Particles, reagents, test kits, and methods for detecting target substances for immunoturbidimetry.

Abstract

This invention provides particles, reagents, and a test kit for immunoturbidimetry that can detect trace components in low-concentration ranges while suppressing changes in their dispersion stability over time in water, as well as a method for detecting target substances. [Solution] Immunoturbidimetry particles containing a first resin and titanium dioxide, comprising a first layer having the first resin and a second layer having the titanium dioxide, wherein the second layer is located inside the first layer, and the first resin has either a structure represented by formula (1-1) or a structure represented by a specific formula. JPEG2026094927000017.jpg5646

Description

[Technical Field]

[0001] The present invention relates to particles for immunoturbidimetry, reagents, test kits, and methods for detecting target substances. [Background technology]

[0002] In recent years, immunoturbidimetric testing has attracted attention as a simple and rapid immunoassay method. Immunoturbidimetric testing is known to involve mixing a dispersion of particles having antibodies or antigens on their surface as ligands with a sample that may contain a target substance (antigen or antibody). When the sample contains the target substance (antibody or antigen), the particles undergo an agglutination reaction. This agglutination reaction can be optically detected as changes in scattered light intensity, transmitted light intensity, absorbance, etc., to identify the presence or absence of disease.

[0003] Traditionally, polystyrene-based latex particles, primarily composed of polystyrene, have been used as particles for immunoturbidimetric methods because they are easy to sensitize (immobilize) with antigens or antibodies, are relatively inexpensive, and their polymerization reactions are easy to control. However, immunoturbidimetric methods using polystyrene latex particles sometimes had the problem of failing to detect trace components in the low-concentration range. Therefore, there was a need to develop particles that were more sensitive than polystyrene latex particles.

[0004] To solve the above problem, it is necessary to increase the absorbance change due to particle aggregation associated with immune complex formation. Since the absorbance change is known to be governed by the refractive index of the components constituting the particle, particle development using high refractive index materials is being pursued. For example, particle development is underway using titanium dioxide, which is well known as a high refractive index material. Patent document 1 proposes immunoturbidimetry particles in which titanium dioxide fine particles are coated with a polyethylene glycol analog. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2017-1222684 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] When titanium dioxide is used as a particle for immunoturbidimetry, in addition to suppressing nonspecific adsorption, it is necessary to stably disperse the particles in water over a long period of time. Therefore, dispersion treatment with resins, i.e., surfactants or coatings, is performed. However, depending on the type of resin used, desorption of the resin may occur, and the dispersion stability of the particles in water may decrease over time. This disclosure has been made in view of the background technologies and challenges. Specifically, the purpose of this disclosure is to provide immunoturbidimetric particles, reagents, test kits, and methods for detecting target substances that can detect trace components in the low-concentration range and suppress changes in dispersion stability in water over time. [Means for solving the problem]

[0007] In other words, this disclosure concerns the following: Particles for immunoturbidimetry containing a first resin and titanium dioxide, The first layer has the first resin, and the second layer has the titanium oxide, The aforementioned second layer is located inside the aforementioned first layer. Particles for immunoturbidimetry, characterized in that the first resin has at least one of the structures represented by the following formula (1-1) and the following formula (1-2). [ka] (In formula (1-1), R 1 This represents a hydrogen atom or a methyl group. R 2 (This indicates a structure having a hydroxyl group or a carboxyl group.) [ka] (In formula (1-2), R 3 represents a hydrogen atom or a methyl group. R 4 represents a structure having a hydroxy group or a structure having a carboxy group.)

[0008] Moreover, the reagent according to the present disclosure is a reagent characterized in that the particles for immunoturbidimetry are dispersed in an aqueous solution. Further, the test kit according to the present disclosure is a test kit characterized by including the above reagent and a container containing the above reagent. Moreover, the method for detecting a target substance according to the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnosis, and is characterized by mixing the above reagent and a specimen that may contain the target substance. Further, the method for detecting a target substance according to the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnosis, and includes a step of mixing a specimen that may contain the target substance with the above reagent to obtain a mixed solution, a step of irradiating the mixed solution with light, and a step of detecting at least one of transmitted light and scattered light from the light irradiated on the mixed solution.

Advantages of the Invention

[0009] According to the present disclosure, it is possible to provide particles for immunoturbidimetry containing titanium oxide that can detect trace components in a low concentration range and suppress the change over time in the dispersion stability in water.

Modes for Carrying Out the Invention

[0010] Hereinafter, embodiments of the present disclosure will be described in detail, but the technical scope of the present disclosure is not limited to these embodiments. The particles for immunoturbidimetry according to the present disclosure are particles for immunoturbidimetry containing a first resin and titanium oxide, having a first layer having the first resin and a second layer having titanium oxide, the second layer being inside the first layer, and the first resin having at least one of the structure represented by the following formula (1-1) and the structure represented by the following formula (1-2). [Chemical formula] (In formula (1-1), R 1 represents a hydrogen atom or a methyl group. R 2 represents a structure having a hydroxy group or a structure having a carboxy group.) [Chemical formula] (In formula (1-2), R 3 represents a hydrogen atom or a methyl group. R 4 represents a structure having a hydroxy group or a structure having a carboxy group.)

[0011] The inventors have found that in particles for immunoturbidimetry containing titanium oxide, by arranging a specific structure on the surface layer of the particles, a uniformly and firmly highly hydrophilic resin layer can be formed on the surface layer of the titanium oxide-containing particles. As a result, it has become possible to detect trace components in a low concentration region, suppress the change over time in the aqueous dispersion stability, and suppress non-specific adsorption.

[0012] The particles for immunoturbidimetry according to the present disclosure have a first layer containing a first resin having at least one of the structure represented by formula (1-1) and the structure represented by formula (1-2) on the outside of the second layer containing titanium oxide. The structure of the resin is not particularly limited as long as it has the structure represented by formula (1-1) or (1-2), but this structure is a vinyl polymer having a carboxy group or a hydroxy group in the side chain. By having a carboxyl group or a hydroxy group in the side chain, it is considered that hydrogen bonds can be formed with titanium oxide and good interaction can be achieved. Furthermore, by being a vinyl polymer, it is considered that there is no exposure of titanium oxide and uniform coating can be achieved. That is, by having the structure represented by formula (1-1) or formula (1-2), the coating resin (first resin) can interact well with titanium oxide and can be uniformly coated without exposure of titanium oxide. Therefore, desorption of the coating resin can be suppressed, reduction in the aqueous dispersion stability of the particles for immunoturbidimetry can be suppressed, and non-specific adsorption can be suppressed. Specific examples of the structure represented by formula (1-1) or formula (1-2) are shown in the following formulas (1-A-1) to (1-A-12), but are not limited thereto.

Chemical formula

[0013] Furthermore, it is preferable that the first resin has at least one of the structure represented by the following formula (2) and the structure represented by the following formula (3).

Chemical formula

[0014] Resins having structures shown in formulas (1-1) and (1-2), as well as structures shown in formulas (2) and (3), are vinyl polymers and contain alkoxysilanes. This is preferable because the interaction with titanium dioxide further suppresses the exposure of titanium dioxide, resulting in a uniform coating.

[0015] The immunoturbidimetric particles according to this disclosure are not particularly limited as long as they have the structure shown in formula (1-1) or formula (1-2). For example, they can be obtained by carrying out a polymerization reaction, i.e., seed polymerization, by coexisting titanium dioxide-containing particles and monomers in an aqueous medium and adding a polymerization initiator. The monomer is not particularly limited, but for example, it can be obtained by using glycidyl (meth)acrylate as a monomer, carrying out seed polymerization, and then carrying out a ring-opening reaction of the glycidyl group. In addition, the monomer may be used alone or as a mixture of two or more. The monomers to be mixed are not particularly limited, but if they have the structure shown in formula (2) or formula (3), they can be obtained by mixing monomers from which the structure shown in formula (2) or formula (3) is derived and carrying out seed polymerization by copolymerization of two or more monomers. The monomers from which the structure shown in formula (2) or formula (3) is derived are not particularly limited as long as they have the structure from which the structure shown in formula (2) or formula (3) is derived. Examples include vinyltrimethoxysilane, vinyltriethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane. These can be used individually or in combination of two or more.

[0016] Furthermore, the particles for immunoturbidimetry according to this disclosure are not particularly limited as long as they contain titanium dioxide in the second layer, but may also have a third layer containing a second resin inside the second layer containing titanium dioxide. The second resin according to this disclosure is not particularly limited, but is preferably high in refractive index (1.5 or higher). Preferred specific structures for this purpose include styrene structures, fluorene structures, dinaphthothiophene structures, naphthalene structures, anthracene structures, and phenanthrene structures.

[0017] Particles having a third layer containing a second resin inside a second layer containing titanium dioxide can be obtained, for example, by a hydrolysis reaction carried out in the presence of the second resin, a metal oxide precursor, an oxygen-containing organic solvent, and water, i.e., by the sol-gel method.

[0018] The preferred range for volume-average particle size of the particles used in immunoturbidimetry according to this disclosure is 150 nm to 1000 nm, and more preferably 180 nm to 800 nm. Furthermore, the preferred range for particle size distribution of the particles used in immunoturbidimetry is a value of 1.00 to 1.25 for the ratio of volume-average particle size to number-average particle size.

[0019] The titanium dioxide content in the immunoturbidimetric particles according to this disclosure is preferably 10% by mass or more and 98% by mass or less, and more preferably 20% by mass or more and 90% by mass or less. When the titanium dioxide content is 98% by mass or less, the titanium dioxide is less likely to be exposed, and nonspecific adsorption can be further suppressed. Furthermore, when the content is 10% by mass or more, the effect of including titanium dioxide is fully exhibited, and it is excellent for detecting trace components in the low concentration range.

[0020] The immunoturbidimetric particles according to this disclosure may be pre-sensitized particles without ligands attached, or they may be affinity particles having ligands further attached to their surface. The affinity particles have a selective or specific high affinity for the target substance due to the ligand attached to the particle surface. In particular, it is preferable that the ligand is attached to the surface of the particle by chemical bonding.

[0021] In this disclosure, a ligand is a compound that specifically binds to a receptor on a particular target substance. The binding site of the ligand to the target substance is fixed, and it has a selective or specific high affinity. Examples include antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, and nucleic acids, but ligands in this disclosure are not limited to these. Examples of nucleic acids include deoxyribonucleic acid. In this disclosure, affinity particles have a selective or specific high affinity for the target substance. It is preferable that the ligand in this disclosure is an antibody, an antigen, or a nucleic acid.

[0022] Furthermore, the reagents relating to this disclosure are reagents in which the immunoturbidimetric particles relating to this disclosure are dispersed in an aqueous solution. The reagent is not particularly limited in its target of testing. Examples of reagents include in vitro diagnostic medical devices, such as antigen detection reagents for testing antigens and antibody detection reagents for testing antibodies. The reagent may consist of pre-sensitized particles without ligands dispersed in an aqueous solution, with the expectation that the user will attach a desired ligand, such as an antibody, to the particles. Alternatively, it may consist of affinity particles with a ligand already attached, dispersed in an aqueous solution, with a specific target substance in mind.

[0023] The reagents relating to this disclosure comprise immunoturbidimetric particles (pre-sensitization particles or affinity particles) relating to this disclosure and a dispersion medium for dispersing the immunoturbidimetric particles. To the extent that the objectives of this disclosure can be achieved, the reagents relating to this disclosure may also include third substances such as solvents and blocking agents in addition to the immunoturbidimetric particles relating to this disclosure. Two or more types of third substances such as solvents and blocking agents may be included in combination. Examples of dispersion media used include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the dispersion media included in the reagents are not limited to these.

[0024] The test kit according to this disclosure comprises the reagent according to this disclosure and a container containing the reagent. In addition to the reagent described above (hereinafter referred to as Reagent 1), the test kit may also include a reaction buffer (hereinafter referred to as Reagent 2). Reagent 1 and Reagent 2, or either one, may contain a sensitizer. Furthermore, in addition to Reagent 1 and Reagent 2, the test kit according to this disclosure may also include a positive control, a negative control, a serum diluent, a primary antibody, and a secondary antibody. As the medium for the positive control and negative control, serum, physiological saline, or a solvent may be used, provided that the target substance that can be measured is not present.

[0025] The method for detecting a target substance according to this disclosure is a method for detecting a target substance in a sample by in vitro diagnostics, and may involve mixing the reagent according to this disclosure with a sample that may contain the target substance. Alternatively, the method for detecting a target substance according to this disclosure is a method for detecting a target substance in a sample by in vitro diagnostics, and may include the steps of: obtaining a mixture by mixing the reagent according to this disclosure with a sample that may contain the target substance; irradiating the mixture with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture.

[0026] (Method for measuring the titanium dioxide content in particles used for immunoturbidimetry) This disclosure describes an example of a method for measuring the titanium dioxide content in particles used for immunoturbidimetry. The titanium dioxide content in the particles is measured using a thermomass spectrometer. For example, STA200 (manufactured by Hitachi High-Tech Science) is used. 5 mg to 10 mg of the cured material is weighed into an aluminum pan and measured under a nitrogen atmosphere. The temperature conditions are to hold at 30°C for 30 minutes, then increase the temperature from 30°C to 500°C at a rate of 10°C / min, and then hold at 500°C for 10 minutes. The titanium dioxide content in the particles can be measured from the mass decrease of the resin component at 300°C.

[0027] (Method for measuring volume-average particle size and particle size distribution in an aqueous dispersion of particles for immunoturbidimetry) An example of a method for measuring the volume-average particle size (Dv) of particles for immunoturbidimetry in this disclosure is described. The Dv of particles present in an aqueous dispersion is measured by dynamic light scattering. For example, a zetasizer (zetasizer ultra: Malvern Panalytical) is used and the measurement is performed at 25°C. Furthermore, the particle size distribution of the particles used for immunoturbidimetry in this disclosure is calculated by measuring the number-average particle size (Dn) using the dynamic light scattering method described above, and then calculating it as the ratio of Dv to Dn (Dv / Dn). [Examples]

[0028] The present disclosure will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0029] [Examples of particle production] (Resin-containing third layer formation process) A mixture was prepared by weighing 12.68 g of styrene (St: Kishida Chemical Co., Ltd.), 0.23 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.), and 1512.02 g of deionized water into a 2 L four-neck separable flask. This mixture was maintained at 70°C while stirring at 140 rpm, and the inside of the four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Next, a solution prepared separately by dissolving 0.55 g of V-50 (Fujifilm Wako Pure Chemical Corporation) in 20 g of deionized water was added to the mixture to initiate soap-free polymerization. After 23 hours of reaction from the start of polymerization, a dispersion of third-layer forming resin particles 1, consisting of a copolymer of styrene and divinylbenzene, was obtained. A portion of this dispersion was taken and evaluated using dynamic light scattering (Zetasizer Ultra: Malvern Panalytical), and the volume-average particle size was found to be 190 nm.

[0030] (Titanium dioxide-containing second layer formation process) Third layer forming resin particle 1 was prepared with deionized water to a solid content concentration of 0.6%. 20 g of this dispersion was mixed with 404.50 g of ethanol (Kishida Chemical Co., Ltd.) containing 0.2% polyvinylpyrrolidone K-30 (PVP K-30: Kishida Chemical Co., Ltd.), and the mixture was maintained at 70°C while stirring at 140 rpm. Next, a solution prepared by mixing 5.0 ml of titanium(IV)n-butoxide monomer (TBOT: Kishida Chemical Co., Ltd.), which had been prepared separately, with 197.50 g of ethanol was added to the above mixture to initiate the sol-gel reaction. The reaction was allowed to proceed for 24 hours from the start of the sol-gel reaction, and the particles dispersed in the above mixture were separated by centrifuge and redispersed in ethanol. Further separation of the particles from the above dispersion by centrifuge and redispersion of the particles in deionized water was repeated twice to purify the mixture and obtain second layer forming particle 1. The second layer-forming particles 1 were stored in an aqueous dispersion adjusted to a final concentration of 5.0% by mass. A portion of this dispersion was taken, and the dynamic light scattering of the second layer-forming particles 1 was evaluated, revealing a volume-average particle size of 200 nm. Furthermore, the metal oxide content was evaluated using differential thermal-thermogravimetric analysis (NEXTA STA200RV: Hitachi High-Tech Corporation), which showed it to be 45% of the particle weight.

[0031] (Resin-containing first layer formation process) A dispersion of second-layer-forming particles 1 with a solid content of 0.2% was prepared using deionized water to a volume of 149.55 g. 0.135 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) and 0.015 g of 3-methacryloxypropyltrimethoxysilane (MPS: Shin-Etsu Chemical Co., Ltd.) were added, and the mixture was maintained at 70°C while stirring at 100 rpm. The four-necked separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Then, a solution of 0.03 g of V-50 dissolved in 0.3 g of deionized water, which had been prepared separately, was added to the above mixture to initiate shell formation. After stirring continued for 18 hours after the start of the reaction, a dispersion containing first-layer-forming particles 1 was obtained.

[0032] (Process for imparting reactive functional groups) An aqueous solution containing pre-prepared mercaptosuccinic acid (MSA: Wako Pure Chemical Industries, Ltd.) was added to a dispersion containing the first-layer formed particle 1 (the total number of moles of MSA was equal to the number of moles of the glycidyl methacrylate). Triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, the mixture was heated to 70°C while stirring at 800 rpm, and maintained at this temperature for 18 hours to obtain a particle dispersion. The particles were separated from the dispersion using a centrifuge, and the particles were further purified by repeating the process of redispersing them in ion-exchanged water eight times to obtain particle 1. The aqueous dispersion was stored in a state where particle 1 was finally adjusted to 5.0% by mass.

[0033] (Preparation of particle 2) In the resin-containing first layer formation step, instead of adding 0.135 g of glycidyl methacrylate and 0.015 g of 3-methacryloxypropyltrimethoxysilane, 0.15 g of glycidyl methacrylate was added. Otherwise, the dispersion of particle 2 was obtained using the same procedure as for particle 1.

[0034] (Preparation of particle 3) 0.6 g of titanium dioxide particles TTO-55 (Ishihara Sangyo Co., Ltd.), 10.1 g of 28% aqueous ammonia (Kanto Chemical Co., Ltd.), 45 g of ethanol (Kishida Chemical Co., Ltd.), 44 g of pure water, and 0.3 g of 3-methacryloxypropyltrimethoxysilane (MPS: Shin-Etsu Chemical Co., Ltd.) were mixed. Using a TK homomixer (Primix Co., Ltd.), the mixture was dispersed at 12,000 rpm for 1 hour to obtain a titanium dioxide microparticle dispersion. The titanium dioxide microparticles and supernatant were separated from the dispersion using a centrifuge, and then the supernatant was redispersed with an equal mass of ion-exchanged water to obtain a purified titanium dioxide microparticle dispersion.

[0035] (Resin-containing first layer formation process) A purified titanium dioxide fine particle dispersion, whose concentration was adjusted by adding 80g of pure water, was deoxygenated using a nitrogen flow. 1.74g of styrene (St: Kishida Chemical Co., Ltd.) and 0.174g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.) were added, and the temperature was raised to 70°C. The styrene layer was initiated by adding a solution of 0.04g of ammonium persulfate (Kishida Chemical Co., Ltd.) dissolved in 1g of deionized water to the above mixture. After stirring for 18 hours, 0.3g of glycidyl methacrylate and a solution of 0.01g of ammonium persulfate (Kishida Chemical Co., Ltd.) dissolved in 1g of deionized water were added. The reaction was continued for another 12 hours to obtain a dispersion containing the first layer-forming particles 3.

[0036] (Imparting of reactive functional groups) A dispersion of particle 3 was obtained using the same method as for particle 1, except that particle 3 was used instead of particle 1, the first layer forming particle. The obtained particles 1 to 3 have the structure shown by formula (1-A-9).

[0037] [Example of comparative particle production] (Preparation of comparison particles) Titanium tetraethoxide was added to an acetonitrile / ethanol solution to prepare a 0.1 M titanium tetraethoxide solution. Ethanol and 0.1 M aqueous ammonia were mixed with this solution and stirred at room temperature for 60 minutes to allow sufficient hydrolysis. After hydrolysis, the mixture was stirred at 80°C for more than 3 hours, heated under reflux, and then centrifuged to adjust the concentration to approximately 20% solid content to obtain a titanium dioxide particle dispersion. Next, 5 ml of water was added to 1 g of a copolymer of polyoxyethylene-monoallyl-monomethyl ether and maleic anhydride (NOF Corporation). The solution obtained after hydrolysis and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed with ultrapure water to concentrations of 50 mg / ml and 50 mM, respectively. 4-aminosalicylic acid (Wako Pure Chemical Industries, Ltd.) was mixed into the prepared solution to a concentration of 0.1 M and reacted by shaking at room temperature for 24 hours. After the reaction, the resulting solution was transferred to a Spectra / Pore CE dialysis tube (molecular weight cutoff = 3500, Spectrum Laboratories, Inc.) and dialyzed at room temperature for 24 hours. After dialyzation, dimethylformamide (DMF: Wako Pure Chemical Industries, Ltd.) was added to the freeze-dried powder to a concentration of 25 mg / ml and mixed to obtain a 4-aminosalicylic acid-bound PEG solution. Next, using DMF, the 4-aminosalicylic acid-bound PEG solution was adjusted to a final concentration of 0.6 mg / ml and the titanium dioxide particle dispersion to a final solid content of 0.5%, resulting in 20 ml of reaction solution. This reaction solution was heated at 130°C for 16 hours. After the reaction was complete, the reaction vessel was cooled to below 50°C, DMF was removed using an evaporator, and then distilled water was added to obtain comparative particles with the dispersant bound to the particle surface.

[0038] (Production of affinity particles) For the dispersion of particle 1, 300 μL of the dispersion (3 mg of particle solids) was diluted with deionized water to a solid content concentration of 1.0% by mass, and this was placed in a 1.5 mL microtube. To this, 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Tokyo Chemical Industries, Ltd.) and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium (Tokyo Chemical Industries, Ltd.) were added. This mixture was stirred at room temperature for 30 minutes to obtain an activated particle dispersion containing carboxyl groups (activated particle dispersion). After centrifugal washing, 270 μL of pH 5.5 phosphate buffer-physiological saline (PBS) was added, and the particles with activated carboxyl groups were dispersed using ultrasound. To this, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.12 mg of antibody) was added, and the mixture was stirred at room temperature for 3 hours to obtain antibody-sensitized affinity particles 1. Affinity particles 2 and 3, as well as the comparative affinity particle, were prepared using the same experimental procedure as for affinity particle 1, except that the particle types were changed to particles 2 and 3, and the comparative particle, respectively.

[0039] (Preparation of the first reagent) The first reagent was prepared by dissolving 50 mM HEPES, 0.05% by mass TritonX-100, and 1.0% by mass sodium chloride (Kishida Chemical Co., Ltd.) in deionized water.

[0040] (Preparation of the second reagent) Affinity particles 1 were centrifuged and washed, then redispersed in 500 μL of buffer (HEPES buffer) containing 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (Triton X-100, Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​dissolved in deionized water. Subsequently, the mixture was mixed and diluted with HEPES buffer until affinity particles 1 comprised 0.1% by mass to obtain the second reagent 1. Second reagent 2, second reagent 3, and comparative second reagent were prepared using the same experimental procedure as for second reagent 1, except that the types of particles were changed to affinity particles 2, affinity particles 3, and comparative affinity particles, respectively.

[0041] (Measurement of change in absorbance) Samples were prepared with ferritin concentrations of 0.0 ng / mL (physiological saline) and 100 ng / mL. 15 μL of each sample was prepared, and 60 μL of the first reagent 1 was added to each to create a mixed solution. This mixture was incubated at 37°C for 290 seconds. Next, 30 μL each of the second reagents 1-3 and comparative second reagent were added to the mixture, and the absorbance was measured after 42 seconds of stirring. Furthermore, this mixture was allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance measured after 42 seconds of stirring was defined as the absorbance change ΔABS. Absorbance measurements were performed using an Eppendorf BIOSPECTROMETER spectrophotometer at a wavelength of 572 nm. As a result, all of the second reagents showed a difference in absorbance change ΔABS between the ferritin concentration of 0.0 ng / mL (physiological saline) and the ferritin concentration of 100 ng / mL, indicating favorable results.

[0042] (Calculation of the water dispersion stability index) A mixture was prepared by mixing 15 μL of lipic acid solution, consisting of triolein, lecithin, free fatty acids, bovine albumin, and Tris buffer, with 60 μL of the first reagent 1. This mixture was incubated at 37°C for 290 seconds. Next, 30 μL each of the second reagents 1-3 and comparative second reagent were mixed into the mixture, and the absorbance was measured after 42 seconds of stirring. Furthermore, these mixtures were allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance measured after 42 seconds of stirring was defined as the absorbance change ΔABS. Absorbance measurements were performed using an Eppendorf BIOSPECTROMETER spectrophotometer at a wavelength of 572 nm. After storing each second reagent at 4°C for 30 days, the same evaluation was performed, and the water dispersion stability index was calculated using the following formula with the initial absorbance change ΔABS (initial) and the absorbance change ΔABS after 30 days (after 30 days). Table 2 shows the physical properties and water dispersion stability index evaluations of the affinity particles used in each example and comparative example. Dispersion stability index in water = |ΔABS(after 30 days)-ΔABS(initial)| / ΔABS(initial) The evaluation was based on the value of the water dispersion stability index, as follows: A: The water dispersion stability index is less than 0.05. B: Dispersion stability index in water is 0.05 or higher and less than 0.20 C: Dispersion stability index in water is 0.20 or higher.

[0043] [Table 1]

[0044] As described above, the immunoturbidimetry particles 1 to 3 of this disclosure exhibit excellent dispersion stability in water. On the other hand, Comparative Example 1 exhibited poor dispersion stability in water.

[0045] This embodiment includes the following configurations and methods. (Composition 1) Particles for immunoturbidimetry containing a first resin and titanium dioxide, The first layer has the first resin, and the second layer has the titanium oxide, The aforementioned second layer is located inside the aforementioned first layer. Particles for immunoturbidimetry, characterized in that the first resin has at least one of the structures represented by the following formula (1-1) and the following formula (1-2). [ka] (In formula (1-1), R 1 This represents a hydrogen atom or a methyl group. R 2 (This indicates a structure having a hydroxyl group or a carboxyl group.) [ka] (In formula (1-2), R 3 This represents a hydrogen atom or a methyl group. R 4 (This indicates a structure having a hydroxyl group or a carboxyl group.) (Configuration 2) The immunoturbidimetric particle according to configuration 1, characterized in that the first resin further has at least one of the structures represented by the following formula (2) and the following formula (3). [ka] (In formula (2), R 5 This represents a hydrogen atom or a methyl group. n1 is an integer between 1 and 3 (inclusive), m1 is an integer between 0 and 2 (inclusive), and n1 + m1 is 3. *1 independently indicates bonding to a titanium atom or a silicon atom, or to a hydrogen atom, a methyl group, or an ethyl group. 6 (Each of these independently represents either a methyl group or an ethyl group.) [ka] (In formula (3), R 7 This represents a hydrogen atom or a methyl group. R 8 This represents a single bond, a phenylene group, or an alkylene group having 3 or fewer carbon atoms. n2 represents an integer between 1 and 3 (inclusive), m2 represents an integer between 0 and 2 (inclusive), and n2 + m2 is 3. *2 independently indicates bonding to a titanium atom or a silicon atom, or to a hydrogen atom, a methyl group, or an ethyl group. 9 Each of these independently represents either a methyl group or an ethyl group. (Composition 3) Particles for immunoturbidimetry according to configuration 1 or 2, characterized in that the titanium dioxide content in the particles is 10% by mass or more and 98% by mass or less. (Composition 4) A particle for immunoturbidimetry according to any one of configurations 1 to 3, characterized in that its volume-average particle size is 150 nm or more and 1000 nm or less. (Composition 5) A particle for immunoturbidimetry according to any one of configurations 1 to 4, characterized in that it has a third layer containing a second resin inside the second layer having titanium dioxide. (Composition 6) A particle for immunoturbidimetry according to any one of configurations 1 to 5, characterized in that it further has a ligand on its surface. (Composition 7) A reagent characterized in which immunoturbidimetric particles described in any of configurations 1 to 6 are dispersed in an aqueous solution. (Composition 8) A test kit characterized by comprising the reagent described in configuration 7 and a container containing the reagent. (Method 9) A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in configuration 7 with a specimen that may contain the target substance. (Method 10) A method for detecting a target substance in a specimen by in vitro diagnostics, comprising the steps of: mixing a specimen potentially containing the target substance with the reagent described in configuration 7 to obtain a mixture; irradiating the mixture with light; and detecting at least one of transmitted light and scattered light from the light irradiated onto the mixture.

Claims

1. Particles for immunoturbidimetry containing a first resin and titanium dioxide, The first layer has the first resin, and the second layer has the titanium oxide, The aforementioned second layer is located inside the aforementioned first layer. Particles for immunoturbidimetry, characterized in that the first resin has at least one of the structures represented by the following formula (1-1) and the following formula (1-2). 【Chemistry 1】 (In formula (1-1), R １ This represents a hydrogen atom or a methyl group. R ２ (This indicates a structure having a hydroxyl group or a carboxyl group.) 【Chemistry 2】 (In formula (1-2), R ３ This represents a hydrogen atom or a methyl group. R ４ (This indicates a structure having a hydroxyl group or a carboxyl group.)

2. The immunoturbidimetric particle according to claim 1, characterized in that the first resin further has at least one of the structures represented by the following formula (2) and the following formula (3). 【Transformation 3】 (In formula (2), R ５ This represents a hydrogen atom or a methyl group. n1 represents an integer between 1 and 3 (inclusive), m1 represents an integer between 0 and 2 (inclusive), and n1 + m1 is 3. *1 independently indicates bonding to a titanium atom or a silicon atom, or to a hydrogen atom, a methyl group, or an ethyl group. ６ (Each of these independently represents either a methyl group or an ethyl group.) 【Chemistry 4】 (In formula (3), R ７ This represents a hydrogen atom or a methyl group. R ８ This represents a single bond, a phenylene group, or an alkylene group having three or fewer carbon atoms. n² represents an integer between 1 and 3 (inclusive), m² represents an integer between 0 and 2 (inclusive), and n² + m² equals 3. *2 each independently represents being bonded to a titanium atom or a silicon atom, or represents a hydrogen atom, a methyl group, or an ethyl group. R ９ each independently represents a methyl group or an ethyl group.)

3. The particles for immunoturbidimetry according to claim 1, characterized in that the titanium dioxide content in the particles is 10% by mass or more and 98% by mass or less.

4. Particles for immunoturbidimetry according to claim 1, characterized in that the volume-average particle size is 150 nm or more and 1000 nm or less.

5. The immunoturbidimetric particle according to claim 1, characterized in that it has a third layer containing a second resin inside the second layer having titanium dioxide.

6. Particles for immunoturbidimetry according to claim 1, characterized in that they further have a ligand on their surface.

7. A reagent characterized in that the immunoturbidimetry particles described in claim 1 or 6 are dispersed in an aqueous solution.

8. A test kit comprising the reagent described in claim 7 and a container containing the reagent.

9. A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in claim 7 with a specimen that may contain the target substance.

10. A method for detecting a target substance in a specimen by in vitro diagnostics, comprising the steps of: mixing a specimen potentially containing the target substance with the reagent described in claim 7 to obtain a mixture; irradiating the mixture with light; and detecting at least one of transmitted light and scattered light from the light irradiated onto the mixture.

Images