Particles, reagents, test kits, and detection methods for immunoturbidimetry.

Abstract

This invention provides particles, reagents, test kits, and detection methods for immunoturbidimetry used for highly sensitive detection of target substances, including metal oxides. [Solution] Particles for immunoturbidimetry containing a metal oxide, characterized in that the refractive index of the particles is 1.70 or more and 2.25 or less, the volume-average particle size of the particles is 200 nm or more and 400 nm or less, and the product of the refractive index and the volume-average particle size is 340 nm or more and 780 nm or less.

Description

Technical Field

[0001] The present disclosure relates to particles for immunoturbidimetry, reagents, test kits, and detection methods.

Background Art

[0002] As a simple and rapid immunoassay method, immunoturbidimetry using particles can be mentioned. In this method, a dispersion of particles bound with a ligand having an affinity for a target substance is mixed with a specimen that may contain the target substance. At this time, since an aggregation reaction of the particles occurs according to the amount of the target substance contained in the specimen, the target substance can be qualitatively or quantitatively determined by optically detecting this aggregation reaction as a change amount such as scattered light intensity, transmitted light intensity, absorbance, etc. As a measure for improving the sensitivity for measuring a target substance in a low concentration range by immunoturbidimetry, methods such as increasing the particle size and increasing the refractive index have been carried out. As a method for increasing the refractive index, a method using a metal oxide having a high refractive index as a particle carrier has been proposed. In Patent Document 1, particles for immunoturbidimetry using amorphous titanium oxide having an average particle size of 80 nm to 140 nm are disclosed. Further, in Patent Document 2, particles for immunoturbidimetry using composite particles of titanium oxide fine particles and polystyrene particles are disclosed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] Further improvements in sensitivity were needed in immunoturbidimetry. However, according to the disclosure in Patent Document 1, sensitivity decreased when the particle size was 150 nm or larger. In addition, the titanium dioxide content of the particles disclosed in Patent Document 2 remained at 20% by mass or less. [Means for solving the problem]

[0005] Until now, no optimal design method has been proposed for using metal oxides with such high refractive indices as particle carriers. As a result of diligent research by the inventors, they discovered that when using metal oxide particles, there is a range of combinations in which both particle size and refractive index are increased that are effective for immunoturbidimetry, leading to this disclosure.

[0006] A first aspect of this disclosure for solving the above problem is: Particles for immunoturbidimetry containing metal oxides, The refractive index of the aforementioned particles is 1.70 or more and 2.25 or less. The volume-average particle size of the aforementioned particles is 200 nm or more and 400 nm or less. The particles for immunoturbidimetry are characterized in that the product of the refractive index and the volume-average particle size is 340 nm or more and 780 nm or less.

[0007] Furthermore, a second aspect of this disclosure is: The reagent is characterized in that the aforementioned immunoturbidimetric particles are dispersed in an aqueous solution.

[0008] Furthermore, a third aspect of this disclosure is: The test kit is characterized by comprising the aforementioned reagent and a container containing the reagent.

[0009] In addition, a fourth aspect of this disclosure is: A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent with the specimen which may contain the target substance.

[0010] Finally, the fifth aspect of this disclosure is: 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 a reagent to obtain a mixture; irradiating the mixture with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture. [Effects of the Invention]

[0011] According to this disclosure, in immunoturbidimetry, the absorbance is increased due to the agglutination reaction, and the detection of the target substance is made more sensitive. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows an example of an X-ray diffraction pattern according to this embodiment. [Modes for carrying out the invention]

[0013] The embodiments of this disclosure will be described in detail below, but the scope of the art is not limited to these embodiments. The immunoturbidimetric particles of this disclosure are particles containing metal oxides. Examples of metal oxides include titanium dioxide, aluminum oxide, zirconium oxide, and silica. Among these, titanium dioxide, which has a relatively low specific gravity and a high refractive index, is preferred. Titanium dioxide is not particularly limited, but can be produced by methods such as the sulfuric acid method, the chlorine method, or the sol-gel method. In the sol-gel method, a metal oxide containing titanium dioxide can be obtained by carrying out a hydrolysis reaction in the presence of a metal oxide precursor, an oxygen-containing organic solvent, and water.

[0014] Examples of metal oxide precursors include metal chlorides, metal acetates, metal alkoxides, and metal hydroxides. Among these, metal alkoxides, metal acetates, and metal hydroxides are preferred from the viewpoint of by-product impurities (e.g., chlorides). In particular, metal alkoxides represented by the chemical formula Mx(OR)y (where M represents a metal element, R represents an alkyl group, and x and y are each independently integers between 1 and 4), metal hydroxides represented by the chemical formula Mx(OH)y·nH2O (where M represents a metal element, x and y are each independently integers between 1 and 4, and n is an integer of 1 or more), and compounds containing the aforementioned metal alkoxides and / or metal hydroxides are preferred.

[0015] Examples of titanium-containing metal oxide precursors include titanium methoxide, titanium ethoxide, titanium-diisopropoxide bis(2,4-pentanedione), titanium-diisopropoxide bis(ethylacetoacetate), titanium-n-butoxide, titanium isopropoxide, titanium methoxypropoxide, titanium-n-nonyloxide, titanium-n-propoxide, titanium stearyl oxide, titanium triisostearyl isopropoxide, and titanium trimethylsiloxide.

[0016] The immunoturbidimetric particles of this disclosure have a refractive index of 1.70 or more and 2.25 or less, a volume-average particle size of 200 nm or more and 400 nm or less, and a product of the refractive index and volume-average particle size of 340 nm or more and 780 nm or less. The inventors diligently investigated the optimal design of particles containing metal oxides with a high refractive index for use in immunoturbidimetry. As a result, they found that high sensitivity can be obtained with particles that satisfy the refractive index and volume-average particle size requirements. Here, the refractive index is the value at the wavelength during immunoturbidimetry measurement. Furthermore, the wavelength during immunoturbidimetry measurement preferably includes 500 nm to 750 nm, with 572 nm being the most preferred.

[0017] The following theory is considered for the mechanism by which high sensitivity is obtained. In the particle size range for immunoturbidimetry, Mie scattering makes a large contribution. Based on the theoretical formula for Mie scattering, the particle extinction factor (Q) can be determined from the particle size and refractive index. ext The absorbance of a dispersion of certain particles can be calculated using the following formula (A).

number

[0018] Next, assuming that the particle size increases from the original particle size (r1) to the volume equivalent diameter (r2) when the particles aggregate, the absorbance difference (ΔAbs) during the aggregation reaction can be calculated using the following equation (B).

number

[0019] Based on this formula, the calculation of ΔAbs shows that for particle sizes of 150 nm or less, ΔAbs increases monotonically with respect to the refractive index. On the other hand, for particle sizes between 200 nm and 400 nm, ΔAbs has a maximum value at a certain refractive index, and it is shown that ΔAbs can become negative if the refractive index is too large. A negative value for ΔAbs indicates that the change in absorbance due to the aggregation reaction is negative. The inventors found, through their investigation, that the above formula is consistent with the observed trends.

[0020] <Degree of crystallinity of titanium dioxide> In the titanium dioxide particles for immunoturbidimetry according to this disclosure, the proportion of crystalline titanium dioxide (also referred to as the degree of crystallinity in this disclosure) is preferably 20% to 90%. The degree of crystallinity being within this range allows the refractive index to be controlled within the range defined in this disclosure. Amorphous titanium dioxide can be obtained by a method called the sol-gel method, which involves hydrolysis of a metal oxide precursor, as described above. Amorphous titanium dioxide can be crystallized by sintering or heating in water. Among these methods, heating in water is preferred because it can suppress the fusion of titanium dioxide particles. Furthermore, the degree of crystallinity of titanium dioxide in the particles can be controlled by changing conditions such as heating temperature and time. The crystalline state may have crystal structures such as anatase, brookite, or rutile, and may coexist with the amorphous state.

[0021] <Organic polymer> The immunoturbidimetry particles of this disclosure preferably contain an organic polymer. The inclusion of an organic polymer makes it possible to control the refractive index.

[0022] The first embodiment of the immunoturbidimetric particles of this disclosure contains an organic polymer and a metal oxide, and is characterized by having a first layer having a first organic polymer and a second layer having a metal oxide, the second layer being located outside the first layer. The organic polymer can be obtained by general polymer particle manufacturing methods such as emulsion polymerization, soap-free polymerization, dispersion polymerization, suspension polymerization, phase inversion emulsion, wet pulverization, and dry pulverization. Although not particularly limited, organic polymer particles obtained by emulsion polymerization and soap-free polymerization are preferred because they provide a desired volume-average particle size and a uniform particle size distribution for use in immunoturbidimetric particles. A first embodiment of the immunoturbidimetry particles of this disclosure may have a third layer having a second organic polymer outside a second layer having a metal oxide.

[0023] In the first form of the particles for immunoturbidimetry of the present disclosure, the first layer having the first organic polymer preferably contains a polymer having a structural unit represented by formula (1). The first layer can be a core layer.

Chemical formula

[0024] Formula (1) has a substituted or unsubstituted phenyl group or a naphthyl group in R 2 . The polymer having the structural unit represented by formula (1) has a relatively high refractive index among organic polymers. Therefore, by containing the polymer having the structural unit represented by formula (1), the refractive index can be controlled within the range defined in the present disclosure. <0))

[0025] Formula (1) is preferably a structure represented by formula (1-A). By being formula (1-A), the refractive index of the particles can be increased. In addition, due to the high hydrophobicity of the structure of formula (1-A), a desired volume average particle diameter can be obtained by the emulsion polymerization method and the soap-free polymerization method, and a uniform particle size distribution can be obtained.

Chemical formula

[0026] In the present embodiment, the structure represented by formula (1-A) is obtained by polymerizing the monomer represented by formula (X1).

Chemical formula

[0027] In this embodiment, examples of monomers represented by formula (X1) include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene, but styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferred. These monomers may be used individually or in combination. In this case, styrenes refer to 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, and p-phenylstyrene, etc.

[0028] In the first embodiment of the immunoturbidimetric particles of this disclosure, the first layer having the first organic polymer preferably further has a crosslinked structure. The crosslinked structure is obtained by polymerization using a crosslinkable radical polymerizable monomer, which is a monomer having two or more radical polymerizable unsaturated bonds in one molecule. Examples of such crosslinkable monomers include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, and dipentaerythritol hexamethacrylate, conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate. Alternatively, two or more crosslinkable radical polymerizable monomers may be used. The crosslinked structure is more preferably the structure represented by formula (D). The cross-linked structure makes the particles physically stronger, eliminating concerns about cracking or chipping even when repeated centrifugation is performed during purification. [ka] (Z represents a substituted or unsubstituted phenylene group or naphthalene group; in the case of substitution, the substituent is a methyl group or an ethyl group. Z may differ for each structural unit.)

[0029] Examples of crosslinkable radical polymerizable monomers used to form the crosslinkable structure of formula (D) include 1,2-divinylbenzene, 1,3-divinylbenzene, 1,4-divinylbenzene, 2,6-diethynylnaphthalene, and 2,7-diethynylnaphthalene. These may be used individually or in combination. Among the crosslinkable radical polymerizable monomers exemplified, divinylbenzene is preferred. Although the reason is unclear, when divinylbenzene is used, it exhibits excellent handling properties during radical polymerization reactions and improves the monomer conversion rate during particle formation.

[0030] The water-soluble polymerization initiator used in this embodiment is not particularly limited, but water-soluble azo compounds and water-soluble peroxides are preferably used. In this case, the water-soluble azo compound is preferably any of the following: 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, or 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate. Furthermore, the water-soluble peroxide is preferably one of the following: potassium persulfate, ammonium persulfate, sodium persulfate, tert-butyl hydroperoxide, cumyl hydroperoxide, paramenthane hydroperoxide, or diisopropylbenzene hydroperoxide.

[0031] In the first embodiment of the immunoturbidimetric particles of this disclosure, the second layer comprises a metal oxide. Preferably, the second layer comprising the metal oxide is prepared by a sol-gel method using a metal oxide precursor.

[0032] In the first embodiment of the immunoturbidimetry particles of this disclosure, the third layer having a second organic polymer preferably contains a polymer having a structural unit represented by formula (2). [ka] (R 3 R represents a hydrogen atom or a methyl group. 4 R indicates a group having an epoxy group, a group having a hydroxyl group, or a group having a carboxyl group. 3 and R 4 (These may differ for each structural unit.)

[0033] The structure represented by formula (2-A) is preferable to the structure represented by formula (2-A). The structure represented by formula (2-A) has either a hydroxyl group or a carboxyl group. Therefore, its ability to suppress nonspecific adsorption is equivalent to or better than that of the structure having an epoxy group, which is preferable. [ka] (R 3 R represents a hydrogen atom or a methyl group. 31 and R 32 One of them represents a hydroxyl group, and the other represents a hydroxyl group or the group represented by formula (2-B). [ka] (R 20 R indicates a single bond or a methylene group. 22 , R 23 , R 24 R represents a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group. 22 , R 23 , and R 24 One or more of them contain a hydroxyl group or a carboxyl group.1 * indicates a sulfur atom or imino group. *1 indicates the bonding position with the structure shown in formula (2-A).

[0034] Examples of specific structures of equation (2-A) are shown below in (2-A-1) to (2-A-12), but are not limited to these. [ka]

[0035] The structure represented by formula (2-A) in this embodiment is obtained by reacting a polymer obtained by polymerizing the monomer represented by formula (X2), which will be described later, with formula (X3), which will be described later. [ka] (R 13 R represents a hydrogen or methyl group. 14 (This indicates an ethylene group or a carbonyl group.) [ka] (R 15 R represents an amino group or a thiol group. 16 , R 17 R represents a group having a hydrogen atom, a methyl group, a hydroxyl group, or a carboxyl group. 16 , R 17 At least one of these groups represents a group having a hydroxyl group or a group having a carboxyl group.

[0036] In this embodiment, since the monomer represented by formula (X2) has a glycidyl group in its side chain, the compound represented by formula (X3) can be reacted with the glycidyl group in the polymer of formula (X2), and the carboxyl group or hydroxyl group contained in formula (X3) can be introduced into the third layer. The monomer represented by formula (X2) is not particularly limited, but glycidyl (meth)acrylate is preferred.

[0037] In this embodiment, the monomer represented by formula (X3) is added to the third layer by the reaction of the amino group or thiol group in formula (X3) with the glycidyl group in the polymer of formula (X2), thereby forming formula (2). The monomer represented by formula (X3) is not particularly limited, but examples include mercaptosuccinic acid, aspartic acid, 3-mercapto-1,2-propanediol, 3-amino-1,2-propanediol, 2-amino-1,3-propanediol, ethanolamine, and trishydroxymethylaminomethane.

[0038] The third layer having a second organic polymer in the first embodiment of the immunoturbidimetric particles of the present disclosure preferably further contains repeating units represented by formula (3) or (4). [ka] [ka] (R 51 and R 52 R represents a hydrogen atom or a methyl group. 71 * indicates a single bond, a phenylene group, or an alkylene group with 3 or fewer carbon atoms. n is an integer between 1 and 3, m is an integer between 0 and 2, and n+m is 3. *2 independently indicates a bond to a titanium atom or a silicon atom, or a hydrogen atom, a methyl group, or an ethyl group. R 61 and R 62 Each independently represents either a methyl group or an ethyl group. That is, the structures represented by formulas (3) and (4) may be bonded to the titanium atom of titanium oxide via an oxygen atom, or they may be bonded to the silicon atom of another structure represented by formula (3) or (4) via an oxygen atom. 51 , R 52 , R 61 , R 62 , and R 71 (These may differ for each structural unit.)

[0039] Further inclusion of repeating units that are vinyl polymers represented by formula (3) or (4) and have alkoxysilanes is preferable because the interaction with titanium dioxide further suppresses the exposure of titanium dioxide, resulting in a uniform coating.

[0040] The third layer having a second organic polymer in the first form of the immunoturbidimetric particles of this disclosure is not particularly limited as long as it has a repeating unit represented by formula (2), but 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 a repeating unit represented by formula (3) or (4) is further contained, it can be obtained by mixing monomers from which the repeating unit represented by formula (3) or (4) is derived and performing seed polymerization by copolymerization of two or more monomers. The monomers from which the repeating unit represented by formula (3) or (4) is derived are not particularly limited as long as they have the structure from which the repeating unit represented by formula (3) or (4) is derived, but examples include repeating units derived from vinyltrimethoxysilane, vinyltriethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane. These can also be used individually or in combination of two or more types.

[0041] A second embodiment of the immunoturbidimetric particles of the present disclosure is characterized by containing an organic polymer and a metal oxide, having a first layer having the metal oxide and a second layer having the organic polymer, wherein the second layer is located outside the first layer.

[0042] Examples of metal oxides in the second embodiment of the immunoturbidimetric particles of this disclosure include titanium oxide, aluminum oxide, zirconium oxide, silica, etc., as described in the embodiments above. Among these, titanium oxide, which has a relatively low specific gravity and a high refractive index, is preferred. Titanium oxide can be produced by methods such as the sulfuric acid method, chlorine method, or sol-gel method, although these methods are not particularly limited. In the second embodiment of the immunoturbidimetry particles of this disclosure, the organic polymer preferably contains a polymer having structural units represented by formulas (1), (D), (2), (3), and (4), as described in the embodiments described above.

[0043] <Substances that specifically bind to a target substance (ligands)> A ligand is a compound that specifically binds to a receptor on a particular target substance. The binding site of a ligand to the target substance is fixed, and it has a selective or specific high affinity. Examples include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc., as long as the objectives of this disclosure can be achieved. Specifically, examples of ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, etc.

[0044] The ligand in the immunoturbidimetric particles of this disclosure is preferably an antibody or a virus-derived antigen. The ligand being an antibody or virus-derived antigen enables highly sensitive detection of target substances that bind to the antibody or antigen. In this disclosure, the method for immobilizing the ligand on the particles can be any known method, and the ligand can be immobilized by physically or chemically binding it to the particles. Examples of chemical binding methods include carbodiimide-mediated reactions, NHS ester activation reactions, and methods in which avidin is attached to a carboxyl group and then a biotin-modified ligand is attached.

[0045] The polydispersity index of the particles for immunoturbidimetry in this disclosure is preferably 0.1 or less. A polydispersity index within this range results in less variation in particle size and the absence of large particles, leading to a larger size difference between the aggregated particles and the aggregated bodies after aggregation, thus increasing sensitivity. The method for measuring physical properties in this disclosure is described below.

[0046] <Method for measuring the volume-average particle size> This disclosure describes the method for measuring the volume-average particle size (Dv) of particles. 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 polydispersity index of particles in this disclosure is calculated by the dynamic light scattering method described above.

[0047] <Method for measuring the refractive index of particles> The refractive index of the particles is measured using an Abbemat (Anton Paar). In this disclosure, the refractive index is measured when the particle aqueous dispersion is dispersed to a concentration of 5% by mass. The measurement conditions are 25°C and a measurement wavelength of 589.3 nm. The particle refractive index is calculated from the Lorentz-Lorentz equation using the measured refractive index value, the specific gravity of the dispersion medium, the refractive index, and the specific gravity of the particles.

[0048] <Method for measuring the amount of antibody against particles (amount of antibody sensitization of particles)> In this disclosure, the antibodies bound to the particles were identified by protein quantification using the Protein Assay BCA Kit (Fujifilm Wako Pure Chemical Corporation).

[0049] <Method for measuring the crystallinity of particles> The crystallinity of the particles is measured using X'Pert-Pro (Malvern Panalytical). The X-ray diffraction pattern is measured in the range of 20°≦2θ≦60° under X-ray output conditions of 45kV and 40mA. The crystallinity is calculated as the ratio of the peak area indicating the crystalline component to the total measured peak area. An example of this calculation is shown below. In the case of the particle X-ray diffraction pattern shown in Figure 1, there are peaks around 25°, 31°, 38°, 47°, and 54° that originate from the titanium oxide crystal structure. By separating the baseline and peak components using any method, the crystallinity can be calculated as the ratio of the peak area indicating the crystalline component to the total measured signal area.

[0050] <Reagents> The immunoturbidimetric particles of this embodiment can also be used as a reagent for detecting a target substance via a ligand. While the form of the reagent is not limited, it is preferable to use a reagent in which the immunoturbidimetric particles of this disclosure are dispersed in an aqueous solution.

[0051] <Testing Kit> The reagent containing the immunoturbidimetry particles of this embodiment can also be used as a test kit for detecting a target substance via a ligand. The form of the test kit is not limited, but it preferably comprises the reagent containing the immunoturbidimetry particles of this disclosure and a container containing the reagent. The composition of the reagent is not particularly limited, but as an example, a preferred form is one in which the first reagent includes a buffer and a surfactant, and the second reagent includes a buffer, a surfactant, and particles for immunoturbidimetry.

[0052] <Detection Method> The reagent containing the immunoturbidimetric particles of this embodiment can detect a target substance in a sample for in vitro diagnostic purposes. In this disclosure, "detection" may refer to both qualitative and quantitative detection of the target substance. An example of a method for detecting a target substance using the reagent containing the immunoturbidimetric particles of this embodiment will be described. One example of a detection method involves the following three steps. (1) A step of obtaining a mixture by mixing a sample that may contain a target substance with a reagent containing the immunoturbidimetry particles of this embodiment. (2) Step of irradiating the mixture with light (3) A step of detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture. [Examples]

[0053] The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited to these examples. [Examples of particle production] (Preparation of particle 1) (Process for producing the first layer having the first organic polymer) 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 first layer particles 1 consisting of a copolymer of St and DVB 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.

[0054] (Process for forming the second layer containing titanium dioxide) A 20 g dispersion of the first layer particle 1 with a solid content of 0.6% by mass was prepared using deionized water. This dispersion was mixed with 404.50 g of ethanol (Kishida Chemical Co., Ltd.) containing 0.2% by mass of 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 separately by mixing 5.0 mL of titanium(IV)n-butoxide, monomer (TBOT: Kishida Chemical Co., Ltd.) with 197.50 g of ethanol was added to the above mixture to initiate the sol-gel reaction. After 24 hours of reaction, the second layer forming particle 1 was separated from the mixture by centrifugation and redispersed in ethanol. Furthermore, the second layer-forming particles 1 were separated from the dispersion using a centrifuge, and the process of redispersing particles 1 in ion-exchanged water was repeated twice to purify the second layer-forming particles 1. The aqueous dispersion was then prepared so that the second layer-forming particles 1 accounted for 5.0% by mass and stored. 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. In addition, the metal oxide content was evaluated using differential thermal-thermogravimetric analysis (NEXTA® STA200RV: Hitachi High-Tech Corporation), and it was found to be 45% by mass of the particle mass.

[0055] (Process for forming a third layer having a second organic polymer) A dispersion of second-layer-forming particles 1 with a solid content of 0.2% by mass was prepared in ion-exchanged 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-Methacryloxypropyltrimethoxysillane (MPS: Shin-Etsu Chemical Co., Ltd.) were added, and the mixture was maintained at 70°C while stirring at 100 rpm. The four-neck 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 ion-exchanged water, which had been prepared separately, was added to the mixture to initiate shell formation. After stirring continued for 18 hours, a dispersion containing third-layer-forming particles 1 was obtained.

[0056] (Process for imparting reactive functional groups) An aqueous solution of mercaptosuccinic acid (MSA: Fujifilm Wako Pure Chemical Industries, Ltd.), which had been prepared in advance, was added to a dispersion containing the third layer-forming particle 1. At this time, the aqueous solution was prepared so that the total number of moles of MSA was equal to the number of moles of the glycidyl methacrylate. Next, triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Then, the above was heated to 70°C while stirring at 800 rpm, and maintained in this state for 18 hours to obtain a dispersion containing particle 1.

[0057] (Particle washing process) A dispersion of particle 1 was obtained by separating particle 1 from the above dispersion using a centrifuge and then redispersing it in ion-exchanged water, repeating this process eight times until the particle concentration was finally adjusted to 5.0% by mass.

[0058] (Synthesis of particle 2) Particle 2 was synthesized using the same experimental procedure as for particle 1, except that in the step of forming the third layer containing the second organic polymer in the synthesis method of particle 1, the temperature was kept at 50°C instead of 70°C.

[0059] (Synthesis of particle 3) A dispersion of particle 3 was obtained using the same procedure as for particle 1, except that in the step of forming the second layer containing titanium dioxide in the synthesis method of particle 1, the amount of titanium(IV)n-butoxide,monomer was changed from 5.0 mL to 3.0 mL.

[0060] (Synthesis of particle 4) A dispersion of particle 4 was obtained using the same procedure as for particle 1, except that in the step of forming the second layer containing titanium dioxide in the synthesis method of particle 1, the amount of titanium(IV)n-butoxide,monomer was changed from 5.0 mL to 7.5 mL.

[0061] (Synthesis of particle 5) In the synthesis method for particle 1, a dispersion of particle 5 was obtained by the same procedure as for particle 1, except that the amounts of St, DVB, and V-50 were changed to 71.75 g, 1.30 g, and 3.11 g, respectively, and the stirring speed and reaction time were changed to 200 rpm and 48 hours, respectively.

[0062] The synthesis conditions and physical properties of the obtained particles 1 to 5 are summarized in Table 1. The particles 1 to 5 described above illustrate an example of a first form of the immunoturbidimetric particles of the present disclosure. In each of the particles 1 to 5, the first layer of the particle contains a styrene-divinylbenzene copolymer, i.e., R 10 It had a structural unit represented by formula (1-A) in which is a phenyl group. In addition, the third layer had R 3 is a methyl group, R 4 This includes a structural unit represented by formula (2) having a structural unit represented by any of the following formulas (2-C), (2-D), or (2-E). [ka] [ka] [ka] (*3 indicates the bonding position with the structure shown in equation (2).)

[0063] [Table 1]

[0064] (Synthesis of particle 6) (Process for fabricating the first layer containing metal oxides) 0.6 g of titanium dioxide particles TTO-55 (Ishihara Sangyo Co., Ltd.), 10.1 g of 28% by mass 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-Methacryloxypropyltrimethoxysillane (MPS: Shin-Etsu Chemical Co., Ltd.) were mixed and dispersed for 1 hour at 12000 rpm using a TK homomixer (Primix Co., Ltd.) 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.

[0065] (Process for forming the second layer containing organic polymers) 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. Subsequently, a solution of 0.04g of ammonium persulfate (Kishida Chemical Co., Ltd.) dissolved in 1g of deionized water was added to the above mixture to initiate the formation of the styrene layer. 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, and the reaction was continued for another 12 hours to obtain a dispersion containing particles. Further reactive functional group imparting and particle washing steps similar to those for particle 1 were performed to obtain a dispersion of particle 6. The particle size, titanium dioxide content, degree of crystallinity, and refractive index of the obtained particle 6 were 400 nm, 10 mass%, 80%, and 1.70, respectively.

[0066] The particle 6 described above illustrates an example of a second embodiment of the immunoturbidimetric particle of the present disclosure. The particle 6 had a titanium dioxide-containing layer as a first layer, and a styrene-divinylbenzene copolymer as a second layer outside of that, i.e., R 10 A layer containing a structural unit represented by formula (1-A) in which is a phenyl group, and further outside there, R 3 is a methyl group, R 4The structure had a layer containing a structural unit represented by formula (2), which in turn had a structural unit represented by any of the following formulas (2-C), (2-D), or (2-E).

[0067] [Examples] (Preparation of antibody-sensitized particles) This example and the comparative examples described later show examples of particles for immunoturbidimetry using ferritin as the target substance. For the dispersion of particle 1, 300 μL of the dispersion (3 mg as particle solids), diluted with deionized water to a solid content concentration of 1.0% by mass, was placed in a 1.5 mL microtube. 90 μL of a 5.0% by mass aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Tokyo Chemical Industries, Ltd.) and 90 μL of a 5.0% by mass aqueous solution of N-hydroxysulfosuccinimide sodium (Tokyo Chemical Industries, Ltd.) were added, and the 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.

[0068] To this, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (0.12 mg of antibody) was added and stirred at room temperature for 3 hours to obtain test particles sensitized with the antibody. After centrifugation, these test particles were washed, and 500 μL of PBS was added to obtain antibody-sensitized particle 1.

[0069] (Comparative particle) As comparative examples in this disclosure, polystyrene particles (Immutex-plainP2118, JSR Corporation) and titanium dioxide particles (STS-21, Ishihara Sangyo Co., Ltd.) were used and designated as particle 7 and particle 8, respectively. The particle size and refractive index of the polystyrene particles were 200 nm and 1.62, and the particle size and refractive index of the titanium dioxide particles were 200 nm and 2.70. Antibody-sensitized particles were prepared by conjugating antibodies to these particles using existing methods. Specifically, 1 mL of a dispersion (5 mg as particle solids) diluted with 10 mM HEPES to a solid content concentration of 0.5% by mass was placed in a 1.5 mL microtube, and 80 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (0.4 mg as antibody) was added, and the mixture was stirred at room temperature for 3 hours. After centrifugation of these particles, 10 mM HEPES containing 1% by mass fetal bovine serum albumin was added, and the mixture was stirred at room temperature for 1 hour. After centrifuging these test particles, 500 μL of PBS was added to obtain antibody-sensitized particles 7 and 8.

[0070] (Preparation of the first reagent) The first reagent was prepared by dissolving 50 mM HEPES, 0.05% by mass Triton X-100, and 1.0% by mass sodium chloride (Kishida Chemical Co., Ltd.) in deionized water.

[0071] (Preparation of the second reagent) After centrifuging and washing antibody-sensitized particles 1, they were redispersed in 500 μL of buffer (HEPES buffer) prepared by dissolving 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (Triton X-100: Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​in deionized water. Subsequently, the mixture and dilution with HEPES buffer were performed until antibody-sensitized particles 1 comprised 0.1% by mass to obtain second reagent 1. Second reagents 2 to 8 were prepared using the same experimental procedure as for the preparation of second reagent 1, except that the type of particle was changed from antibody-sensitized particle 1 to antibody-sensitized particles 2 to 8.

[0072] (Measurement of change in absorbance) Absorbance measurements were performed using a BIOSPECTROMETER (Eppendorf) spectrophotometer, with a measurement wavelength of 572 nm. A mixture was prepared by mixing 15 μL of a sample, prepared to have a ferritin concentration of 250 ng / mL, with 60 μL of the first reagent 1, and incubating it at 37°C for 290 seconds. Next, 30 μL of the second reagent 1 was mixed into the mixture, and the absorbance was measured after stirring for 42 seconds. Furthermore, this mixture was allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance after 30 seconds was defined as the change in absorbance (ΔAbs).

[0073] (Calculation of sensitivity index) The value of ΔAbs × 10000 was calculated and used as the ferritin sensitivity index. A higher ferritin sensitivity index is expected to indicate more sensitive detection of the target substance. The evaluation was based on the sensitivity index values ​​as follows. A: ΔAbs × 10000 was greater than 500. B:ΔAbs×10000 was greater than 100 and less than or equal to 500. C:ΔAbs×10000 was a value less than or equal to 100. The results for each are shown in Table 2.

[0074] [Table 2] These results show that the second reagent, using particles 1 to 6 having refractive indices and volume-average particle sizes that satisfy the provisions of this disclosure, exhibits superior detection sensitivity compared to the comparative example.

[0075] This embodiment includes the following configurations and methods. (Composition 1) Particles for immunoturbidimetry containing metal oxides, The refractive index of the aforementioned particles is 1.70 or more and 2.25 or less. The volume-average particle size of the aforementioned particles is 200 nm or more and 400 nm or less. Particles for immunoturbidimetry, characterized in that the product of the refractive index and the volume-average particle size is 340 nm or more and 780 nm or less. (Configuration 2) The immunoturbidimetric particle according to configuration 1, characterized in that the metal oxide is titanium oxide. (Composition 3) The immunoturbidimetric particle according to configuration 2, characterized in that the degree of crystallinity of the titanium dioxide is 20% or more and 90% or less. (Composition 4) Particles for immunoturbidimetry according to any one of configurations 1 to 3, characterized in that the aforementioned particles further contain an organic polymer. (Composition 5) The immunoturbidimetric particle according to configuration 4, characterized in that the organic polymer contains a structure represented by the following formula (1). [ka] (R 1 R represents a hydrogen atom and a methyl group. 2 R represents a structure containing a substituted phenyl group or naphthyl group. 1 and R 2 (These may differ for each structural unit.) (Composition 6) The immunoturbidimetric particle according to configuration 4 or 5, characterized in that the organic polymer further contains a structure represented by the following formula (2). [ka] (R 3 R represents a hydrogen atom or a methyl group. 4 R indicates a group having an epoxy group, a group having a hydroxyl group, or a group having a carboxyl group. 3 and R 4 (These may differ for each structural unit.) (Composition 7) Particles for immunoturbidimetry according to any one of configurations 4 to 6, characterized in that the above formula (2) is represented by the following formula (2-A). [ka] (R 31 and R 32One of the terms represents a hydroxyl group, and the other represents a hydroxyl group or the group represented by formula (2-B). [ka] (R 20 R indicates a single bond or a methylene group. 22 , R 23 , R 24 R represents a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group. 22 , R 23 , and R 24 One or more of them contain a hydroxyl group or a carboxyl group. 1 * indicates a sulfur atom or imino group. *1 indicates the bonding position with the structure shown in formula (2-A). (Composition 8) Particles for immunoturbidimetry according to any one of configurations 1 to 7, characterized in that the polydispersity index of the particles is 0.1 or less. (Composition 9) The particle for immunoturbidimetry according to any one of configurations 1 to 8, characterized in that the particle comprises a first layer having a first organic polymer and a second layer provided outside the first layer and having the metal oxide. (Composition 10) The particle for immunoturbidimetry according to configuration 9, characterized in that the particle further has a third layer provided outside the second layer and having a second organic polymer. (Composition 11) The immunoturbidimetric particle according to any one of configurations 1 to 10, characterized in that the particle further has a ligand. (Composition 12) The immunoturbidimetric particle according to configuration 11, characterized in that the ligand is an antibody or a virus-derived antigen. (Composition 13) A reagent characterized in that the immunoturbidimetric particles described in any one of configurations 1 to 12 are dispersed in an aqueous solution. (Composition 14) A test kit characterized by comprising the reagent described in configuration 13 and a container containing the reagent. (Method 15) A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in configuration 13 with a specimen that may contain the target substance. (Method 16) 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 13 to obtain a mixed solution; irradiating the mixed solution with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixed solution.

Claims

1. Particles for immunoturbidimetry containing metal oxides, The refractive index of the aforementioned particles is 1.70 or more and 2.25 or less. The volume-average particle size of the aforementioned particles is 200 nm or more and 400 nm or less. Particles for immunoturbidimetry, characterized in that the product of the refractive index and the volume-average particle size is 340 nm or more and 780 nm or less.

2. The immunoturbidimetric particle according to claim 1, characterized in that the metal oxide is titanium oxide.

3. The immunoturbidimetric particle according to claim 2, characterized in that the degree of crystallinity of the titanium dioxide is 20% or more and 90% or less.

4. The particles for immunoturbidimetry according to claim 1, characterized in that the particles further contain an organic polymer.

5. The immunoturbidimetric particle according to claim 4, characterized in that the organic polymer contains a structure represented by the following formula (1). 【Chemistry 1】 (R １ R represents a hydrogen atom and a methyl group. ２ R represents a structure containing a substituted phenyl group or naphthyl group. １ and R ２ (These may differ for each structural unit.)

6. The particle for immunoturbidimetry according to claim 5, characterized in that the organic polymer further contains a structure represented by the following formula (2). 【Chemistry 2】 (R ３ R represents a hydrogen atom or a methyl group. ４ R indicates a group having an epoxy group, a group having a hydroxyl group, or a group having a carboxyl group. ３ and R ４ (These may differ for each structural unit.)

7. The immunoturbidimetric particle according to claim 6, characterized in that formula (2) is represented by the following formula (2-A). 【Transformation 3】 (One of R ３１ and R ３２ represents a hydroxy group, and the other represents a hydroxy group or a group represented by formula (2-B).) 【Chemistry 4】 (R ２０ R represents a single bond or a methylene group. ２２ , R ２３ , R ２４ R represents a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group. ２２ , R ２３ , and R ２４ One or more of these contain a hydroxyl group or a carboxyl group. １ * indicates a sulfur atom or imino group. １ (This indicates the bonding position with the structure shown in formula (2-A).)

8. The particle for immunoturbidimetry according to claim 1, characterized in that the polydispersity index of the particle is 0.1 or less.

9. The particle for immunoturbidimetry according to claim 1, characterized in that the particle comprises a first layer having a first organic polymer and a second layer provided outside the first layer and having the metal oxide.

10. The particle for immunoturbidimetry according to claim 9, characterized in that the particle further has a third layer provided outside the second layer and having a second organic polymer.

11. The particle for immunoturbidimetry according to claim 1, characterized in that the particle further has a ligand.

12. The immunoturbidimetric particle according to claim 11, characterized in that the ligand is an antibody or a virus-derived antigen.

13. A reagent characterized in that the immunoturbidimetric particles described in any one of claims 1 to 12 are dispersed in an aqueous solution.

14. A test kit comprising the reagent described in claim 13 and a container containing the reagent.

15. A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in claim 13 with a specimen that may contain the target substance.

16. 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 13 to obtain a mixture; irradiating the mixture with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture.

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