Particles for immunonephelometry, reagent, test kit, and detection method

Abstract

Provided are: particles for immunonephelometry, the particles containing a metal oxide and being used for highly sensitive detection of a target substance; a reagent; a test kit; and a detection method.　The particles for immunonephelometry contain a metal oxide, and are characterized in that: the refractive index of the particles is 1.70-2.25; the volume-average particle size of the particles is 200-400 nm; and the product of the refractive index and the volume-average particle size is 340-780 nm.

Description

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

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

[0002] A simple and rapid immunoassay method is immunoturbidimetry using particles. In this method, a dispersion of particles bound to a ligand with affinity for the target substance is mixed with a sample that may contain the target substance. At this time, an agglutination reaction occurs in the particles depending on the amount of target substance contained in the sample. By optically detecting this agglutination reaction as a change in scattered light intensity, transmitted light intensity, absorbance, etc., the target substance can be qualitatively or quantitatively identified.

[0003] To improve the sensitivity of immunoturbidimetric methods for measuring target substances in the low-concentration range, methods such as increasing particle size and increasing refractive index have been employed. As a method for increasing refractive index, a method using a metal oxide with a high refractive index as a particle carrier has been proposed. Patent Document 1 discloses immunoturbidimetric particles using amorphous titanium oxide with an average particle size of 80 nm to 140 nm. Patent Document 2 discloses immunoturbidimetric particles using composite particles of titanium oxide fine particles and polystyrene particles.

[0004] Japanese Patent Publication No. 2017-122684 Japanese Patent Publication No. 2008-241357

[0005] 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.

[0006] Until now, no optimal design method had 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 there is a combination range in which both particle size and refractive index are increased when using particles made from metal oxides that is effective for immunoturbidimetry, leading to this disclosure.

[0007] A first aspect of this disclosure for solving the above problems is an immunoturbidimetric particle containing a metal oxide, characterized in that the refractive index of the particle is 1.70 or more and 2.25 or less, the volume-average particle size of the particle 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.

[0008] Furthermore, a second aspect of this disclosure is a reagent characterized in that the immunoturbidimetric particles are dispersed in an aqueous solution.

[0009] Furthermore, a third aspect of this disclosure is a test kit characterized by comprising the reagent and a container containing the reagent.

[0010] In addition, a fourth aspect of the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent with a specimen that may contain the target substance.

[0011] Finally, a fifth aspect of the present 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.

[0012] 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.

[0013] This figure shows an example of an X-ray diffraction pattern according to this embodiment.

[0014] The embodiments of this disclosure will be described in detail below, but the scope of the art is not limited to these embodiments.

[0015] The particles for immunoturbidimetry of the present disclosure are particles containing a metal oxide. Examples of the metal oxide include titanium oxide, aluminum oxide, zirconium oxide, silica, and the like. Among these, titanium oxide with a relatively low specific gravity and a high refractive index is preferable. Titanium oxide is not particularly limited, but is produced by a production method such as the sulfuric acid method, the chlorine method, or the sol-gel method. In the sol-gel method, a metal oxide precursor, an oxygen-containing organic solvent, and water are made to coexist, and a hydrolysis reaction is carried out to obtain a metal oxide containing titanium oxide.

[0016] Examples of the metal oxide precursor include metal chlorides, metal acetates, metal alkoxides, metal hydroxides, and the like. Among these, from the viewpoint of by-produced impurities (such as chlorides, etc.), metal alkoxides, metal acetates, and metal hydroxides are preferably used. Among these, particularly, a metal alkoxide represented by the chemical formula Mx(OR)y (where M represents a metal element, R represents an alkyl group, and x and y each independently represent an integer of 1 or more and 4 or less), a metal hydroxide represented by the chemical formula Mx(OH)y·nH ２ O (where M represents a metal element, x and y each independently represent an integer of 1 or more and 4 or less, and n represents an integer of 1 or more), and a compound containing the aforementioned metal alkoxide and / or metal hydroxide are preferable.

[0017] Examples of the metal oxide precursor containing titanium include titanium methoxide, titanium ethoxide, titanium-diisopropoxide bis(2,4-pentanedionate), titanium-diisopropoxide bis(ethyl acetoacetate), titanium-n-butoxide, titanium isopropoxide, titanium methoxypropoxide, titanium-n-nonyloxide, titanium-n-propoxide, titanium stearyloxide, titanium triisostearyl isopropoxide, titanium trimethylsiloxide, and the like.

[0018] The particles for immunoturbidimetry of the present disclosure have a refractive index of 1.70 or more and 2.25 or less, a volume average particle diameter of the particles of 200 nm or more and 400 nm or less, and a product of the refractive index and the volume average particle diameter of 340 nm or more and 780 nm or less.

[0019] As a result of intensive studies on the optimal design of particles containing metal oxides with a high refractive index as particles for immunoturbidimetry, the inventors have found that high sensitivity can be obtained in particles that satisfy the refractive index and volume average particle size. Here, the refractive index is the value at the wavelength during immunoturbidimetry measurement. Further, the wavelength during immunoturbidimetry measurement preferably includes 500 nm or more and 750 nm or less, and 572 nm is most preferable.

[0020] The following theory can be considered for the mechanism by which high sensitivity is obtained. In the particle size range of particles for immunoturbidimetry, the contribution of Mie scattering is large. Based on the theoretical formula of Mie scattering, the attenuation factor (Q ｅｘｔ ) of the particles can be calculated from the particle size and refractive index. The absorbance of a dispersion of certain particles can be calculated by the following formula (A). (I: transmitted light intensity, I ０ : incident light intensity, c: volume fraction, L: optical path length, r: particle radius)

[0021] Next, assuming that the volume equivalent diameter (r １ ) increases from the original particle size (r ２ ) when the particles aggregate, the absorbance difference (ΔAbs) during the aggregation reaction can be calculated as follows in formula (B). (Q ｅｘｔ２ : attenuation coefficient of the particles after aggregation, r ２ : particle size of the particles after aggregation, Q ｅｘｔ１ : attenuation coefficient of the particles before aggregation, r １ : particle size of the particles before aggregation)

[0022] As a result of calculating ΔAbs based on this calculation formula, it is shown that ΔAbs increases monotonically with the refractive index when the particle size is 150 nm or less. On the other hand, when the particle size is 200 nm or more and 400 nm or less, ΔAbs has a maximum at a certain refractive index, and it is shown that ΔAbs may become a negative value when the refractive index is too large. The fact that ΔAbs becomes a negative value means that the absorbance change due to the aggregation reaction becomes a negative value. As a result of the study, the inventors have found that the above calculation formula is consistent with the measured tendency.

[0023] <Crystallization of Titanium Dioxide> In the particles for immunoturbidimetry of the present disclosure, the proportion of titanium dioxide that is crystallized (also referred to as the degree of crystallinity in this disclosure) is preferably 20% or more and 90% or less. By having the degree of crystallinity within the above range, the refractive index can be controlled to the range defined in this disclosure.

[0024] As mentioned above, amorphous titanium dioxide can be obtained by a method called the sol-gel method, which involves hydrolysis of a metal oxide precursor. 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-containing 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.

[0025] <Organic Polymers> The immunoturbidimetry particles of this disclosure preferably contain organic polymers. The inclusion of organic polymers makes it possible to control the refractive index value.

[0026] A 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 grinding, and dry grinding. 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 immunoturbidimetric particles of this disclosure may have a third layer having a second organic polymer located outside the second layer having a metal oxide.

[0027] In the first embodiment of the immunoturbidimetric particles of this disclosure, the first layer having the first organic polymer preferably contains a polymer having a structural unit represented by formula (1). The first layer may be a core layer. (R １ R represents a hydrogen atom or a methyl group. ２ R represents a substituted or unsubstituted phenyl group or naphthyl group, where the substituent is a methyl group or an ethyl group. １ and R ２ (These may differ for each structural unit.)

[0028] Equation (1) is R ２ The polymer has substituted or unsubstituted phenyl groups and naphthyl groups. Polymers having the structural unit represented by formula (1) have a relatively high refractive index among organic polymers. Therefore, by including a polymer having the structural unit represented by formula (1), the refractive index can be controlled to the range specified in this disclosure.

[0029] It is preferable that the structure of formula (1) is represented by formula (1-A). Having the structure of formula (1-A) allows for an increase in the refractive index of the particles. Furthermore, because the structure of formula (1-A) is highly hydrophobic, the desired volume-average particle size can be obtained by emulsion polymerization and soap-free polymerization, and a uniform particle size distribution can be achieved. (R １０ (This represents a phenyl group, a tolyl group, or a naphthyl group.)

[0030] In this embodiment, the structure represented by formula (1-A) is obtained by polymerizing the monomer represented by formula (X1). (R １１ R represents a hydrogen atom or a methyl group. １２ (This indicates a substituted or unsubstituted phenyl group or naphthyl group; in the case of substitution, the substituent is a methyl group or an ethyl group.)

[0031] In this embodiment, examples of monomers represented by formula (X1) include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene, with styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene being 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, p-n-butylstyrene, p-tert-butylstyrene, p-n-hexylstyrene, p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene, p-n-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene, etc.

[0032] In the first embodiment of the particles for immunoturbidimetry 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, and 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. (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.)

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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). (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.)

[0039] The structure represented by formula (2-A) is preferable. 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. (R ３ R represents a hydrogen atom or a methyl group. ３１ and R ３２ Either one represents a hydroxyl group, and the other represents a hydroxyl group or the group represented by formula (2-B). (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).)

[0040] Examples of specific structures of formula (2-A) are shown below in (2-A-1) to (2-A-12), but are not limited to these.

[0041] 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. (R１３ R represents a hydrogen or methyl group. １４ (This indicates an ethylene group or a carbonyl group.) (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.

[0042] 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.

[0043] 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.

[0044] The third layer having the 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). (R ５１ and R ５２ R represents a hydrogen atom or a methyl group. ７１ * represents 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. ２Each of these independently indicates a bond with a titanium atom or a silicon atom, or a hydrogen atom, a methyl group, or an ethyl group. 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 atoms of titanium oxide via an oxygen atom, or they may be bonded to the silicon atoms 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.)

[0045] A vinyl polymer represented by formula (3) or (4), further containing repeating units having alkoxysilane, is preferable because the interaction with titanium dioxide further suppresses the exposure of titanium dioxide, resulting in a uniform coating.

[0046] The third layer having the 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.

[0047] 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.

[0048] 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 is not particularly limited, but can be produced by methods such as the sulfuric acid method, chlorine method, or sol-gel method. As for the organic polymer in the second embodiment of the immunoturbidimetric particles of this disclosure, it is preferable to include a polymer having structural units represented by formulas (1), (D), (2), (3), and (4), as described in the embodiments above.

[0049] <Substances that specifically bind to target substances (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 antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc., but are not limited to these as long as the objectives of this disclosure can be achieved. Specifically, ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')). ２ Examples include F(ab'), Fv, scFv, etc., naturally derived nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, etc.

[0050] 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.

[0051] 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 material after aggregation, thus increasing sensitivity.

[0052] The method for measuring physical properties in this disclosure is described below.

[0053] <Method for Measuring the Volume-Average Particle Size> The method for measuring the volume-average particle size (Dv) in this disclosure is described below. The Dv of particles present in an aqueous dispersion is measured by dynamic light scattering. For example, a Zetasizer (Zetasizer Ultra: Malvern Panalogical) is used and the measurement is performed at 25°C. Furthermore, the polydispersity index of the particles in this disclosure is calculated by the measurement using the dynamic light scattering method described above.

[0054] <Method for measuring the refractive index of particles> The refractive index of particles is measured using Abbemat (Anton Paar). The refractive index in this disclosure 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.

[0055] <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 confirmed by protein quantification using the Protein Assay BCA Kit (Fujifilm Wako Pure Chemical Corporation).

[0056] <Method for Measuring Particle Crystallinity> The crystallinity of particles is measured using X'Pert-Pro (Malvern Panalogical). The X-ray diffraction pattern is measured in the range of 20° ≤ 2θ ≤ 60° under the conditions of X-ray output: 45kV, 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 the calculation is shown below. In the case of the X-ray diffraction pattern of the particle shown in Figure 1, there are peaks originating from the crystalline structure of titanium oxide around 25°, 31°, 38°, 47°, and 54°. The baseline and peak components can be separated by any method, and the crystallinity can be calculated as the ratio of the peak area indicating the crystalline component to the total measured signal area.

[0057] <Reagents> The immunoturbidimetric particles of this embodiment can also be used as reagents for detecting target substances via ligands. The form of the reagent is not limited, but it is preferable to use a reagent in which the immunoturbidimetric particles of this disclosure are dispersed in an aqueous solution.

[0058] <Test 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 is preferable to have the reagent containing the immunoturbidimetry particles of this disclosure and a container that encloses the reagent.

[0059] 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.

[0060] <Detection Method> The reagent containing the immunoturbidimetry 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 immunoturbidimetry particles of this embodiment will be described.

[0061] An example of a detection method includes the following three steps: (1) A step of mixing a sample potentially containing the target substance with a reagent containing the immunoturbidimetric particles of this embodiment to obtain a mixture; (2) A 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.

[0062] The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited to these examples.

[0063] [Particle Preparation Example] (Preparation of Particle 1) (Preparation Process for the First Layer Having the First Organic Polymer) 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 were weighed into a 2 L four-neck separable flask to make a mixture. This mixture was kept 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 above mixture to initiate soap-free polymerization. After 23 hours of reaction from the start of polymerization, a dispersion of the first layer particle 1, consisting of a copolymer of St and DVB, was obtained. A portion of the sample was collected and evaluated using dynamic light scattering (Zetasizer Ultra: Malvern Panalogical), revealing a volume-average particle size of 190 nm.

[0064] (Process for forming the second layer containing titanium dioxide) A dispersion of the first layer particle 1 with a solid content of 0.6% by mass was prepared using deionized water to 20 g. 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. The reaction was allowed to proceed for 24 hours from the start of the sol-gel reaction, and 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 the 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.

[0065] (Process for forming the third layer having a second organic polymer) A dispersion of the 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-Methacryloxypropyltrimethoxysilane (MPS: Shin-Etsu Chemical Co., Ltd.) were added, and the mixture was maintained at 70°C while stirring at 100 rpm. The inside of 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, which had been prepared separately, dissolved in 0.3 g of ion-exchanged water was added to the above mixture to start the formation of the shell. After stirring continued for 18 hours after the start of the reaction, a dispersion containing the third layer-forming particles 1 was obtained.

[0066] (Step 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 used 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.

[0067] (Particle washing process) Particle 1 was separated from the above dispersion using a centrifuge, and the process of redispersing it in ion-exchanged water was repeated eight times until the particle concentration was finally adjusted to 5.0% by mass, thereby obtaining a dispersion of particle 1.

[0068] (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 having the second organic polymer in the synthesis method of Particle 1, the temperature was kept at 50°C instead of 70°C.

[0069] (Synthesis of Particle 3) A dispersion of Particle 3 was obtained by 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.

[0070] (Synthesis of Particle 4) A dispersion of particle 4 was obtained by 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.

[0071] (Synthesis of Particle 5) In the first layer preparation step of the synthesis method for Particle 1, 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. Otherwise, a dispersion of Particle 5 was obtained by the same procedure as for Particle 1.

[0072] The synthesis conditions and physical properties of the obtained particles 1 to 5 are summarized in Table 1. Particles 1 to 5 represent an example of the first form of the immunoturbidimetry particles of this disclosure. In all of 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). (* ３ (This indicates the bonding position with the structure shown in equation (2).)

[0073]

[0074] (Synthesis of Particle 6) (Process for preparing 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-Methacryloxypropyltrimethoxysilane (MPS: Shin-Etsu Chemical Co., Ltd.) were mixed and dispersed at 12,000 rpm for 1 hour using a TK homomixer (Primix Co., Ltd.) to obtain a titanium dioxide fine particle dispersion. The titanium dioxide fine particles and supernatant were separated from the titanium dioxide fine particle dispersion using a centrifuge, and then the supernatant was redispersed with an equal mass of ion-exchanged water to obtain a purified titanium dioxide fine particle dispersion.

[0075] (Process for forming the second layer containing organic polymers) A purified titanium dioxide fine particle dispersion, whose concentration was adjusted by adding 80 g of pure water, was deoxygenated using a nitrogen flow. 1.74 g of styrene (St: Kishida Chemical Co., Ltd.) and 0.174 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.) were added, and the temperature was raised to 70°C. Subsequently, a solution of 0.04 g of ammonium persulfate (Kishida Chemical Co., Ltd.) dissolved in 1 g of deionized water was added to the above mixture to initiate the formation of the styrene layer. After stirring for 18 hours after the start of the reaction, 0.3 g of glycidyl methacrylate and a solution of 0.01 g of ammonium persulfate (Kishida Chemical Co., Ltd.) dissolved in 1 g of deionized water were added, and the reaction was continued for another 12 hours to obtain a dispersion containing particles. Further, a process of imparting reactive functional groups and a particle washing process similar to that 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.

[0076] The particle 6 described above illustrates an example of a second embodiment of the immunoturbidimetric particle of the present disclosure. The particle 6 has 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 ４ The 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).

[0077] (Preparation of antibody-sensitized particles) This example and the comparative examples described later show examples of particles for immunoturbidimetry with ferritin as the target substance.

[0078] 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. 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 a dispersion of particles containing activated carboxyl groups (activated particle dispersion). After centrifugation washing, 270 μL of pH 5.5 phosphate buffer-physiological saline (hereinafter referred to as PBS) was added, and the particles with activated carboxyl groups were dispersed by ultrasound.

[0079] 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 washing of these test particles, 500 μL of PBS was added to obtain antibody-sensitized particle 1.

[0080] (Comparative Particles) As comparative examples of this disclosure, polystyrene particles (Immutex-plain P2118, JSR Corporation) and titanium dioxide particles (STS-21, Ishihara Sangyo Co., Ltd.) were used 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 of 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.

[0081] (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.

[0082] (Preparation of the second reagent) After centrifuging and washing the antibody-sensitized particles 1, they were redispersed in 500 μL of a buffer (HEPES buffer) prepared by dissolving 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (Tritoon X-100: Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​in deionized water. Then, the mixture was mixed and diluted with HEPES buffer until the antibody-sensitized particles 1 accounted for 0.1% by mass to obtain the second reagent 1.

[0083] 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.

[0084] (Measurement of Absorbance Change) Absorbance was measured using a BIOSPECTROMETER spectrophotometer (Eppendorf), with a measurement wavelength of 572 nm. A mixture was prepared by mixing 15 μL of the specified sample, which was 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, stirred, and the absorbance was measured after 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 42 seconds was defined as the absorbance change (ΔAbs).

[0085] (Calculation of Sensitivity Index) The value of ΔAbs × 10000 was calculated and used as the ferritin sensitivity index. A larger ferritin sensitivity index is expected to indicate that the target substance can be detected with higher sensitivity.

[0086] The evaluation was conducted as follows based on the sensitivity index value, and the results for each are shown in Table 2. 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 less than or equal to 100.

[0087]

[0088] 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.

[0089] This disclosure is not limited to the embodiments described above, and various modifications and alterations are possible without departing from the spirit and scope of this disclosure. Accordingly, the following claims are attached to make the scope of this disclosure public.

[0090] This application claims priority based on Japanese Patent Application No. 2024-208144, filed on November 29, 2024, and all of its contents are incorporated herein by reference.

Claims

1. 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.

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

3. Particles for immunoturbidimetry 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 immunoturbidimetric particle according to any one of claims 1 to 3, characterized in that the particle further comprises 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). (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 immunoturbidimetric particle according to claim 4 or 5, characterized in that the organic polymer further contains a structure represented by the following formula (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 particle for immunoturbidimetry according to claim 6, wherein the formula (2) is represented by the following formula (2-A). (One of R ３１ and R ３２ represents a hydroxy group, and the other represents a hydroxy group or a group represented by the formula (2-B).) (R ２０ represents a single bond or a methylene group. R ２２ , R ２３ , R ２４ represent a hydrogen atom, a methyl group, a hydroxy group, a carboxy group, a hydroxymethyl group, or a carboxymethyl group, and one or more of R ２２ , R ２３ , and R ２４ contain a hydroxy group or a carboxy group. Y １ represents a sulfur atom or an imino group. * １ represents the bonding position with the structure represented by the formula (2-A).) 8. Particles for immunoturbidimetry according to any one of claims 1 to 7, characterized in that the polydispersity index of the particles is 0.1 or less.

9. The particle for immunoturbidimetry according to any one of claims 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.

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 any one of claims 1 to 10, 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. The immunoturbidimetric particle according to claim 12, characterized in that the ligand is an anti-ferritin antibody.

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

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

16. A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in claim 14 with a specimen that may contain the target substance.

17. 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 14 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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