triethylamine (Kishida Chemical Co., Ltd.) and 2N aqueous hydrochloric acid) was added. The mixture was then stirred at 200 rpm for 3 hours at room temperature. The amount of 3-amino-1,2-propanediol in the aqueous solution was equal to the amount of glycidyl methacrylate in the aqueous solution. The contents of the 200 ml flask were then adjusted to pH 10 using triethylamine. The temperature was then raised to 70°C and the mixture was stirred at 200 rpm for another 3 hours. This allowed the epoxy groups derived from the glycidyl methacrylate to react with the amino groups of the 3-amino-1,2-propanediol, yielding resin base particles 7. No agglomerates were formed during the chemical reaction. Resin base particles 7 were purified by centrifugation, and the dispersion medium was replaced with a phosphate buffer solution containing 3% BSA for storage (the replacement of the dispersion medium was also performed by centrifugation). Resin base particles 7 were evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number average particle size was found to be 238 nm.

[0057] (Resin base particle 8) Resin base particles 8 were obtained in the same manner as for resin base particles 7, except that the amount of sodium styrenesulfonate was changed from 0.05 g to 0.075 g. Resin base particles 8 were evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 234 nm.

[0058] (Resin base particle 9) Resin base particles 9 were obtained in the same manner as for resin base particles 7, except that the amount of sodium styrenesulfonate was changed from 0.05 g to 0.14 g. Resin base particles 9 were evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.) and found to have a number-average particle size of 228 nm.

[0059] (Resin base particle 10) Resin base particles 10 were obtained in the same manner as for resin base particles 7, except that the amount of sodium styrenesulfonate was changed from 0.05 g to 0.16 g. Resin base particles 10 were evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number average particle size was found to be 228 nm.

[0060] (Resin base particle 11) Resin base particles 11 were obtained by the same procedure as for resin base particles 7, except that the amount of styrene sulfonic acid was changed from 0.05 g to 0.20 g. Resin base particles 11 were evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number average particle size was found to be 216 nm.

[0061] (Resin base particle 12) Resin base particles 12 were obtained by the same procedure as for resin base particles 7, except that the amount of styrene sulfonic acid was changed from 0.05 g to 0.0035 g. Resin base particles 12 were evaluated using a dynamic light scattering method (DLS-8000: Otsuka Electronics Co., Ltd.), and the number average particle size was found to be 228 nm.

[0062] (Resin base particle 13) A 200 ml flask was charged with 3.0 g of styrene (Kishida Chemical Co., Ltd.), 0.04 g of divinylbenzene (Kishida Chemical Co., Ltd.), 0.02 g of styrene sulfonic acid, and 100 g of ion-exchanged water to obtain a mixed solution. The mixed solution was then stirred at 200 rpm while being maintained at 70°C, and nitrogen bubbling was performed for 30 minutes. The nitrogen bubbling was then switched to nitrogen flow.

[0063] Radical polymerization was initiated by adding a separately prepared solution of 0.03 g of potassium peroxodisulfate (Fujifilm Wako Pure Chemical Corporation) dissolved in 3 g of purified water to the above mixture. The mixture was stirred at 200 rpm for 10 hours at 70 °C and then slowly cooled to room temperature. At this point, the contents of the 200 ml flask were sampled and the radical polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was essentially 100%. No agglomerates or other particles formed during the chemical reaction. The resin base particles 13 were purified by centrifugation, and the dispersion medium was replaced with a phosphate buffer solution containing 3% BSA and stored (the dispersion medium replacement was also performed by centrifugation). Dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.) of the resin base particles 13 revealed a number-average particle size of 224 nm.

[0064] (Resin base particle 14) A 200 ml flask was charged with 1.2 g of styrene (Kishida Chemical Co., Ltd.), 1.8 g of glycidyl methacrylate (Kishida Chemical Co., Ltd.), 0.04 g of divinylbenzene (Kishida Chemical Co., Ltd.), and 100 g of ion-exchanged water to obtain a mixed solution. The mixed solution was then stirred at 200 rpm while being maintained at 70°C, and nitrogen bubbling was performed for 30 minutes. The nitrogen bubbling was then switched to nitrogen flow.

[0065] Radical polymerization was initiated by adding a separately prepared solution of 0.06 g of V-50 (Fujifilm Wako Pure Chemical Corporation) dissolved in 3 g of purified water to the above mixture. Two hours after the start of polymerization, 0.3 g of glycidyl methacrylate was added to the radical polymerization reaction field. The mixture was then stirred at 200 rpm for an additional 8 hours at 70 °C, after which it was slowly cooled to room temperature. At this point, the contents of the 200 ml flask were sampled, and the radical polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was essentially 100%.

[0066] Next, a previously prepared aqueous solution of glycine (1 / 10 moles of glycine relative to the moles of glycidyl methacrylate, prepared using triethylamine (Kishida Chemical Co., Ltd.) and 2N hydrochloric acid solution to a pH of 7) was added. The mixture was then stirred at 200 rpm for 3 hours at room temperature, and the contents of the 200 ml flask were adjusted to pH 10 using triethylamine. The temperature was then raised to 70°C and the mixture was stirred at 200 rpm for another 3 hours, allowing the epoxy groups derived from glycidyl methacrylate and the amino groups derived from glycine to chemically react. Next, the contents of the 200 ml flask were adjusted to pH 1.5 using 2N hydrochloric acid solution and then stirred at 70°C for another 3 hours at 200 rpm. This chemically reacted the unreacted epoxy groups with water, converting them to glycols, yielding resin base particles 14 having carboxyl groups as reactive functional groups. During the chemical reaction, aggregates formed, which were removed by decantation. The resin base particles 14 were then purified by centrifugation, and the dispersion medium was replaced with a phosphate buffer solution and stored (the replacement of the dispersion medium was also performed by centrifugation). The resin base particles 14 were evaluated using dynamic light scattering (DLS-8000: Otsuka Electronics Co., Ltd.), and the number-average particle size was found to be 222 nm.

[0067] An example of preparing a dispersion of activated resin particles is described below. The sensitization rates of the antibodies used for activation are shown in Table 1.

[0068] [Example of preparation of activated resin particle dispersion liquid activated by chemical adsorption] For each of Resin Base Particles 1 to 6 and Resin Base Particle 14, 180 μL of a 0.017% by mass water suspension was placed in a 1.5 mL microtube. 90 μL of a 0.1% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 0.1% aqueous solution of N-hydroxysulfosuccinimide sodium were added. The mixture was stirred at room temperature for 30 minutes to obtain a dispersion of resin particles with activated carboxy groups.

[0069] After centrifugation and washing, 270 μL of 10 mM HEPES at pH 7.2 was added, and the resin particles with activated carboxy groups were dispersed by ultrasonication. 5 μL of a 4.9 mg / mL dispersion of clone C5 (Funakoshi Corporation), a monoclonal mouse anti-human C-reactive protein (CRP antibody), was added to the mixture. Stirring at room temperature for 3 hours yielded activated resin particles (hereafter referred to as activated resin particles) through antibody sensitization to the carboxyl-activated resin particles. After centrifugation and washing, each activated resin particle was dispersed in 10 mM HEPES buffer (pH 7.9) supplemented with 0.05% Tween 20 to prepare an activated resin particle dispersion. The CRP antibody sensitization rate for each activated resin particle is also shown in Table 1. Note that for activated resin particle dispersion 14, 0.5 mg / mL BSA was further added to prepare the dispersion because the dispersibility was insufficient.

[0070] [Example of preparation of activated resin particle dispersion liquid activated by physical adsorption] Resin base particles 7 to 13 were each dispersed in 270 μL of 10 mM HEPES (pH 7.2) to a particle concentration of 0.017% by mass. The resulting solution was placed in a 1.5 mL microtube, and 5 μL of a 4.9 mg / mL dispersion of CRP antibody clone C5 (Funakoshi Co., Ltd.) was added and stirred at room temperature for 1 hour. After centrifugally washing these resin base particles, a blocking treatment was performed by adding a 1 mg / mL BSA aqueous solution to obtain activated resin particles. After centrifugally washing the activated resin particles, activated resin particle dispersions 7 to 13 were prepared, respectively. The CRP antibody sensitization rate for each activated resin particle is also shown in Table 1. To evaluate the CRP antibody sensitization rate, a resin particle dispersion was simultaneously prepared by performing the same procedure without adding the CRP antibody.

[0071] [Table 1]

[0072] [Preparation of buffer solutions 1 to 26] As shown in Table 2, buffer solutions 1 to 26 were prepared by mixing each buffer and additive in a predetermined amount. The pH of each buffer solution is also shown in Table 2. Aqueous sodium hydroxide solution or aqueous hydrochloric acid solution was used to adjust the pH. [Table 2]

[0073] [Performance evaluation] Using the resin base particles, activated resin particle dispersion, and buffer solution shown in the above Production Examples, Evaluations 1 and 2 described below were carried out. Tables 3 and 4 show the mass ratio W(A) / W(B) of the content W(A) of the thiourea compound to the content W(B) of the resin particles in the total amount of the particle dispersion and buffer solution.

[0074] (Evaluation 1: Evaluation of non-specific adsorption to normal human specimens) Resin base particles 1 to 14 were dispersed in 10 mM pH 7.9 HEPES (containing 0.05% Tween 20) to a concentration of 0.1% by mass to prepare a dispersion (Liquid P). Next, 55 μL of a specimen dilution solution (Liquid Q) consisting of human normal specimen A (serum specimen, 5 μL) and one of buffer solutions 1 to 25 (50 μL) in Table 2, and 55 μL of a specimen dilution solution (Liquid Q') consisting of human normal specimen A (serum specimen, 5 μL) and buffer solution 26 (50 μL) were prepared.

[0075] Next, for Examples 1 to 32 and Comparative Examples 1 to 4, the mixed solution obtained by stirring the Q solution or the Q' solution was allowed to stand at 37°C for 5 minutes. After standing, the P solution (50µL) was added to the Q solution (55µL) or the Q' solution (55µL), and immediately thereafter, the absorbance at a wavelength of 572nm was measured. Here, the mixed solution obtained by adding the P solution to the Q solution was designated the PQ solution, and the mixed solution obtained by adding the P solution to the Q' solution was designated the PQ' solution. After that, each mixed solution was allowed to stand for another 5 minutes at 37°C, and the absorbance at a wavelength of 572nm was measured again, and the change in absorbance ΔABS was calculated.

[0076] For Examples 33 and 34, Solution P (50 μL) was mixed with Solution Q (55 μL) or Solution Q' (55 μL) at 25° C., and the absorbance was measured immediately afterwards. After allowing to stand for another 5 minutes at 37° C., the absorbance at a wavelength of 572 nm was measured again, and the change in absorbance ΔABS was calculated.

[0077] Absorbance measurements were performed using an Eppendorf Biospectrometer. The reduction in ΔOD of the PQ solution relative to the ΔOD of the PQ' solution, which contained no additives other than Tween 20, was evaluated as the reduction rate. This allowed for a comparative evaluation of whether combinations of each resin base particle dispersion and buffer solution containing each additive could suppress nonspecific agglutination between the sample and the resin base particles. A high reduction rate is interpreted as suppressing nonspecific adsorption. High nonspecific agglutination may lead to false positives in normal samples when used as a latex agglutination test reagent. The threshold values ​​in Table 3 were determined based on the noise risk when attempting to detect low concentrations of target substances using latex agglutination. A rank E improvement rate of less than 10% raises the risk of false positives in normal samples. The normal human sample A used in Evaluation 1 was previously evaluated and found to exhibit nonspecific adsorption to sulfur-containing resin base particles. Furthermore, the same ranking tendency as in Evaluation 1 was observed for the antibody-sensitized activated resin particle dispersion. A: 70% or more B: 50% or more but less than 70% C: 30% or more but less than 50% D: 10% or more but less than 30% E: Less than 10%

[0078] [Table 3]

[0079] (Evaluation 2: Evaluation of standard serum) The CRP standard serum was diluted with HEPES buffer (pH 7.2) to a concentration of 0.75 mg / dL to prepare a CRP sample solution. 1 μL of this CRP sample solution was mixed with 50 μL of each of the buffer solutions used in preparing Q solution in Evaluation 1 to prepare a mixture (hereinafter referred to as R1+) and keep it at 37°C. As a control, 1 μL of HEPES buffer (pH 7.2) was mixed with 50 μL of each of the buffer solutions used in preparing Q solution in Evaluation 1 to prepare a mixture (hereinafter referred to as R1-) and keep it at 37°C.

[0080] Next, R1+ was mixed with 50 μL of each of the activated resin particle dispersions 1 to 14 shown in Table 1 (particle concentration 0.1% by mass, referred to as R2), which had been thoroughly dispersed again using ultrasonic waves before use. Similarly, R2 and R1- were mixed.

[0081] The absorbance at 572 nm of the mixed solution (volume 101 μL) immediately after stirring was measured using an Eppendorf Biospectrometer. The mixed solution was then allowed to stand at 37°C for 5 minutes, after which the absorbance at 572 nm was measured again, and the change in absorbance, ΔABS × 10,000, was calculated.

[0082] A large value for R1- indicates that the resin particles are undergoing aggregation due to nonspecific adsorption or osmotic aggregation. In this case, if the particles are used in specimen testing as latex agglutination particles, there is a concern that normal samples may be interpreted as false positives due to noise. It was confirmed that no such aggregation was observed in any of the combinations containing activated resin particles (activated resin particle dispersions 1-12) and buffer solutions (buffer solutions 1-22) evaluated in this example. On the other hand, the combinations containing activated resin particles (activated resin particle dispersion 14) or buffer solutions (buffer solutions 23-25) evaluated in the comparative example showed large values, indicating aggregation. Resin particles with a higher R+ value are expected to be able to detect low concentrations of target substances with high sensitivity when used as particles for latex agglutination testing in specimen testing. The results are summarized in Table 4. If the R+ value is less than 1000 (rank E), it may not be possible to detect sufficiently low concentrations of target substances, and there is a concern that it may be interpreted as a false negative. AA: 1750 or more A: 1500 or more and less than 1750 B: 1350 or more and less than 1500 C: 1200 or more and less than 1350 D: 1000 or more and less than 1200 E: Less than 1000

[0083] [Table 4]

[0084] The disclosure of this embodiment includes the following configurations and methods. (Configuration 1) Contains a thiourea compound and resin particles, An in vitro diagnostic test reagent, wherein the resin particles contain elemental sulfur. (Configuration 2) The test reagent according to the above aspect 1, wherein the thiourea compound comprises a compound represented by the following formula (1): [ka] (In formula (1), R 1 ~R 4 each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an allyl group, an acetyl group, or a group represented by the following formula (2): [ka] (In formula (2), * indicates the bonding position in the compound represented by formula (1).) (Configuration 3) 3. The test reagent according to claim 1, wherein the content of the thiourea compound in the test reagent is 0.0005M or more and 2M or less. (Configuration 4) 4. The test reagent according to any one of configurations 1 to 3, wherein the thiourea compound contains thiourea. (Configuration 5) 5. The test reagent according to any one of configurations 1 to 4, characterized in that it contains at least one buffer selected from the group consisting of MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPSO, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, and CAPS. (Configuration 6) 6. The test reagent according to any one of configurations 1 to 5, further comprising a chelating agent. (Configuration 7) 7. The test reagent according to any one of configurations 1 to 6, wherein the amount of sulfur element in the resin particles as determined by an X-ray photoelectron analyzer is 0.1 atm % or more and 8.0 atm % or less. (Configuration 8) 8. The test reagent according to any one of configurations 1 to 7, wherein the amount of sulfur element in the resin particles as determined by an X-ray photoelectron analyzer is 0.5 atm % or more and 6.0 atm % or less. (Configuration 9) 9. The test reagent according to any one of configurations 1 to 8, wherein the resin particles have an antibody or an antigen. (Configuration 10) 10. The test reagent according to any one of configurations 1 to 9, wherein the resin particles have a polymer having a structure derived from at least one of styrenes and (meth)acrylates in its main chain. (Configuration 11) 11. The test reagent according to any one of configurations 1 to 10, wherein the resin particles have a polymer having at least one of a sulfide group and a thiol group. (Configuration 12) 12. The test reagent according to any one of configurations 1 to 11, wherein the resin particles comprise a polymer having a hydroxy group. (Configuration 13) 13. The test reagent according to any one of configurations 1 to 12, wherein the resin particles have a polymer having at least one of a carboxy group and a structure derived from a carboxy group. (Configuration 14) 14. The test reagent according to any one of configurations 1 to 13, characterized in that the mass ratio W(A) / W(B) is 0.2 or more and 450 or less, where W(A) is the content of the thiourea compound in the test reagent and W(B) is the content of the resin particles in the test reagent. (Configuration 15) The test reagent according to any one of configurations 1 to 14, characterized in that the test reagent is a test reagent composed of two or more liquids including a buffer solution and a resin particle dispersion, the buffer solution does not contain resin particles but contains the thiourea compound, and the resin particle dispersion contains the resin particles. (Configuration 16) 16. The test reagent according to claim 15, wherein the pH of the buffer solution is 6.0 or higher and 9.0 or lower. (Configuration 17) 17. The test reagent according to any one of configurations 1 to 16, characterized in that it is used in a test by latex agglutination. (Configuration 18) A method for detecting a target substance in a specimen by in vitro diagnosis, comprising a step of mixing a test reagent according to any one of configurations 1 to 17 with a specimen that may contain the target substance.

Claims

1. Contains a thiourea compound and resin particles, An in vitro diagnostic test reagent, wherein the resin particles contain elemental sulfur.

2. 2. The test reagent according to claim 1, wherein the thiourea compound comprises a compound represented by the following formula (1): 【Chemistry 1】 (In formula (1), R １ ~R ４ each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an allyl group, an acetyl group, or a group represented by the following formula (2): 【Chemistry 2】 (In formula (2), * indicates the bonding position in the compound represented by formula (1).)

3. 2. The test reagent according to claim 1, wherein the content of the thiourea compound in the test reagent is 0.0005 M or more and 2 M or less.

4. 2. The test reagent according to claim 1, wherein the thiourea compound comprises thiourea.

5. 2. The test reagent according to claim 1, comprising at least one buffer selected from the group consisting of MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPSO, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, and CAPS.

6. The test reagent according to claim 1 , further comprising a chelating agent.

7. 2. The test reagent according to claim 1, wherein the amount of sulfur element in the resin particles as determined by an X-ray photoelectron analyzer is 0.1 atm % or more and 8.0 atm % or less.

8. 2. The test reagent according to claim 1, wherein the amount of sulfur element in the resin particles as determined by an X-ray photoelectron analyzer is 0.5 atm % or more and 6.0 atm % or less.

9. The test reagent according to claim 1 , wherein the resin particles have an antibody or an antigen.

10. 2. The test reagent according to claim 1, wherein the resin particles comprise a polymer having a structure derived from at least one of styrenes and (meth)acrylates in its main chain.

11. 2. The test reagent according to claim 1, wherein the resin particles comprise a polymer having at least one of a sulfide group and a thiol group.

12. 2. The test reagent according to claim 1, wherein the resin particles contain a polymer having a hydroxy group.

13. 2. The test reagent according to claim 1, wherein the resin particles have a polymer having at least one of a carboxy group and a structure derived from a carboxy group.

14. The test reagent according to claim 1, characterized in that, when the content of the thiourea compound in the test reagent is W(A) and the content of the resin particles in the test reagent is W(B), the mass ratio W(A) / W(B) is 0.2 or more and 450 or less.

15. The test reagent according to claim 1, characterized in that the test reagent is composed of two or more liquids, including a buffer solution and a resin particle dispersion, the buffer solution does not contain resin particles but contains the thiourea compound, and the resin particle dispersion contains the resin particles.

16. 16. The test reagent according to claim 15, wherein the buffer solution has a pH of 6.0 or more and 9.0 or less.

17. 2. The test reagent according to claim 1, which is used in a test by latex agglutination.

18. A method for detecting a target substance in a specimen by in vitro diagnosis, comprising the step of mixing a test reagent according to any one of claims 1 to 17 with a specimen that may contain the target substance.

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