Particle, colored particle, sensitization particle, measurement reagent, and measurement method

Abstract

The present disclosure provides a particle which exhibits excellent solvent resistance and is useful in, for example, an immunological measurement method. The present disclosure also provides: a colored particle, a sensitization particle and a measurement reagent obtained using such a particle; and a measurement method in which said sensitization particle is used. A surface of the particle of the present disclosure includes a group derived from a monomer represented by formula (1) or includes a group derived from a monomer represented by formula (2) and a group derived from a monomer represented by formula (3). Formula (2): CH2=C(R1)-C(=O)-O-(R4O)m-H. Formula (3): CH2=C(R1)-C(=O)-O-(R5-C(=O)-O)p-M.

Description

Particles, colored particles, sensitized particles, measuring reagents, and measuring methods

[0001] This disclosure relates to particles, colored particles, sensitized particles, measurement reagents, and measurement methods.

[0002] Particles are used in a wide variety of fields, including clinical testing. For example, in clinical testing, immunological assays utilizing antigen-antibody reactions are widely used as a method for quantifying trace amounts of test substances in a sample, and one such assay method involves using particles carrying antigens or antibodies.

[0003] Regarding conventional particles, for example, Patent Document 1 discloses a method for producing microparticle solid polymer beads that are insoluble in organic solvents, and Patent Document 2 discloses latent curing agent particles for epoxy resins that have excellent solvent resistance and other properties, and a method for producing the same.

[0004] JP-A-4-253707 JP-A-5-310888

[0005] Through diligent research, the inventors discovered that colored particles containing coloring agents are useful for a wide range of applications, including use as fluorescent labels in clinical tests. On the other hand, they found that if the solvent resistance of the particles before coloring agents is low, it is difficult to produce colored particles, and even if they can be produced, there are problems such as low fluorescence intensity.

[0006] This disclosure is made in view of the above-mentioned circumstances and aims to provide particles that have excellent solvent resistance, particularly to N,N-dimethylformamide, and are useful for, for example, immunological assays. This disclosure also aims to provide colored particles, sensitized particles, and assay reagents using such particles, as well as assay methods using said sensitized particles.

[0007] Disclosure 1 is a particle having a monomer-derived group represented by the following formula (1) on its surface.

[0008]

[0009] In the formula, R １ R represents a hydrogen atom or a methyl group. ２ O represents an oxyalkylene group with 2 to 10 carbon atoms. n represents R ２represents the average number of added moles of the oxyalkylene group represented by O, which is a number from 1 to 20. When n is 2 or more, R ２ O may be the same as or different from each other. R ３ represents a linear hydrocarbon chain having 1 to 10 carbon atoms or a hydrocarbon chain containing a benzene ring. M represents a hydrogen atom or a salt residue.

[0010] The present disclosure 2 is the particle according to the present disclosure 1, which has an average particle diameter of 50 to 1000 nm.

[0011] The present disclosure 3 is the particle according to the present disclosure 1 or 2, which further contains a group derived from a monomer represented by the following formula (2) on the surface. CH ２ =C(R １ )-C(=O)-O-(R ４ O) ｍ -H (2) In the formula, R １ represents a hydrogen atom or a methyl group. R ４ O represents an oxyalkylene group having 2 to 10 carbon atoms. m represents the average number of added moles of the oxyalkylene group represented by R ４ O, which is a number from 1 to 20. When m is 2 or more, R ４ O may be the same as or different from each other.

[0012] The present disclosure 4 is the particle according to any one of the present disclosures 1 to 3, which has a structural unit derived from the following (a) and / or (b), wherein the total amount of the monomer represented by the above formula (1), the following (a) and the following (b) is 50% by mass or more based on 100% by mass of the total amount of all monomers constituting the particle. (a) a monomer having a polymerizable group and an aryl group (b) a monomer having a polymerizable group and a sulfonic acid (salt) group, provided that (a) and (b) above do not include a monomer falling within the scope of the monomer represented by the above formula (1), and (a) above does not include a monomer falling within the scope of (b) above.

[0013] The present disclosure 5 is the particle according to any one of the present disclosures 1 to 4, which comprises a core portion and a shell portion covering at least a part of the core portion, and has a group derived from the monomer represented by the above formula (1) on the surface of the shell portion.

[0014] Disclosure 6 is a particle of Disclosure 3 or 4, comprising a core portion and a shell portion covering at least a part of the core portion, wherein the surface of the shell portion has a monomer-derived group represented by formula (1) and a monomer-derived group represented by formula (2).

[0015] Disclosure 7 is a particle of any one of Disclosures 1 to 4, comprising a shell portion and a hollow portion surrounded by the shell portion, and having a monomer-derived group represented by formula (1) on the surface of the shell portion.

[0016] Disclosure 8 is a particle of Disclosure 3 or 4, comprising a shell portion and a hollow portion surrounded by the shell portion, wherein the surface of the shell portion has a monomer-derived group represented by formula (1) and a monomer-derived group represented by formula (2).

[0017] Disclosure 9 is a particle having a monomer-derived group represented by the following formula (2) and a monomer-derived group represented by the following formula (3) on its surface. CH ２ = C(R １ )-C(=O)-O-(R ４ O) ｍ -H (2) CH ２ = C(R １ )-C(=O)-O-(R ５ -C (=O) -O) ｐ -M (3) where, R １ R represents a hydrogen atom or a methyl group, either identical or distinct. ４ O represents an oxyalkylene group with 2 to 10 carbon atoms. m represents R ４ This represents the average number of moles of oxyalkylene groups represented by O, and is a number between 1 and 20. Note that if m is 2 or greater, R ４ O may be the same or different from each other. ５ represents an alkylene group with 1 to 10 carbon atoms. p is (R ５ This represents the average number of moles added to a group represented as -C(=O)-O), and is an integer between 1 and 10. M represents a hydrogen atom or a salt residue.

[0018] The present disclosure 10 is a particle of the present disclosure 9, comprising a core portion and a shell portion covering at least a part of the core portion, wherein the surface of the shell portion has a monomer-derived group represented by formula (2) and a monomer-derived group represented by formula (3).

[0019] The present disclosure 11 is a particle of the present disclosure 9, comprising a shell portion and a hollow portion surrounded by the shell portion, wherein the surface of the shell portion has a monomer-derived group represented by formula (2) and a monomer-derived group represented by formula (3).

[0020] Disclosure 12 is a particle according to any one of Disclosures 1 to 11, having a surface carboxyl group content of 10 to 3000 μeq / g.

[0021] Disclosure 13 is one of the particles from Disclosures 1 to 12, wherein when the absorbance of the aqueous suspension of the particles at any of the measurement wavelengths of 400 nm, 550 nm, or 700 nm is set to 100%, the absorbance of an N,N-dimethylformamide suspension containing the particles at the same particle concentration as the aqueous suspension at the same measurement wavelength is 1% or more.

[0022] Disclosure 14 is a colored particle comprising any one of the particles described in Disclosures 1 to 13 and a coloring agent.

[0023] Disclosure 15 is a colored particle of Disclosure 14 in which the coloring agent contains a fluorescent dye.

[0024] Disclosure 16 is a particle of Disclosure 15, wherein the fluorescent dye is a cyanine-based dye.

[0025] Disclosure 17 is a sensitized particle obtained by binding a substance with specific affinity for the substance to be measured or a substance similar to the substance to be measured to any one of the colored particles of Disclosures 14 to 16.

[0026] Disclosure 18 is a sensitized particle of Disclosure 17, the surface of which is treated with bovine serum albumin.

[0027] Disclosure 19 is a measurement reagent comprising sensitized particles of Disclosure 17 or 18 dispersed in a buffer solution.

[0028] Disclosure 20 is a method for measuring the above-mentioned target substance in a sample by an immunoassay using sensitized particles according to Disclosure 17 or 18.

[0029] Disclosure 21 is the method of Disclosure 20, wherein the immunological measurement method is immunochromatography.

[0030] This disclosure provides particles that exhibit excellent solvent resistance, particularly to N,N-dimethylformamide, and are useful for applications such as immunological assays. Furthermore, this disclosure also provides colored particles, sensitized particles, and assay reagents using such particles, as well as assay methods using the sensitized particles.

[0031] This is the absorption spectrum of each suspension containing particle 1. This is the absorption spectrum of each suspension containing particle 4. This is the absorption spectrum of each suspension containing particle 31. This is the absorption spectrum of each suspension containing particle 32. This is the absorption spectrum of each suspension containing particle 20. This is a schematic diagram of the configuration of the immunochromatographic test strip for PSA measurement used in the example, viewed from the side. This is a cross-sectional SEM image of colored particle CX4.

[0032] (Particles) One aspect of the present disclosure is a particle having a group derived from the monomer represented by formula (1) (also referred to as monomer (1)) on its surface. This particle is also referred to as "particle A". By having a group derived from monomer (1) on its surface, particle A can exhibit excellent solvent resistance. Particle A is particularly excellent in its resistance to N,N-dimethylformamide (also referred to as DMF).

[0033] Another aspect of the present disclosure is a particle having a group derived from a monomer represented by formula (2) (also referred to as monomer (2)) and a group derived from a monomer represented by formula (3) (also referred to as monomer (3)) on its surface. This particle is also referred to as "particle B". By having both the group derived from monomer (2) and the group derived from monomer (3) on its surface, particle B can exhibit excellent solvent resistance that cannot be exhibited if only one of these groups is present. Particle B also exhibits particularly excellent resistance to DMF.

[0034] Particles A and B described above preferably have an average particle diameter of 50 to 1000 nm (i.e., 0.05 to 1 μm). When the average particle diameter is within this range, for example, when the particles are used for measurement by immunoturbidimetric method, the amount of optical change due to aggregation can be obtained more appropriately, resulting in higher sensitivity. Furthermore, when the particles are colored and used for immunological measurement, the reaction results can be appropriately obtained as optical signals such as absorbance or fluorescence, or as a visible change in color intensity, resulting in higher sensitivity. In addition, the preparation of sensitized particles becomes easier.

[0035] The lower limit of the average particle diameter is more preferably 100 nm or more, even more preferably 150 nm or more, and particularly preferably 180 nm or more. The upper limit of the average particle diameter is more preferably 800 nm or less, even more preferably 550 nm or less, and particularly preferably 530 nm or less. Note that particle A and particle B may be smaller particles. For example, the average particle diameter may be 500 nm or less, less than 500 nm, or 480 nm or less.

[0036] In this specification, average particle size refers to the volume-average particle size obtained by measurement using a laser diffraction / scattering particle size distribution analyzer (e.g., Beckman Coulter's "LS 13 320"). The measurement parameters are set appropriately according to the solvent used and the composition of the particles being measured. The validity of the measurement parameters is confirmed by measuring standard polystyrene particles (certified by a third-party organization) commercially available from Beckman Coulter and others, and ensuring that the particle size is within the manufacturer's specifications.

[0037] The particles A and B described above are preferably used in clinical tests, and more preferably in immunological assays. Although immunological assays will be described later, the particles A and B are even more preferably used in latex immunoturbidimetry (also known as LTIA). LTIA includes both sandwich methods and competitive methods. Furthermore, the particles A and B used in immunological assays using the LTIA method are included in preferred embodiments of this disclosure.

[0038] Particle A has a carboxyl group (which may be a carboxylate) derived from monomer (1) on its surface. Particle B also has a carboxyl group (which may be a carboxylate) derived from monomer (3) on its surface. In this case, specific affinity substances, as described later, can be chemically bonded to particle A or particle B. This homogenizes the orientation of the specific affinity substances, improving the specific reaction rate, significantly increasing detection sensitivity, and improving convergence. High convergence means that the error in the measurement results due to nonspecific reactions is small, and that the results correlate more closely with the true value or the reference measurement method.

[0039] The carboxyl group content on the surface of particle A and particle B is preferably 10 to 3000 μeq / g (i.e., 10 to 3000 nmol / mg), respectively. This facilitates the chemical bonding of specific affinity substances to the particles, for example. The carboxyl group content is more preferably 15 μeq / g or more, even more preferably 30 μeq / g or more, particularly preferably 50 μeq / g or more, and even more preferably 70 μeq / g or more. The carboxyl group content is also more preferably 1500 μeq / g or less, and even more preferably 1000 μeq / g or less.

[0040] The carboxyl group content on the particle surface is measured using a potentiometric automatic titrator (for example, the "AT-710" manufactured by Kyoto Electronics Manufacturing Co., Ltd.).

[0041] The absorbance Abs of particle A at any of the measurement wavelengths of 400 nm, 550 nm, or 700 nm is set to 100% for the aqueous suspension a1. Preferably, the absorbance Abs of DMF suspension a2 at the same measurement wavelength is 1% or more, more preferably 2% or more, even more preferably 5% or more, and particularly preferably 7% or more. Furthermore, when the absorbance Abs of aqueous suspension a1 at any of the measurement wavelengths of 400 nm, 550 nm, or 700 nm is set to 100%, the absorbance Abs of DMF suspension a2 at the same measurement wavelength is preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less. Aqueous suspension a1 is a suspension consisting of the particle A and pure water. DMF suspension a2 is a suspension consisting of the particle A and an N,N-dimethylformamide solution (referred to as DMF solution), and the particle concentration is the same as that of aqueous suspension a1. The DMF solution consists of DMF and pure water, and the volume ratio of DMF to pure water in the solution is DMF:water = 99.2:0.8.

[0042] In other words, the above particle A preferably has an absorbance of 1% or more, more preferably 2% or more, even more preferably 5% or more, particularly preferably 7% or more, and also preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less. In particular, for the above-mentioned particles A, it is preferable that the absorbance Abs of the DMF suspension a2 at a measurement wavelength of 400 nm is 1% or more, more preferably 2% or more, even more preferably 5% or more, particularly preferably 7% or more, and also preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less.

[0043] The absorbance Abs of the particles B at the measurement wavelength of 400 nm, 550 nm, or 700 nm is set to 100% for the aqueous suspension b1. Preferably, the absorbance Abs of the DMF suspension b2 at the same measurement wavelength is 1% or more, more preferably 2% or more, even more preferably 5% or more, and particularly preferably 7% or more. Furthermore, when the absorbance Abs of the aqueous suspension b1 at the measurement wavelength of 400 nm, 550 nm, or 700 nm is set to 100%, the absorbance Abs of the DMF suspension b2 at the same measurement wavelength is preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less. Aqueous suspension b1 is a suspension consisting of the particles B and pure water. DMF suspension b2 is a suspension consisting of the particles B and a DMF solution, with the particle concentration being the same as that of the aqueous suspension b1. As described above, the DMF solution consists of DMF and pure water, and the volume ratio of DMF to pure water in the solution is DMF:water = 99.2:0.8.

[0044] In other words, the above particle B preferably has an absorbance of 1% or more, more preferably 2% or more, even more preferably 5% or more, particularly preferably 7% or more, and also preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less. In particular, the above-mentioned particle B preferably has an absorbance Abs of 1% or more, more preferably 2% or more, even more preferably 5% or more, particularly preferably 7% or more, and also preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less.

[0045] Particles that meet the absorbance conditions described above are highly preferable because they have excellent solvent resistance and can suitably encapsulate colorants, for example, as will be described later.

[0046] Absorbance measurements will be performed using a Shimadzu UV-Vis spectrophotometer "UV-1850". The external environment during measurement will be a temperature of 25°C and a humidity of 50% RH.

[0047] The absorbance Abs of the tetrahydrofuran suspension (also referred to as THF suspension) at the measurement wavelength of any of the above particles A and B is preferably 1% or more, when the absorbance Abs of the aqueous suspension a1 or b1 at any of the measurement wavelengths of 400 nm, 550 nm, or 700 nm is set to 100%, and the absorbance Abs of the acetone suspension at the measurement wavelength is also preferably 1% or more. When the absorbance Abs of the aqueous suspension a1 or b1 at the measurement wavelength is set to 100%, the absorbance Abs of the THF suspension or acetone suspension at the measurement wavelength is more preferably 2% or more, even more preferably 5% or more, particularly preferably 7% or more, and preferably 80% or less, more preferably 50% or less, and even more preferably 20% or less. The THF suspension is a suspension consisting of the above particles and a tetrahydrofuran solution (also referred to as THF solution), and the particle concentration is the same as that of the aqueous suspension a1 or b1. The acetone suspension is a suspension consisting of the above particles and an acetone solution, and the particle concentration is the same as that of the above aqueous suspension a1 or b1. The THF solution consists of THF and pure water, and the volume ratio of THF to pure water in the solution is THF:water = 99.2:0.8. The acetone solution consists of acetone and pure water, and the volume ratio of acetone to pure water in the solution is acetone:water = 99.2:0.8.

[0048] The particles A and B described above may be non-magnetic particles. To say that a particle is non-magnetic is equivalent to saying that it substantially does not contain magnetic material. More specifically, the amount of magnetic material may be less than 1% by mass or less than 0.1% by mass in 100% by mass of the total amount of particles A or B. The magnetic material refers to an iron oxide-based substance, specifically ferrite or magnetite.

[0049] The specific compositions of particle A and particle B described above will be explained in detail below. In this specification, (meth)acryloyl group means methacryloyl group and / or acryloyl group, and (meth)acrylic acid means methacrylic acid and / or acrylic acid.

[0050] (Particle A) Particle A contains a group on its surface derived from a monomer represented by the following formula (1). The monomer (1) that gives the group may be one type of monomer or two or more types of monomers.

[0051]

[0052] In the formula, R １ R represents a hydrogen atom or a methyl group. ２ O represents an oxyalkylene group with 2 to 10 carbon atoms. n represents R ２ This represents the average number of moles of oxyalkylene groups represented by O, and is a number between 1 and 20. Note that if n is 2 or greater, R ２ O may be the same or different from each other. ３ represents a linear hydrocarbon chain having 1 to 10 carbon atoms, or a hydrocarbon chain containing a benzene ring. M represents a hydrogen atom or a salt residue.

[0053] In the above formula (1), R ２ The number of carbon atoms in the oxyalkylene group represented by O is 2 to 10, but from the viewpoint of improving solvent resistance, 2 to 5 is preferred, 2 to 3 is more preferred, and 2 is even more preferred. n is a number from 1 to 20, but from the viewpoint of improving solvent resistance, 1 to 15 is preferred, 1 to 10 is more preferred, and 1 to 5 is even more preferred.

[0054] R ３ This represents a linear hydrocarbon chain having 1 to 10 carbon atoms, or a hydrocarbon chain containing a benzene ring.

[0055] R ３ When represents a linear hydrocarbon chain, the number of carbon atoms in the hydrocarbon chain is 1 to 10. From the viewpoint of improving solvent resistance, the number of carbon atoms is preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 2. Furthermore, the linear hydrocarbon chain is preferably an alkylene group.

[0056] R ３When represents a hydrocarbon chain containing a benzene ring, the number of carbon atoms other than those constituting the benzene ring is preferably 0 to 10, more preferably 0 to 5, and 0 (i.e., R ３ It is even more preferable that (where represents a phenylene group or a naphthylene group). ３ If R is a phenylene group, ３ R may be any of o-phenylene, m-phenylene, or p-phenylene groups, but o-phenylene is preferred. ３ If R is a naphthylene group, ３ The group may be either an α-naphthylene group or a β-naphthylene group. Of the phenylene group and naphthylene group, the phenylene group is preferred.

[0057] M represents a hydrogen atom or a salt residue. That is, monomer (1) has a carboxyl group or a carboxylate salt group. A carboxylate salt group means a salt in which the proton of the carboxyl group is replaced with any cation. Examples of the salt include sodium salt, potassium salt, lithium salt, ammonium salt, and the like.

[0058] The monomer (1) is preferably at least one selected from the group consisting of 2-methacryloyloxyethyl succinic acid (also referred to as 2-methacryloyloxyethyl succinic acid monoester), poly(ethylene glycol) methacrylate succinic acid monoester, acryloyloxyethyl phthalic acid (also referred to as 2-acryloyloxyethyl phthalic acid monoester), and salts thereof. When monomer (1) is a salt of the above compound, it is particularly preferably a sodium salt. Particles A having such monomer (1)-derived groups on their surface exhibit even greater solvent resistance. For example, when colored particles are prepared using particle A as described later, the colored particles will have higher fluorescence intensity. Furthermore, when these colored particles are used in immunological measurements, the reaction results can be more appropriately obtained as optical signal amounts such as absorbance and fluorescence, or as visible changes in color intensity, resulting in even higher sensitivity. The preparation of sensitized particles also becomes easier.

[0059] The average number of moles of ethylene glycol (i.e., oxyethylene groups) added to poly(ethylene glycol) succinate monoester methacrylate corresponds to n above, but 5 is the most preferred.

[0060] The blending ratio of the above monomer (1) is preferably set so that the carboxyl groups (which may be carboxylate salts) on the particle surface are within the preferred range described above. For example, of the total amount of all monomers constituting the above particle A, it is preferable that the above monomer (1) is 0.1 to 40% by mass, more preferably 1.5 to 30% by mass, even more preferably 5 to 20% by mass, and particularly preferably 10 to 15% by mass.

[0061] Preferably, the particle A described above also contains a hydroxyl group or a cyclic ether group on its surface. In this case, the hydrophilicity of particle A is improved, and the balance between hydrophobicity and hydrophilicity on the particle surface becomes better. As a result, for example, when sensitized particles are obtained using particle A, the adsorption of substances other than specific affinity substances onto the particles is sufficiently suppressed, and the convergence is further improved.

[0062] The hydroxyl group or cyclic ether group on the particle surface is preferably derived from a monomer (also referred to as monomer (c)) containing a polymerizable group and a hydroxyl group and / or a cyclic ether group. That is, the particle A is preferably further containing a group derived from monomer (c) containing a polymerizable group and a hydroxyl group and / or a cyclic ether group. The monomer (c) that gives the group may be one monomer or two or more monomers. The monomer (c) may also include monomers that have a polymerizable group and a functional group (e.g., a sulfonic acid group) whose group before proton dissociation contains a hydroxyl group (or a cyclic ether group) and which can generate a negative charge upon proton dissociation.

[0063] With respect to the monomer (c) described above, the polymerizable group is preferably a radically polymerizable polymerizable group, and more preferably a vinyl group or a (meth)acryloyl group. Furthermore, the number of hydroxyl groups or cyclic ether groups (or the total number if both are present) in the monomer (c) is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2 in a single monomer.

[0064] Preferred examples of the above monomer (c) include, for example, the monomer represented by the following formula (4), the monomer represented by the following formula (5), and the monomer having a polymerizable group and a sulfobetaine structure.

[0065] CH ２ = C(R １ )-(C(=O)-O) ｎ -X-Y (4) where, R １ represents a hydrogen atom or a methyl group. n is 0 or 1. X is an alkylene group having 1 to 10 carbon atoms, or -(R ４ O) ｍ R represents an alkylene glycol chain. ４ O represents an oxyalkylene group with 1 to 10 carbon atoms. m represents R ４ This represents the average number of moles of oxyalkylene groups represented by O, and is a number between 1 and 20. Note that if m is 2 or greater, R ４ O may be the same or different from each other. Y represents a hydrogen atom or a cyclic ether group.

[0066] In formula (4) above, when X represents an alkylene group, the number of carbon atoms is 1 to 10, but in order to achieve a better balance between hydrophobicity and hydrophilicity of the particle surface, the number of carbon atoms is preferably 1 to 8, and more preferably 1 to 5. Also, when X represents an alkylene glycol chain, R ４ The oxyalkylene group represented by O has 1 to 10 carbon atoms, but to achieve a better balance between hydrophobicity and hydrophilicity on the particle surface, the number of carbon atoms is preferably 1 to 8, and more preferably 1 to 5. m is a number from 1 to 20, but from a similar viewpoint, it is preferably 1 to 15, and more preferably 1 to 10.

[0067] Preferred examples of monomers represented by the above formula (4) include, for example, 2-hydroxyethyl (meth)acrylate, monoethylene glycol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, β-methallyl alcohol, glycidyl (meth)acrylate, and the like.

[0068] CH ２ = C(R １ )-C(=O)-OR ６ -A (5) where R１ R represents a hydrogen atom or a methyl group. ６ represents an alkylene group with 2 to 10 carbon atoms. A represents a phosphorylcholine group.

[0069] Examples of monomers represented by the above formula (5) include 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate.

[0070] Regarding the monomer having the polymerizable group and the sulfobetaine structure described above, the polymerizable group is as described above. The sulfobetaine structure is R ７ A positively charged ammonium group ((R)) is formed via any group represented by ８ ) (Caution ９ ) (Caution １０ ) N ＋ ) and a negatively charged sulfonic acid group (-SO ３ － ) and the structure includes R ７ For example, R is a hydrocarbon group having 1 to 10 carbon atoms. ８ , R ９ and R １０ These are preferably hydrocarbon groups having 1 to 10 carbon atoms, and are either identical or different from each other. ８ A double or triple bond is formed between the nitrogen atom (N) and R ９ Ya R １０ It is not necessary for it to exist.

[0071] Examples of monomers having the polymerizable group and sulfobetaine structure include 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonic acid.

[0072] Among these, monomer (c) is preferably a monomer represented by formula (4) above, and more preferably a monomer represented by formula (2) below. That is, particle A is particularly preferably further containing a group derived from the monomer represented by formula (2) below. Such particle A can more fully exhibit the above effects due to containing a hydroxyl group or a cyclic ether group, and for example, when colored particles are prepared using particle A as described later, the colored particles will have a higher fluorescence intensity. Furthermore, when the colored particles are used in immunological measurements, the reaction results can be obtained more appropriately as optical signal amounts such as absorbance and fluorescence, or as visible changes in color intensity, resulting in even higher sensitivity. The preparation of sensitized particles also becomes easier.

[0073] CH ２ = C(R １ )-C(=O)-O-(R ４ O) ｍ -H (2) R in equation １ , R ４ O and m are as described above. Note that in equation (2), R ４ The number of carbon atoms in the oxyalkylene group represented by O is preferably 2 to 10. In particular, from the viewpoint of achieving a good balance between hydrophobicity and hydrophilicity of the particle surface, the number of carbon atoms is more preferably 2 to 8, and even more preferably 2 to 5.

[0074] Preferred examples of monomers represented by formula (2) above include, for example, 2-hydroxyethyl (meth)acrylate, monoethylene glycol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, and the like.

[0075] When particle A contains a group derived from monomer (c), it is preferable that the proportion of monomer (c) in 100% by mass of the total amount of all monomers constituting particle A is 0.1 to 60% by mass. In particular, it is preferable that particle A further contains a group derived from monomer (2) on its surface, and that the proportion of monomer (2) in 100% by mass of the total amount of all monomers constituting particle A is 0.1 to 60% by mass, more preferably 1 to 50% by mass, even more preferably 1.5 to 40% by mass, and particularly preferably 5 to 35% by mass. When colored particles are produced using such particle A, the colored particles will have an even higher fluorescence intensity.

[0076] The above particle A also preferably has structural units derived from (a) and / or (b) below: (a) a monomer having a polymerizable group and an aryl group; (b) a monomer having a polymerizable group and a sulfonic acid (salt) group. However, (a) and (b) above do not include monomers corresponding to monomer (1), and (a) above does not include monomers corresponding to (b). (a) and (b) above also do not include monomers corresponding to monomer (2).

[0077] Regarding (a) above, the polymerizable group is preferably a polymerizable group that can be radically polymerized, and more preferably a vinyl group. Examples of aryl groups include a phenyl group, a naphthyl group, and an anthracenyl group. Among these, a phenyl group is preferred as the aryl group from the viewpoint of making it easier to control the volume-average particle size of particle A. Having an aryl group in particle A makes it possible to more effectively detect, optically, the change in turbidity when a sensitized particle is obtained using particle A and a substance that binds to the sensitized substance (e.g., an antigen or antibody) supported on the surface of the particle binds to it.

[0078] Specific examples of (a) above include, for example, styrene monomers such as styrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, 4-vinylbenzoic acid, divinylbenzene, and vinyltoluene; naphthalene monomers such as 1-vinylnaphthalene, 2-vinylnaphthalene, α-naphthyl (meth)acrylate, and β-naphthyl (meth)acrylate; and so on. Among these, styrene and / or 1-vinylnaphthalene are preferred. That is, (a) above preferably contains styrene and / or 1-vinylnaphthalene, and more preferably contains styrene. When particle A has a structural unit derived from (a), (a) that provides the structural unit may be one monomer or two or more monomers.

[0079] The above (b) comprises a polymerizable group and a sulfonic acid group and / or a sulfonate group. The polymerizable group is preferably a radically polymerizable group, and more preferably a vinyl group. The sulfonate group means a salt in which the proton of the sulfonic acid group is substituted with any cation. The number of sulfonic acid groups (or salts thereof) in the above (b) is preferably one or two per monomer, and more preferably one, from the viewpoint of easily controlling the surface charge of the particle A.

[0080] The above (b) preferably further contains an aryl group, and more preferably the sulfonic acid group (or salt thereof) of the above (b) substitutes for any hydrogen atom of the aryl group. Specific examples of aryl groups are as described above, with the phenyl group being particularly preferred.

[0081] Specific examples of (b) above include, for example, styrene sulfonic acid, divinylbenzene sulfonic acid, o-methylstyrene sulfonic acid, p-methylstyrene sulfonic acid, ethylstyrene sulfonic acid, vinylnaphthalene sulfonic acid, and salts thereof. Examples of these salts include sodium salts, potassium salts, lithium salts, and ammonium salts.

[0082] The above (b) may also be, for example, sodium 2-methyl-2-propene-1-sulfonate, potassium 3-sulfopropyl methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid, vinyl sulfonic acid, sodium vinyl sulfonate, 2-acrylamido-2-methylpropanesulfonic acid, sodium 2-acrylamido-2-methylpropanesulfonate, etc. Among these, styrene sulfonates are preferred, and sodium styrene sulfonate is more preferred. That is, the above (b) preferably contains styrene sulfonates, and more preferably contains sodium styrene sulfonate. If the above particle A has a structural unit derived from the above (b), the above (b) that gives the structural unit may be one monomer or two or more monomers.

[0083] It is preferable that the total amount of monomer (1), (a), and (b) in the total amount of all monomers constituting particle A is 50% by mass or more, more preferably 55% by mass or more, and even more preferably 60% by mass or more.

[0084] Furthermore, the ratios of (a) and (b) above to 100% by mass of the total amount of all monomers constituting the particle A are preferably 0 to 99.9% by mass and 0 to 10% by mass, more preferably 30 to 99% by mass and 0 to 5% by mass, and even more preferably 50 to 98% by mass and 0 to 2% by mass, respectively.

[0085] From the viewpoint of exhibiting the above-mentioned effects, the particle A is preferably a core-shell type particle having a group derived from monomer (1) on its surface, and more preferably a core-shell type particle having a group derived from monomer (1) and a hydroxyl group or a cyclic ether group on its surface. More specifically, the particle A comprises a core portion and a shell portion covering at least a part of the core portion, and it is preferable that the surface of the shell portion has a group derived from monomer (1), and more preferably that the surface of the shell portion has a group derived from monomer (1) and a group derived from monomer (2).

[0086] For example, from the viewpoint of manufacturing efficiency, if the particle A is a core-shell type particle, it is preferable that the core portion be made of a non-crosslinked polymer. A non-crosslinked polymer is a polymer that does not have a crosslinked structure in its polymer structure. In this case, since the particle A made of a non-crosslinked polymer has a lower particle density, it is expected that the dispersion stability in solution will be improved.

[0087] The particle A, which is a core-shell type particle, is preferably manufactured by performing multiple polymerization steps. For example, the method for manufacturing the particle A is preferably a method that includes a first polymerization step of polymerizing a first monomer component, and a second polymerization step of adding a second monomer component to the reactant obtained in the first polymerization step and polymerizing it further.

[0088] The first monomer component preferably contains at least one of (a) and (b) above, and more preferably contains both (a) and (b). For example, the blending amounts of (a) and (b) based on 100% by mass of the total amount of the first monomer component are preferably 60 to 100% by mass and 0 to 40% by mass, respectively, and more preferably 90 to 100% by mass and 0 to 10% by mass, respectively.

[0089] The second monomer component added in the second polymerization step preferably contains monomer (1), and more preferably contains monomer (1) and monomer (2). The second monomer component may further contain at least one of (a) and (b). The blending amounts of monomer (1) and monomer (2) are preferably set so that the respective content relative to the total amount of all monomers constituting particle A (in this case, the total amount of the first monomer component and the second component) is within the range described above.

[0090] During polymerization, it is preferable not to use surfactants, which are widely used as emulsifiers (for example, alkali metal salts of alkylbenzenesulfonic acid). If surfactants are used, residual surfactants may remain in the resulting particles, potentially interfering with the sensitization (immobilization) of antigens or antibodies to the particle surface. However, by performing the polymerization process without using surfactants, this possibility can be eliminated. For example, if particle A is used as the sensitized particle, the amount of specific affinity substances (e.g., antigens or antibodies) adsorbed by the particle will be sufficient, and higher sensitivity may be obtained.

[0091] As for the polymerization method, any conventionally known method can be used, except that the polymerization step is carried out in an aqueous medium without the use of a surfactant. For example, one method involves adding the above monomer components and polymerization initiator to a reaction vessel containing an aqueous medium as a solvent, and heating it while stirring under a nitrogen atmosphere.

[0092] The above aqueous medium may be water, or it may be a mixed solvent of water and an organic solvent. For example, the mixed solvent may be water and a monohydric alcohol having 1 to 4 carbon atoms ("C"). １－４ Examples include mixed solvents containing (also called "alcohol"), and C １－４ It is preferable that it contains 7.5 to 25% by weight of alcohol. １－４ Examples of alcohols include linear alcohols such as methanol and ethanol; and branched alcohols such as isopropyl alcohol and t-butyl alcohol. Among these, water and / or ethanol are preferred as aqueous media.

[0093] As the polymerization initiator mentioned above, known radical initiators are preferably used. Specifically, examples include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; azo compounds such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), and 2,2'-azobis-2,4-dimethylvaleronitrile; and organic peroxides such as benzoyl peroxide, di-t-butyl peroxide, lauroyl peroxide, and t-butylperoxy-2-ethylhexanoate. Among these, persulfates are preferred, and potassium persulfate is more preferred.

[0094] The amount of polymerization initiator used is not particularly limited, but is usually 0.01 to 1 part by mass per 100 parts by mass of the total amount of monomer components.

[0095] The polymerization temperature is preferably 50 to 100°C, and more preferably 60 to 85°C. The polymerization time depends on conditions such as the monomer composition, concentration, and polymerization initiator, but is usually 5 to 50 hours.

[0096] The above-mentioned particle A is obtained in a suspended state in water or a mixed solvent. The concentration of particle A in the suspension is not particularly limited, but is usually 1 to 20% by mass. Using such a suspension makes it easier to prepare, for example, the sensitized particles described later.

[0097] The particle A described above is also preferably a hollow particle. More specifically, the particle A comprises a shell portion and a hollow portion surrounded by the shell portion, and preferably has a group derived from monomer (1) on the surface of the shell portion, and more preferably has a group derived from monomer (1) and a group derived from monomer (2) on the surface of the shell portion. If the particle A is such a hollow particle, it is preferable from the viewpoint of coloration and from the viewpoint of suppressing natural sedimentation. In this specification, a hollow particle means a particle having a cavity (i.e., a hollow portion) inside the particle, and is not limited to particles in which air is present in the hollow portion, but may also be a particle in which a gas other than air is present in the hollow portion, or a particle in which various solvents are present in the hollow portion. The hollow portion means a portion that is less dense than the shell portion and is composed of part or all of the core portion, and part or all of the core portion may be a complete cavity.

[0098] The hollow particles A can be produced, for example, by obtaining core-shell type particles A as described above, and then immersing the particles in an organic solvent to swell them. That is, the method for producing the hollow particles A preferably includes a first polymerization step of polymerizing a first monomer component, a second polymerization step of adding a second monomer component to the reactant obtained in the first polymerization step and polymerizing it further, and a swelling step of immersing the polymer obtained in the second polymerization step in an organic solvent to swell it. The first polymerization step and the second polymerization step are as described above in the method for obtaining core-shell type particles A. The swelling step is the same as step 1 (step 1 which may be included in the method for obtaining colored particles) described later. It is thought that hollow particles can be obtained by the swelling step, in which the core portion of the core-shell type particles dissolves in the organic solvent or the core portion is made less dense.

[0099] (Particle B) Particle B has a group derived from monomer (2) and a group derived from monomer (3) on its surface. Note that monomers (2) and (3) that give these groups may each be one type of monomer or two or more types of monomers.

[0100] Details and preferred embodiments of the above monomer (2) are provided by reference to the description of particle A above. The proportion of monomer (2) in the total amount of all monomers constituting particle B is preferably 0.1 to 60% by mass, more preferably 0.5 to 50% by mass, even more preferably 1 to 40% by mass, and particularly preferably 1.5 to 36% by mass.

[0101] CH ２ = C(R １ )-C(=O)-O-(R ５ -C (=O) -O) ｐ -M (3) where, R １ R represents a hydrogen atom or a methyl group, either identical or distinct. ５ represents an alkylene group with 1 to 10 carbon atoms. p is (R ５ This represents the average number of moles added to a group represented as -C(=O)-O), and is an integer between 1 and 10. M represents a hydrogen atom or a salt residue.

[0102] In the above formula (3), R ５ represents an alkylene group having 1 to 10 carbon atoms. From the viewpoint of improving solvent resistance, the number of carbon atoms is preferably 2 to 8, and more preferably 2 to 5. Also, p is an integer from 1 to 10, but is preferably 2 to 8, and more preferably 2 to 5.

[0103] M represents a hydrogen atom or a salt residue. That is, the monomer (3) has a carboxyl group or a carboxylate salt group. Examples of the salt include sodium salt, potassium salt, lithium salt, ammonium salt, etc., but sodium salt is preferred.

[0104] The blending ratio of the above monomer (3) is preferably set so that the carboxyl groups (which may be carboxylate salts) on the particle surface are within the preferred range described above. For example, of the total amount of all monomers constituting the above particle B, it is preferable that the proportion of the above monomer (3) is 0.1 to 40% by mass, more preferably 1 to 30% by mass, and even more preferably 1.5 to 25% by mass.

[0105] The particle B described above preferably also has structural units derived from (a) and / or (b). Here, (a) does not include monomers corresponding to monomer (2) and monomer (3), and (b) also does not include monomers corresponding to monomer (2) and monomer (3). Details of (a) and (b) and preferred embodiments are as described above.

[0106] It is preferable that the total amount of monomer (2), monomer (3), (a), and (b) in the total amount of all monomers constituting particle B is 50% by mass or more, more preferably 55% by mass or more, and even more preferably 60% by mass or more. Furthermore, the ratios of (a) and (b) to the total amount of all monomers constituting particle B is preferably 0 to 99.9% by mass and 0 to 10% by mass, more preferably 30 to 99% by mass and 0 to 5% by mass, and even more preferably 50 to 98% by mass and 0 to 2% by mass, respectively.

[0107] From the viewpoint of exhibiting the above-mentioned effects, it is preferable that the particle B is a core-shell type particle having a group derived from monomer (2) and a group derived from monomer (3) on its surface. More specifically, it is preferable that the particle B comprises a core portion and a shell portion covering at least a part of the core portion, and that the surface of the shell portion has a group derived from monomer (2) and a group derived from monomer (3).

[0108] If the particle B is a core-shell type particle, the core portion is preferably composed of a non-crosslinked polymer. Furthermore, the core-shell type particle B is preferably manufactured by performing multiple polymerization steps. For example, the method for manufacturing the particle B is preferably a method that includes a first polymerization step of polymerizing a first monomer component, and a second polymerization step of adding a second monomer component to the reactant obtained in the first polymerization step and polymerizing it further.

[0109] The first monomer component preferably contains at least one of (a) and (b) above, and more preferably contains both (a) and (b). For example, the blending amounts of (a) and (b) based on 100% by mass of the total amount of the first monomer component are preferably 60 to 100% by mass and 0 to 40% by mass, respectively, and more preferably 90 to 100% by mass and 0 to 10% by mass, respectively.

[0110] The second monomer component added in the second polymerization step preferably includes monomer (2) and monomer (3). The second monomer component may also further include at least one of (a) and (b). The blending amounts of monomer (2) and monomer (3) are preferably set so that the respective amounts of each monomer relative to the total amount of all monomers constituting particle B (in this case, the total amount of the first monomer component and the second component) are within the ranges described above.

[0111] Similar to the method for producing particle A as described above, it is preferable not to use surfactants, which are widely used as emulsifiers, during polymerization. The polymerization method, usable aqueous media, polymerization initiator and its amount, as well as the polymerization temperature and polymerization time, are as described above.

[0112] The above-mentioned particle B is obtained in a suspended state in water or a mixed solvent. The concentration of particle B in the suspension is not particularly limited, but is usually 1 to 20% by mass. Using such a suspension makes it easier to prepare sensitized particles, for example, as described later.

[0113] The particle B is also preferably a hollow particle. More specifically, the particle B comprises a shell portion and a hollow portion surrounded by the shell portion, and preferably the surface of the shell portion has groups derived from monomer (2) and groups derived from monomer (3). If the particle B is a hollow particle, it is preferable from the viewpoint of both coloring and suppression of natural sedimentation.

[0114] The hollow particles B can be produced, for example, by obtaining core-shell type particles B as described above and then immersing the particles in an organic solvent to cause swelling. That is, the method for producing the hollow particles B is preferably a method comprising: a first polymerization step of polymerizing a first monomer component; a second polymerization step of adding a second monomer component to the reaction product obtained in the first polymerization step and further performing polymerization; and a swelling step of immersing the polymer obtained in the second polymerization step in an organic solvent to cause swelling. The first polymerization step and the second polymerization step are as described above in the production method for obtaining core-shell type particles B. The swelling step is the same as step 1 described later (a step that can be included in the production method for obtaining colored particles). It is considered that hollow particles are obtained through the swelling step because the core portion of the core-shell type particles dissolves in the organic solvent or the density of the core portion is reduced.

[0115] (Colored Particles) Another aspect of the present disclosure relates to colored particles. The colored particles comprise the particles A and / or the particles B, and a colorant. The colorant is preferably included inside the particles A or the particles B.

[0116] Examples of the colorant include pigments, dyes and the like. The colorant may be used alone or in combination of two or more thereof.

[0117] The colorant is preferably a fluorescent dye. That is, the colorant preferably comprises a fluorescent dye. In this case, the colored particles are useful, for example, as fluorescent labels in biochemical tests.

[0118] Examples of the fluorescent dye include cyanine dyes, perylene dyes, luciferin dyes, coumarin dyes, rare earth complex dyes, and the like. Among these, from the viewpoint of being more advantageous for fluorescent labeling, the fluorescent dye is preferably a cyanine dye. As the cyanine dye, for example, Cy5-based dyes are preferable. Examples of Cy5-based dyes include Sulfo-Cy5 acid, Cy5 acid and DiIC １８ (5) is preferable, Cy5 acid and DiIC １８ (5) is more preferable, DiIC １８ (5) is most preferable.

[0119] A manufacturing method for obtaining the above-mentioned colored particles preferably includes, for example, a step 1 of immersing the particles A and / or particles B in an organic solvent to swell them, and a step 2 of immersing the swollen particles obtained in step 1 in a coloring agent solution containing a coloring agent. The above manufacturing method also preferably further includes a step 3 of replacing the organic solvent with water after step 2.

[0120] The organic solvent used in step 1 is preferably an organic solvent in which the particles exhibit excellent solvent resistance. Specifically, for example, the organic solvent preferably contains at least one selected from the group consisting of DMF, acetone, tetrahydrofuran (also called THF), ethyl acetate, toluene, and chloroform, more preferably contains at least one selected from the group consisting of DMF, acetone, and THF, and even more preferably contains DMF. Since particles A and B have particularly excellent solvent resistance in DMF, when DMF is used in step 1, the dissolution of the particles in DMF is sufficiently suppressed, and colored particles are more preferably obtained.

[0121] In step 1 described above, the time for immersing the particles in the organic solvent is preferably 0.1 to 3 hours, and more preferably 0.5 to 2 hours, considering factors such as manufacturing efficiency and swelling efficiency.

[0122] The coloring agent solution used in step 2 is a solution containing a coloring agent and an organic solvent. The organic solvent used here is also preferably an organic solvent in which the particles exhibit excellent solvent resistance, and it is more preferable that the same organic solvent used in step 1 is used.

[0123] In the above-mentioned coloring agent solution, the concentration of the coloring agent is not particularly limited, but for example, it is preferably 0.1 to 50 mM in molar concentration, and more preferably 0.2 to 10 mM.

[0124] Step 2 described above is preferably a step of immersing the swollen particles in a coloring agent solution. The immersion time of the swollen particles in the coloring agent solution is preferably 0.1 to 3 hours, and more preferably 0.5 to 2 hours, considering, for example, manufacturing efficiency and the adsorption efficiency of the coloring agent.

[0125] Step 1 and Step 2 described above may be performed simultaneously. That is, the above manufacturing method may include Step 4, in which particles A and / or particles B are immersed in a coloring agent solution containing a coloring agent. The coloring agent solution is as described above. The immersion time in Step 4 is preferably, for example, 0.1 to 6 hours, and more preferably 1 to 4 hours. The manufacturing method including Step 4 preferably further includes Step 3, in which the organic solvent is replaced with water, after Step 4.

[0126] Step 3 described above is the step of replacing the organic solvent with water. The water used here is, for example, pure water. In this step, the replacement with water can be done using a generally accepted method.

[0127] The above-mentioned colored particles preferably have an average particle diameter of 50 to 2000 nm. When the average particle diameter is within this range, for example, when the above-mentioned colored particles are used in an immunological assay, the reaction results can be appropriately obtained as optical signals such as absorbance or fluorescence, or as a visible change in color intensity, resulting in higher sensitivity and easier preparation of sensitized particles. The lower limit of the average particle diameter is more preferably 100 nm or more, and even more preferably 200 nm or more. The upper limit of the average particle diameter is more preferably 1500 nm or less, and even more preferably 1000 nm or less. The above-mentioned average particle diameter is the average particle diameter of the colored particles measured when the colored particles are suspended in pure water.

[0128] (Sensitized Particles) Another aspect of the present disclosure is sensitized particles. These sensitized particles are formed by binding a specific affinity substance for the substance to be measured or an analogue of the substance to be measured to the colored particles of the present disclosure described above. In this specification, this "specific affinity substance for the substance to be measured or an analogue of the substance to be measured" is also referred to as "specific affinity substances." Furthermore, the inventors have also discovered sensitized particles formed by binding specific affinity substances to particles A and / or particles B of the present disclosure described above. When these sensitized particles are used in immunological assays, detection sensitivity is improved, and the influence of nonspecific reactions is suppressed, resulting in excellent convergence. Hereinafter, the particles constituting the sensitized particles (i.e., at least one of the colored particles, particles A and particles B described above) will also be referred to as particle Z.

[0129] The specific affinity substances are not particularly limited as long as they are ordinary immunoserological reagents (i.e., reagents used in immunological agglutination and agglutination inhibition reactions) or substances commonly used in biochemical assays. In particular, the specific affinity substances are preferably substances that can be used in antigen-antibody reactions. For example, the specific affinity substance may be an antibody against the substance to be measured, or if the substance to be measured is an antibody, the specific affinity substance may be an antigen. As will be described later, immunological measurements can be performed using the sandwich method with the above-mentioned sensitized particles, or immunological measurements can be performed using the competitive method. In the case of the competitive method, a substance similar to the substance to be measured can be used as the specific affinity substance. In the case of latex immunoturbidimetry, the sandwich method is sometimes called an immunological agglutination reaction, and the competitive method is sometimes called an immunological agglutination inhibition reaction.

[0130] Substances usable in the above antigen-antibody reaction include, for example, antigens or antibodies such as sugars, proteins, nucleic acids, nucleoproteins, hormones such as estrogen, and lipids. Specifically, antigens include, for example, various antigens, receptors, enzymes, etc. More specifically, examples include β2-microglobulin, C-reactive protein (CRP), human fibrinogen, ferritin, rheumatoid factor (RA), α-fetoprotein (AFP), prostate-specific antigen (PSA), mycoplasma antigen, HBs antigen, etc. Antibodies include, for example, antibodies against various toxins and pathogens, more specifically, examples include anti-streptolysin O antibodies, anti-estrogen antibodies, β2-microglobulin antibodies, syphilis treponema antibodies, antibodies against syphilis lipid antigens, anti-HBs antibodies, anti-HBc antibodies, anti-Hbe antibodies, anti-PSA antibodies, anti-CRP antibodies, etc. Note that the above antibodies include not only the immunoglobulin molecule itself, but also, for example, F(ab') ２ Antibody fragments like the one shown may be used. Furthermore, either polyclonal or monoclonal antibodies may be used as the antibody. The method for obtaining the antibody can also be the commonly used method.

[0131] In this specification, the terms “antigen-antibody reaction,” “antigen,” and “antibody” may include, in addition to their usual meanings, any of the above concepts and forms that can bind to each other on the surface of sensitized particles by specific binding reactions, and should not be interpreted restrictively.

[0132] The sensitized particles described above are formed by binding specific affinity substances to the particle Z, but it is preferable that the specific affinity substances are chemically bonded to the surface of the particle Z. In other words, it is preferable that the bond between the particle Z and the specific affinity substances is mainly chemical. This improves the sensitivity of the sensitized particles and also improves their focusing ability.

[0133] Sensitized particles in which the two substances are bonded primarily by chemical bonding exhibit significantly higher sensitivity than sensitized particles in which the two substances are bonded primarily by physical adsorption. In addition to this difference in sensitivity, the presence of carboxyl groups on the surface of the particle Z (preferably also the content of carboxyl groups on the surface) suggests that the particle Z and the specific affinity substances are bonded primarily by chemical bonding, rather than primarily by physical adsorption. It should be noted that, to the extent that it does not hinder the effects of this disclosure, sensitized particles in which the particle Z and the specific affinity substances are physically adsorbed may be included as part of the sensitized particles.

[0134] A chemical bonding method is preferred for supporting specific affinity substances on the above-mentioned particles Z. Specifically, for example, after activating the carboxyl groups of the above-mentioned particles Z, the particles with activated carboxyl groups are brought into contact with the specific affinity substances in an aqueous medium, thereby supporting (sensitizing) the specific affinity substances on the above-mentioned particles Z.

[0135] In the above-described sensitized particles, the amount of specific affinity substances loaded varies depending on the type of specific affinity substance used, and the experimentally optimal amount can be appropriately set. In this specification, the terms "loading," "sensitization," and "immobilization" have their usual meanings and are used synonymously.

[0136] The sensitized particles described above may be coated (blocked) with bovine serum albumin (also known as BSA) or the like, if necessary. This further improves the convergence of the measurement. The surface of the sensitized particles being treated with BSA in this manner is also a preferred form of sensitized particles according to this disclosure.

[0137] The sensitized particles may be dispersed in a buffer solution. A measurement reagent in which the sensitized particles are dispersed in a buffer solution in this manner is also one embodiment of the present disclosure.

[0138] The buffer solution described above may also be a diluent used to dilute the sample to be measured. The pH of the buffer solution is, for example, 4.0 to 10.0. Examples of the buffer solution described above include phosphate buffer, glycine buffer, Tris buffer, borate buffer, citrate buffer, Good's buffer, etc.

[0139] The sensitized particles, measurement reagents, or diluents described above may contain sensitizers as needed to improve measurement sensitivity and promote the antigen-antibody reaction. However, the sensitized particles and measurement reagents of this disclosure exhibit excellent sensitivity and convergence even without sensitizers. For example, the sensitizer may be 0 to 10% by mass or 0 to 5% by mass of 100% by mass of the total amount of the measurement reagent. Examples of sensitizers include alkylated polysaccharides such as methylcellulose and ethylcellulose, pullulan, polyvinylpyrrolidone, and bipolar polymers.

[0140] The above-mentioned sensitized particles, measurement reagents, or diluents may contain proteins such as albumin (bovine serum albumin, oocyte albumin), casein, gelatin, or their degradation products, amino acids, synthetic polymer compounds, or surfactants, taking into consideration the suppression of nonspecific agglutination reactions that may occur due to substances other than the target substance present in the sample, and the enhancement of the stability of the measurement reagents. For example, the amount of the substance that suppresses nonspecific agglutination reactions may be 0 to 10% by mass or 0 to 5% by mass of the total amount of the above-mentioned measurement reagents.

[0141] The above-mentioned sensitized particles and measurement reagents are preferably used in immunological assays, and more preferably in LTIA assays. LTIA assays include both sandwich assays and competitive assays. For example, the above-mentioned sensitized particles and measurement reagents can be combined with diluents (buffers) and standard substances used in the assay to form a measurement kit.

[0142] (Measurement Method) Another aspect of the present disclosure is a measurement method. This measurement method is a method for measuring a target substance in a sample by an immunoassay using the sensitized particles (or the measurement reagent) of the present disclosure described above.

[0143] As described above, the sensitized particles of this disclosure are preferably formed by chemically bonding the above-mentioned specific affinity substances to the surface of the colored particles, particle A and / or particle B. When using such sensitized particles, the measurement method is preferably a method in which the substance to be measured is captured by the specific affinity substances chemically bonded to the surface of the sensitized particles. Immunological measurement methods are measurement methods that utilize antigen-antibody reactions.

[0144] The immunological assay used in the above measurement method is preferably, for example, latex immunoturbidimetry (LTIA). The immunological assay is also preferably an immunochromatographic method that utilizes the property of liquid moving on a membrane carrier such as a nitrocellulose membrane due to capillary action. Immunochromatographic methods are sometimes referred to as lateral flow assays. Immunological assays, as a classification method separate from the LTIA method, include sandwich methods and competitive methods.

[0145] In the sandwich method described above, for example, sensitized particles sensitized with a specific affinity substance are reacted with the object to be measured in the sample and a second specific affinity substance immobilized on a solid-phase material to form a composite on the solid-phase material in which the object to be measured is sandwiched between the sensitized particles and the second specific affinity substance. The measurement is performed by detecting this composite. The solid-phase material is not particularly limited and can be latex particles containing the above-mentioned particles Z, membranes, microplates, microchannels, magnetic particles, etc. If the solid-phase material is latex particles, the amount of optical change due to the aggregation of the composite in a chain may be measured, or if it is a membrane, microplate, microchannel, or magnetic particle, the amount of sensitized particles that have formed the composite on the solid-phase material may be measured by absorbance, fluorescence amount, or visual inspection. The sensitized particles may be dispersed in a buffer solution (liquid) or in a dry state (solid). For example, a measurement method in which dried sensitized particles are redissolved in a solution containing the substance to be measured and reacted with a second specific affinity substance using a membrane as the solid phase material is known as immunochromatography.

[0146] In the above competitive method, for example, sensitized particles sensitized with a specific affinity substance are reacted with the target substance in the sample and an analog of the target substance immobilized on a solid-phase material to form a first composite consisting of the sensitized particles and the analog of the target substance. At the same time, the target substance in the sample also competitively reacts with the sensitized particles to form a second composite. Subsequently, the second composite and unreacted sensitized particles are removed from the solid-phase material by washing with a buffer solution, and the amount of the target substance can be measured by measuring the amount of sensitized particles captured on the solid-phase material. That is, the amount of the target substance can be calculated using a calibration curve based on the relationship that the greater the amount of the target substance, the less sensitized particles are measured on the solid-phase material. The solid-phase material is not particularly limited and can include membranes, microplates, microfluidics, magnetic particles, etc.

[0147] The competitive method described above also involves reacting sensitized particles, which have been sensitized with a substance similar to the substance to be measured, with the substance to be measured in the sample and a specific affinity substance immobilized on a solid-phase material. In this case, the sensitized particles are captured by the specific affinity substance on the solid-phase material, and at the same time, the substance to be measured in the sample is competitively captured by the specific affinity substance on the solid-phase material. That is, the greater the amount of the substance to be measured, the less sensitized particles are captured on the solid-phase material. By washing the solid-phase material with a buffer solution or the like to remove unreacted sensitized particles and measuring the amount of sensitized particles on the solid-phase material, the amount of the substance to be measured can be calculated. Examples of solid-phase materials are as described above.

[0148] When using latex immunoturbidimetry in the above competitive method, a solid phase material is not required. For example, sensitized particles sensitized with a substance similar to the substance to be measured are reacted with the substance to be measured and a specific affinity substance not immobilized on a solid phase material. The substance to be measured competitively inhibits the agglutination reaction between the sensitized particles and the specific affinity substance. In other words, the amount of the substance to be measured can be calculated based on the relationship that the greater the amount of the substance to be measured, the smaller the amount of optical change due to the agglutination reaction.

[0149] In any case, the measurement method of this disclosure may use the above-mentioned sensitized particles in any known immunological assay, and the immunological assay is not limited to the assay described above.

[0150] In the LTIA method described above, the amount of the target substance in a sample can be measured by optically measuring the degree of aggregation that occurs due to the reaction between the target substance in the sample and the specific affinity substances supported on the sensitized particles. Preferably, the sensitized particles used in the LTIA method are particles A and / or particles B to which specific affinity substances are bound.

[0151] For the above optical measurements, optical instruments capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc., or optical instruments equipped with multiple detection methods, can be used. Typically, any automated biochemical analyzer widely used in clinical testing can be used.

[0152] The method for optically measuring the degree of aggregation described above is not particularly limited. Examples include the turbidimetry method, which captures the formation of aggregation as an increase in turbidity; the method for capturing the formation of aggregation as a change in particle size distribution or average particle diameter; and the integrating sphere turbidity method, which measures the change in forward scattered light due to the formation of aggregation using an integrating sphere and compares the ratio with the transmitted light intensity.

[0153] Measurement methods include, for example, rate assays, which involve obtaining at least two measurements at different time points and determining the degree of aggregation based on the increase (rate of increase) between these measurement points, and endpoint assays, which involve obtaining one measurement at a certain time point (usually considered to be the endpoint of the reaction) and determining the degree of aggregation based on this measurement. Among these, the LTIA method is preferable to use an endpoint assay based on turbidimetric methods due to its simplicity and speed. In this specification, the terms "immunoturbidimetric" and "immunoturbidimetric method" are to include all of the above concepts and forms and should not be interpreted restrictively.

[0154] In immunoassay methods using colored particles (for example, immunoassay methods other than LTIA), the amount of the target substance in the sample can be measured by optically measuring the amount of sensitized particles trapped on the solid phase material according to the amount of the target substance in the sample. For the optical measurement, optical instruments capable of detecting reflected light intensity, transmitted light intensity, absorbance, fluorescence, etc., can be used. It is preferable to use optical instruments that are suitable according to the optical properties of the colored particles (for example, absorption wavelength, excitation wavelength, fluorescence wavelength). If the colored particles are of a visible color, the amount or presence of the target substance in the sample may be measured or determined semi-quantitatively or qualitatively by observing changes in the intensity of the color visually.

[0155] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited to these examples. The methods for measuring the carboxyl group content and average particle size on the particle surface are as described above. Absorbance measurements were performed using a UV-1850 ultraviolet-visible spectrophotometer manufactured by Shimadzu Corporation. All experimental procedures were carried out under conditions of 23°C and 50% RH.

[0156] [Test Example 1: Particle Production and Solvent Resistance Evaluation] (Production Example 1-1) In a glass reaction vessel (capacity 2 L) equipped with a stirrer, reflux condenser, temperature detector, nitrogen inlet tube, and jacket, 1100 g of ultrapure water was added along with styrene and sodium p-styrenesulfonate in the amounts shown in Table 1. After replacing the inside of the vessel with nitrogen gas, the temperature was raised to 70°C while stirring at a speed of 210 rpm. After raising the temperature, 0.15 g of potassium persulfate was added and polymerization was carried out for 1 hour (first polymerization step). Subsequently, styrene and HO-MS as monomer i were added in the amounts shown in Table 1 and polymerization was carried out for 17 hours (second polymerization step). After polymerization was completed, the obtained solution was filtered through paper filter paper and particles were removed. Subsequently, the solution was dialyzed for 48 hours using a dialysis membrane to obtain particle 1. Table 1 shows the amount of each monomer added in the first and second polymerization steps (in grams) and the proportion of each monomer in the total amount of all monomers constituting particle 1 (in mass%).

[0157] (Production Example 1-2) Particle 2 was obtained in the same manner as in Production Example 1-1, except that the amount of each monomer added in the first polymerization step was as shown in Table 1, and HEMA was further added as monomer ii in the amount shown in Table 1 in the second polymerization step.

[0158] (Production Examples 1-3 to 1-5, 1-11 to 1-21) Except that the amount of each monomer added in the first polymerization step was the amount shown in Table 1, and the amount of each monomer added in the second polymerization step was the amount shown in Table 1, particles 3 to 5 and particles 11 to 21 were obtained in the same manner as in Production Example 1-2.

[0159] (Production Examples 1-6 to 1-10) Except that the amount of each monomer added in the first polymerization step was as shown in Table 1, the compound shown in Table 1 was used instead of HEMA as monomer ii in the second polymerization step, and the amount of each monomer added in the second polymerization step was as shown in Table 1, particles 6 to 10 were obtained in the same manner as in Production Example 1-2.

[0160] (Production Examples 1-22, 1-23, 1-29, 1-31 and 1-32) Particles 22, 23, 29, 31 and 32 were respectively obtained in the same manner as in Production Example 1-1, except that the addition amounts of each monomer added in the first polymerization step were changed to the amounts shown in Table 1, the compound described in Table 1 was used instead of HO-MS as monomer i in the second polymerization step, and the addition amounts of each monomer added in the second polymerization step were changed to the amounts shown in Table 1.

[0161] (Production Examples 1-24 to 1-28 and 1-30) Particles 24 to 28 and Particle 30 were respectively obtained in the same manner as in Production Example 1-2, except that the addition amounts of each monomer added in the first polymerization step were changed as described in Table 1, the compound described in Table 1 was used instead of HO-MS as monomer i in the second polymerization step, and the addition amounts of each monomer added in the second polymerization step were changed to the amounts shown in Table 1.

[0162]

[0163] The abbreviations in Table 1 are as follows. HO-MS: "Light Ester HO-MS(N)" manufactured by Kyoeisha Chemical Co., Ltd. (2-methacryloyloxyethyl succinic acid) PEG-COOH: a compound represented by the following formula CH ２ =C(CH ３ )-CO-O-(CH ２ CH ２ O) ５ -CO-CH ２ -CH ２ -COOH M-5300: "ARONIX (Registered Trademark) M-5300" manufactured by Toagosei Co., Ltd. (ω-carboxy-polycaprolactone (n≈2) monoacrylate) M-5400: "ARONIX (Registered Trademark) M-5400" manufactured by Toagosei Co., Ltd. (acryloyloxyethyl phthalic acid) HEMA oxidized product: a compound represented by the following formula CH ２ =C(CH ３ )-CO-O-CH ２-COOH MA: Methacrylic acid HEMA: 2-Hydroxyethyl methacrylate sulfobetaine: 4-[(3-Methacrylamidopropyl)dimethylammonio]butane-1-sulfonic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) MPC: 2-Methacryloyloxyethyl phosphorylcholine GMA: Glycidyl methacrylate

[0164] For each particle obtained in the above manufacturing example, the average particle size and the carboxyl group content on the particle surface were measured. The results are shown in Table 2.

[0165]

[0166] 1. Solvent Resistance Test 1 (DMF) For each particle shown in Table 3 from the particles obtained in the above manufacturing example, the solvent resistance (resistance to DMF) was evaluated according to the following procedure. As DMF, "N,N-dimethylformamide (for spectroscopic analysis)" (purity: 99.9% by mass, product number: 048-28325) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used.

[0167] (1) Preparation of Pure Water Particle Suspension The particles obtained in the above production example were suspended in pure water (particle concentration: 50.0 mg / mL, 200 μL). 800 μL of pure water was added to this suspension, and the particles were dispersed using an ultrasonic disperser (the ultrasonic disperser and ultrasonic dispersion conditions are as follows). This operation prepared a pure water suspension with a particle concentration of 10.0 mg / mL. Subsequently, this suspension was diluted 200 times with pure water to prepare a pure water suspension with a particle concentration of 50 μg / mL. This suspension is referred to as pure water particle suspension (1). Ultrasonic disperser: Branson, ultrasonic homogenizer Sonifier® SFX150 Ultrasonic dispersion conditions: pulse 1 second, total 10 seconds, amplitude 30%

[0168] (2) Preparation of DMF particle suspension A suspension (particle concentration: 50.0 mg / mL, 200 μL) of the particles obtained in the above production example was suspended in pure water. 800 μL of DMF was added to this suspension, and the particles were dispersed using an ultrasonic disperser (the ultrasonic disperser and ultrasonic dispersion conditions were as described in 1(1) above). After mixing by inversion, the particles were separated into a supernatant using a centrifuge (the centrifuge and centrifugation conditions were as described below). After centrifugation, 800 μL of the supernatant was removed, and 800 μL of fresh DMF was added, and the particles were dispersed using an ultrasonic disperser (the ultrasonic disperser and ultrasonic dispersion conditions used here were also as described in 1(1) above). This centrifugation and ultrasonic dispersion was repeated again to prepare a DMF suspension with a particle concentration of 10.0 mg / mL (DMF:pure water (volume ratio) = 99.2:0.8). Next, this suspension was diluted 50-fold with DMF solution (DMF:pure water (volume ratio) = 99.2:0.8) to prepare a DMF suspension with a particle concentration of 200 μg / mL. This suspension is referred to as DMF particle suspension (2). Centrifuge: Kubota Shoji Co., Ltd., Micro Refrigerated Centrifuge Model 3740. Centrifuge conditions: 20630 g, 25°C, 10 minutes

[0169] (3) Preparation of pure water-returned particle suspension A DMF suspension with a particle concentration of 10.0 mg / mL, prepared by the same method as described in 1(2) above, was mixed by inversion and then separated into particle components and supernatant using a centrifuge. After centrifugation, 800 μL of the supernatant was removed, and 800 μL of fresh pure water was added to disperse the particles using an ultrasonic disperser (the ultrasonic disperser and ultrasonic dispersion conditions used here were also as described in 1(1) above). This centrifugation and ultrasonic dispersion was repeated twice (i.e., centrifugation and ultrasonic dispersion were performed a total of three times). In this way, a pure water-returned suspension with a particle concentration of 10.0 mg / mL (DMF:pure water (volume ratio) = 0.2:99.8) was prepared (this will be called suspension X). Subsequently, this suspension X was diluted to a predetermined ratio (see below) using a DMF-containing aqueous solution (DMF:pure water (volume ratio) = 0.2:99.8) to prepare a pure water suspension. This suspension is referred to as the pure water-reconstituted particle suspension (3). In the example using particle 1, particle 4, or particle 20, the above suspension X was diluted 200 times with the above DMF-containing aqueous solution to prepare the pure water-reconstituted particle suspension (3) (particle concentration of 50 μg / mL) (since particles that do not dissolve in the solvent tend to have higher absorbance, the absorbance was measured with a lower particle concentration). In the example using particle 31 or particle 32, the above suspension X was diluted 50 times with the above DMF-containing aqueous solution to prepare the pure water-reconstituted particle suspension (3) (particle concentration of 200 μg / mL).

[0170] (4) Measurement of absorbance The absorbance (Abs) was measured at 400 nm, 550 nm, and 700 nm measurement wavelengths for each of the pure water particle suspension (1), DMF particle suspension (2), and pure water returned particle suspension (3) obtained above. The measured values ​​for each absorbance are shown in the "Abs" column of Table 3. Due to the limitations of the UV-Vis spectrophotometer "UV-1850" used, each absorbance was converted to the absorbance when the particle concentration was corrected to 1000 μg / mL, and this converted value is listed in the "Abs corrected value" column. Furthermore, the Abs correction value of the DMF particle suspension (2) was calculated when the Abs correction value of the pure water particle suspension (1) was set to 100%, and this value was entered in the "Abs ratio" column for the DMF particle suspension (2) in Table 3. Similarly, the Abs correction value of the pure water-returned particle suspension (3) was calculated when the Abs correction value of the pure water particle suspension (1) was set to 100%, and this value was entered in the "Abs ratio" column for the pure water-returned particle suspension (3) in Table 3.

[0171] For reference, the absorption spectra (uncorrected measurements) obtained from the above measurements are shown in Figures 1 to 5. Figures 1 to 5 are the absorption spectra of each suspension containing particle 1, particle 4, particle 31, particle 32, and particle 20, respectively. In Figures 1 to 5, (i) is the absorption spectrum of the pure water particle suspension (1) containing the above particles, (ii) is the absorption spectrum of the DMF particle suspension (2) containing the above particles, and (iii) is the absorption spectrum of the pure water-reconstituted particle suspension (3) containing the above particles. Note that in Figures 3 and 4, the absorbance of the DMF particle suspension (2) is approximately 0% at wavelengths of approximately 280 nm or higher (see absorption spectrum (ii)), and the absorbance of the pure water-reconstituted particle suspension (3) is approximately 0% at wavelengths of approximately 230 nm or higher (see absorption spectrum (iii)).

[0172] (5) Evaluation of Solvent Resistance For each of the pure water particle suspensions (1), DMF particle suspension (2), and pure water returned particle suspension (3) obtained above, the presence or absence of dissolved particles in the suspension was evaluated by visual observation. The results are shown in Table 3. In Table 3, particles that were dissolved in the suspension are indicated as "dissolved," and particles that were not dissolved in the suspension are indicated as "-."

[0173]

[0174] Particles 1, 4, and 20 are particles that contain groups derived from the above monomer (1) on their surface. In contrast, particles 31 and 32 are particles that contain groups derived from carboxyl group-containing monomers other than the above monomer (1) on their surface. Comparing the results of visual solvent resistance evaluation for both types of particles, it can be seen that particles 1, 4, and 20 did not dissolve in DMF, while particles 31 and 32 dissolved in DMF. Furthermore, comparing the absorbance of both types of particles, for particles 1, 4, and 20, the absorbance of the DMF particle suspension (2) was 1% or more, more specifically 7% or more, when the absorbance of the pure water particle suspension (1) was set to 100% at each measurement wavelength, whereas for particles 31 and 32, the absorbance of the DMF particle suspension (2) was a very low value of 0 to 0.1% when the absorbance of the pure water particle suspension (1) was set to 100% at each measurement wavelength. In particles 1, 4, and 20, the absorbance increased when DMF was replaced with pure water, but in particles 31 and 32, no increase in absorbance was observed when DMF was replaced with pure water (see absorbance of pure water-returned particle suspension (3)).

[0175] Therefore, it was found that particles 1, 4, and 20 have significantly better resistance to DMF than particles 31 and 32. Although not shown in the table, particle 28 was also evaluated for solvent resistance in the same way, and showed a similar trend to particles 1, 4, and 20. In other words, particle 28 was also found to have excellent resistance to DMF. Furthermore, when particles prepared in Production Example 1-28 without using HEMA (i.e., particles obtained in the same way as in Production Example 1-28 except that HEMA was not added) were evaluated in the same way, the particles dissolved in the DMF suspension, resulting in poor solvent resistance.

[0176] For reference, the contact angle with water was measured for each of particle 1 and particle 4 using the following method. The results are shown in Table 4. "n=1" represents the result of the first measurement. (Method for measuring the contact angle) The contact angle with water (pure water) was measured using a contact angle measuring device (for example, "DMo-602" manufactured by Kyowa Interface Science Co., Ltd.) based on the droplet method. Specifically, first a particle suspension containing 5% by mass of the above particles in solid content was prepared. This suspension was dropped onto a glass plate and dried at 95°C for 2 hours using "WFO-420" manufactured by Tokyo Rikakikai Co., Ltd. to create a thin film of particles on the glass plate. Under conditions of 23°C and 50% RH humidity, 2 μL of pure water was dropped vertically onto the surface of the obtained thin film. An image was taken after 40,000 ms, and the angle between the droplet and the thin film surface was calculated based on the obtained image. The measurement was performed three times, and the average value was taken as the above contact angle.

[0177] The contact angle data in the table above shows that particle 4, in particular, has high hydrophilicity. This high hydrophilicity is thought to contribute to its resistance to organic solvents (DMF).

[0178] 2. Solvent Resistance Test 2 (THF and Acetone) For each particle shown in Table 5 from the particles obtained in the above manufacturing example, the solvent resistance (resistance to THF or acetone) was evaluated according to the following procedure. Tetrahydrofuran (product code: 202-08741, purity: area fraction 99.5% or higher) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used as THF, and acetone (product code: 016-00346, purity: mass fraction 99.5% or higher) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used as acetone.

[0179] (1) Preparation of pure water particle suspension A pure water particle suspension (1) with a particle concentration of 50 μg / mL was prepared in the same manner as in 1(1) above for solvent resistance test 1.

[0180] (2) Preparation of THF particle suspension A THF suspension with a particle concentration of 200 μg / mL was prepared in the same manner as in 1(2) above of Solvent tolerance test 1, except that THF was used instead of DMF. This suspension is referred to as THF particle suspension (2).

[0181] (3) Preparation of acetone particle suspension For some of the particles in Table 5, an acetone suspension with a particle concentration of 200 μg / mL was prepared in the same manner as in 1(2) above of Solvent Resistance Test 1, except that acetone was used instead of DMF. This suspension is referred to as acetone particle suspension (3).

[0182] (4) Measurement of absorbance The absorbance (Abs) was measured for each of the pure water particle suspension (1), THF particle suspension (2), and acetone particle suspension (3) obtained above at measurement wavelengths of 400 nm, 550 nm, and 700 nm. The measured values ​​for each absorbance are shown in the "Abs" column of Table 5. As in 1(4) above, each absorbance was converted to the absorbance when the particle concentration was corrected to 1000 μg / mL, and this converted value is recorded in the "Abs corrected value" column. Furthermore, the Abs correction value of the THF particle suspension (2) was calculated when the Abs correction value of the pure water particle suspension (1) was set to 100%, and this value was entered in the "Abs ratio" column for the THF particle suspension (2) in Table 5. Similarly, the Abs correction value of the acetone particle suspension (3) was calculated when the Abs correction value of the pure water particle suspension (1) was set to 100%, and this value was entered in the "Abs ratio" column for the acetone particle suspension (3) in Table 5.

[0183] (5) Evaluation of solvent resistance For each of the pure water particle suspension (1), THF particle suspension (2), and acetone particle suspension (3) obtained above, whether or not the particles were dissolved in the suspension was evaluated by visual observation in the same manner as in 1(5) above. The results are shown in Table 5.

[0184]

[0185] Table 5 confirms that particles 1, 4, and 20 exhibit superior resistance not only to DMF but also to THF and acetone. Although not shown in the table, particle 28 also showed similar tendencies to particles 1, 4, and 20.

[0186] [Test Example 2: Production of Colored Particles and Evaluation of Fluorescence Intensity] (Production Example 2-1) A pure water suspension of particle 1 obtained in Production Example 1-1 (particle concentration: 162.1 mg / mL, 61.7 μL) was added to a container containing DMF (938.3 μL), and after mixing by inversion, the particle component and supernatant were separated using a centrifuge (centrifuge and centrifugation conditions were as described in 1(2) above). After centrifugation, 800 μL of the supernatant was removed, and 800 μL of fresh DMF was added to disperse the particles using an ultrasonic disperser (ultrasonic disperser and ultrasonic dispersion conditions were as described in 1(1) above). This centrifugation and ultrasonic dispersion were repeated again. After that, centrifugation was performed and 800 μL of the supernatant was removed. 600 μL of fresh DMF was added to the residue after the supernatant was removed, and the particles were dispersed using an ultrasonic disperser. This dispersion was swelled for 1 hour while being stirred with a shaker (shaker and conditions were as described below). Subsequently, 200 μL of a dye solution containing the cyanine dye Cy5 and DMF (Cy5 concentration: 2.5 mM) was added to the container and stained for 1 hour while stirring with a shaker. After that, the particles were separated into a supernatant using a centrifuge, 800 μL of the supernatant was removed, and 800 μL of fresh hydrated DMF (DMF:pure water (volume ratio) = 9:1) was added, and the particles were dispersed using an ultrasonic disperser. This operation was repeated three times. After that, the solution was replaced with pure water and centrifuged to obtain colored particles C1. Shaker: Tokyo Rikakikai Co., Ltd., high-speed shaker, model CM-1000. Shaking conditions: 1200 rpm. Note: As the Cy5 dye, DiIC from MedChemExpress was used. １８ (5) (Product name: DiD perchlorate, Product number: HY-D1028, CAS number: 127274-91-3) was used.

[0187] (Production Examples 2-2 to 2-12) Each colored particle was obtained in the same manner as in Production Example 2-1, except that the particles shown in Table 6 were used as raw materials instead of particle 1. However, when particle 31 obtained in Production Example 1-31 or particle 32 obtained in Production Example 1-32 were used, the particles dissolved in the DMF when immersed in the DMF, making it impossible to perform subsequent processing, and thus colored particles could not be produced (Production Examples 2-11 and 2-12).

[0188] The fluorescence intensity of each of the colored particles C1 to C10 obtained in the above manufacturing example was measured according to the method described below. The results are shown in Table 6. Note that the fluorescence intensity ratios (%) shown in Table 6 are relative values ​​with the fluorescence intensity of colored particle C1 set to 100%.

[0189] (Fluorescence Intensity Measurement) The colored particles were diluted with pure water to prepare a particle concentration of 10 μg / mL. The fluorescence intensity of this particle solution was measured using a spectrofluorometer (JASCO Corporation, FP-8550). The measurement conditions were as follows: Measurement temperature: 25°C Excitation wavelength: 638 nm Excitation bandwidth: 10 nm Fluorescence wavelength: 688 nm Fluorescence bandwidth: 20 nm

[0190]

[0191] [Test Example 3: Evaluation of Non-Specific Adsorption of Particles] In the following, unless otherwise specified, the "%" preceding each material refers to the mass-to-volume concentration ((w / v)%).

[0192] 1. Preparation of colored particles Colored particle CX1 was prepared in the same manner as in Production Example 2-1 of Test Example 2, and colored particle CX4 was prepared in the same manner as in Production Example 2-4 of Test Example 2. The fluorescence intensity of each obtained colored particle was measured in the same manner as described above in Test Example 2. The results are shown in Table 7. The fluorescence intensity ratio (%) shown in Table 7 is the relative value when the fluorescence intensity of colored particle CX1 is set to 100%.

[0193]

[0194] 2. Preparation of Antibody-Sensitized Particles 0.2% (2 mg / mL) colored particles CX1 suspended in 20 mM MES-NaOH (pH 6.5) were mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in an amount equivalent to 1 molar of the carboxyl groups on the surface of the colored particles. The mixture was stirred at 25°C for 10 minutes to activate the carboxyl groups. The suspension of colored particles with activated carboxyl groups was centrifuged, and the supernatant was discarded. An equal amount of 20 mM MES-NaOH (pH 6.5) was added to the discarded supernatant and ultrasonically dispersed. To the ultrasonically dispersed suspension of colored particles, a 20 mM MES-NaOH (pH 6.5) solution containing anti-PSA monoclonal antibody 1 was added in a mass ratio of 20 μg of antibody per 1 mg of colored particles. The mixture was stirred at 25°C for 2 hours to chemically bind the anti-PSA monoclonal antibody 1 to the colored particles. Chemically bonded colored particles were centrifuged, and the supernatant was replaced with 25 mM Tris-HCl buffer (pH 7.4) containing 1% BSA, 150 mM NaCl, 0.05% Tween 20, and 0.05% proclin 300, followed by sonication. This procedure was repeated twice to prepare antibody-sensitized particles S1 by sensitizing colored particles CX1 with anti-PSA monoclonal antibody 1. Similarly, antibody-sensitized particles S4 were prepared by sensitizing colored particles CX4 with anti-PSA monoclonal antibody 1 using the same procedure as above, except that colored particles CX4 were used instead of colored particles CX1. The centrifuge and centrifugation conditions were as described in Test Example 1, 1(2) above, and the sonic disperser and sonic dispersation conditions were as follows. Ultrasonic Disperser: Manufactured by Nippon Seiki Seisakusho, Ultrasonic Generator Model US-50. Ultrasonic dispersion conditions: Tuning dial 5.5, 1 second x 10 times.

[0195] The materials used to prepare the antibody-sensitized particles are as follows: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: Thermo Scientific (registered trademark), "Pierce (registered trademark) EDC, No-Weigh (registered trademark) Format", Product number: A35391 Anti-PSA monoclonal antibody 1: Medix Biochemica, "Anti-h PSA 8301 SPRN-5", Catalog number: 100102 Tween 20: Merck KGaA, "TWEEN (registered trademark) 20", Product number: P1379 proClin 300: Merck KGaA, "ProClin (registered trademark) 300", Product number: 48914-U

[0196] 3. Preparation of Antibody-Immune-Phase Membrane A PBS (phosphate-buffered saline) solution containing 2 mg / mL anti-PSA monoclonal antibody 2 and 2.5% sucrose was prepared and used as the antibody coating solution. Using an immunochromatography dispenser (dispensing platform XYZ3050, BioDot), the antibody coating solution was applied to a nitrocellulose membrane at a rate of 1.0 μL / cm in a line and dried to form the antibody-immobilized portion, thereby preparing the antibody-immobilized membrane. Anti-PSA monoclonal antibody 2 was obtained using commercially available human PSA antigen (Prostate Specific Antigen, Lee Biosolutions) as the immunogen, by a method well known to those skilled in the art.

[0197] 4. Preparation of Test Strips An antibody-immobilized membrane and an absorbent pad were attached to a plastic adhesive sheet and cut to a width of 3.5 mm to obtain an immunochromatographic test strip for PSA measurement (see Figure 6). Figure 6 is a schematic diagram of the configuration of the immunochromatographic test strip for PSA measurement used in this example, viewed from the side.

[0198] 5. Measurement of signal intensity Female human plasma without PSA was prepared with commercially available PSA antigen added at concentrations of 0.05 ng / mL, 0.25 ng / mL, 0.5 ng / mL, and 1 ng / mL, respectively, as well as female human plasma without PSA, resulting in a total of five concentrations of samples. The antibody-sensitized particles S1 obtained in step 2 above were diluted with 20 mM Tris-HCl buffer (pH 7.5) containing 1.33% casein, 4% sucrose, and 0.05% sodium azide to a particle concentration of 0.78 μg / mL. 40 μL of this solution was taken, 10 μL of the sample was added and mixed, and then allowed to stand at room temperature for 5 minutes. The test strip prepared in step 4 above was immersed in the resulting mixture and allowed to stand at room temperature for 10 minutes to allow the mixture to spread on the membrane. After allowing the test strip to stand for 10 minutes, it was removed and continuously irradiated with excitation light at a wavelength of 638 nm while scanning the membrane. Fluorescence at wavelengths of 695–712 nm was continuously measured. The difference between the fluorescence intensity of the antibody-solid phase and the fluorescence intensity around the antibody-solid phase was calculated and used as the signal intensity measurement value (unit: RLU) corresponding to the amount of antibody-sensitized particles bound to the antibody-solid phase. The same measurement procedure was performed twice for each of the five concentrations of samples (these measurements are listed in the "Measured Value" column of Table 8), and the average of the two measurements was calculated for each PSA concentration. This calculated value is listed in the "Average Value" column of Table 8.

[0199] The above measurements were also performed when antibody-sensitized particle S4 was used instead of antibody-sensitized particle S1. The percentage of the relative value of the average measurement value when using antibody-sensitized particle S4 to the average measurement value when using antibody-sensitized particle S1 was calculated and recorded in the "Relative Value" column of Table 8.

[0200]

[0201] Antibody-sensitized particles S4 obtained using colored particles CX4 showed a decrease in measured values ​​in PSA-free samples and an increase in measured values ​​in PSA-added samples compared to antibody-sensitized particles S1 obtained using colored particles CX1 (see Table 8). Since colored particles CX4 have a higher fluorescence intensity than colored particles CX1 (see Table 7), it is thought that the measured values ​​increased in PSA-added samples. On the other hand, in PSA-free samples, the sandwich reaction of the antibody-solid phase does not occur in principle, so it is thought that the measured values ​​did not increase. The signal intensity measured in PSA-free samples is thought to be due to unintended nonspecific reactions, but it is thought that such nonspecific reactions were suppressed with colored particles CX4, so despite the high fluorescence intensity of colored particles CX4, the signal intensity on the test strip was lower than when using colored particles CX1.

[0202] [Test Example 4: Observation of Particle Cross Section] The particle cross section of the colored particle CX4 used in Test Example 3 was observed using an SEM. Specifically, the observation was performed using the following procedure: (i) First, the sample (colored particle CX4) was freeze-dried using a freeze-dryer (FD-3STU-S, manufactured by Nippon Techno Service Co., Ltd.) under reduced pressure of 4 Pa, gradually increasing the temperature from -55°C to 20°C. This obtained a dried powder. (ii) Next, carbon tape for SEM observation was attached to the observation sample stage, and the dried powder was applied onto the carbon tape. (iii) Next, the cross section of the dried powder was prepared by cutting it for 1 hour at an accelerating voltage of 3 kV using an ion milling apparatus (IB-19500CP, manufactured by JEOL Ltd.). (iv) Next, the dried powder was treated with conductivity by osmium deposition for 10 seconds using a vapor deposition apparatus (Neoc-STB, manufactured by Meiwa Forsis). (v) Subsequently, the surface shape was observed using a scanning electron microscope (Regulus® 8220, manufactured by Hitachi, Ltd.). The observation was performed with an acceleration voltage of 3 kV and secondary electron imaging, and the observation magnification was 50,000x.

[0203] Figure 7 shows the cross-sectional SEM image obtained above. From Figure 7, it can be seen that the colored particle CX4 is a particle comprising a shell portion and a hollow portion surrounded by the shell portion, and that the thickness of the shell portion is approximately 100 nm.

[0204] (i): Absorption spectrum of pure water particle suspension (1) (ii): Absorption spectrum of DMF particle suspension (2) (iii): Absorption spectrum of pure water returned particle suspension (3) 1: Absorbent pad 2: Nitrocellulose membrane 3: Antibody-solidified portion 4: Plastic adhesive sheet

Claims

1. Particles comprising a group derived from a monomer represented by the following formula (1) on a surface, or comprising a group derived from a monomer represented by the following formula (2) and a group derived from a monomer represented by the following formula (3) on a surface. CH ２ =C(R １ )-C(=O)-O-(R ４ O) ｍ -H (2) CH ２ =C(R １ )-C(=O)-O-(R ５ -C(=O)-O) ｐ -M (3) In the formulas, R １ represents a hydrogen atom or a methyl group. R ２ O represents an oxyalkylene group having 2 to 10 carbon atoms. n represents the average number of added moles of the oxyalkylene group represented by R ２ O, and is a number from 1 to 20. When n is 2 or more, R ２ O groups may be the same as or different from each other. R ３ represents a linear hydrocarbon chain having 1 to 10 carbon atoms or a hydrocarbon chain containing a benzene ring. M represents a hydrogen atom or a salt residue. R ４ O represents an oxyalkylene group having 2 to 10 carbon atoms. m represents the average number of added moles of the oxyalkylene group represented by R ４ O, and is a number from 1 to 20. When m is 2 or more, R ４ O groups may be the same as or different from each other. R ５ represents an alkylene group having 1 to 10 carbon atoms. p represents the average number of added moles of the group represented by (R ５ -C(=O)-O), and is an integer from 1 to 10.

2. The particle according to claim 1, wherein the average particle diameter is 50 to 1000 nm.

3. The particle according to claim 1, wherein the particle further comprises a monomer-derived group represented by formula (2) on its surface, in the case where the particle has a monomer-derived group represented by formula (1) on its surface.

4. The particle according to any one of claims 1 to 3, wherein the particle has a group derived from the monomer represented by formula (1) on its surface, and the particle has structural units derived from (a) and / or (b) below, and the sum of the monomer represented by formula (1), (a) below, and (b) below is 50% by mass or more of the total amount of all monomers constituting the particle. (a) A monomer having a polymerizable group and an aryl group (b) A monomer having a polymerizable group and a sulfonic acid (salt) group, provided that (a) and (b) do not include monomers corresponding to the monomer represented by formula (1), and (a) does not include monomers corresponding to (b).

5. A particle according to any one of claims 1 to 4, comprising a core portion and a shell portion covering at least a part of the core portion, wherein if the particle has a monomer-derived group represented by formula (1) on its surface, the surface of the shell portion has at least a monomer-derived group represented by formula (1), and if the particle has a monomer-derived group represented by formula (2) and a monomer-derived group represented by formula (3) on its surface, the surface of the shell portion has at least a monomer-derived group represented by formula (2) and a monomer-derived group represented by formula (3).

6. The particle according to claim 3, comprising a core portion and a shell portion covering at least a part of the core portion, wherein the surface of the shell portion has a monomer-derived group represented by formula (1) and a monomer-derived group represented by formula (2).

7. A particle according to any one of claims 1 to 4, comprising a shell portion and a hollow portion surrounded by the shell portion, wherein if the particle has a monomer-derived group represented by formula (1) on its surface, the surface of the shell portion has at least a monomer-derived group represented by formula (1), and if the particle has a monomer-derived group represented by formula (2) and a monomer-derived group represented by formula (3) on its surface, the surface of the shell portion has at least a monomer-derived group represented by formula (2) and a monomer-derived group represented by formula (3).

8. The particle according to claim 3 or 4, comprising a shell portion and a hollow portion surrounded by the shell portion, wherein the surface of the shell portion has a monomer-derived group represented by formula (1) and a monomer-derived group represented by formula (2).

9. The particle according to any one of claims 1 to 8, wherein the content of carboxyl groups on the surface is 10 to 3000 μeq / g.

10. The particle according to any one of claims 1 to 9, wherein when the absorbance of the aqueous suspension of the particle at any of the measurement wavelengths of 400 nm, 550 nm, or 700 nm is set to 100%, the absorbance of an N,N-dimethylformamide suspension containing the particle at the same particle concentration as the aqueous suspension at the measurement wavelength is 1% or more.

11. Colored particles comprising the particles described in any one of claims 1 to 10 and a coloring agent.

12. The coloring agent comprises a fluorescent dye, as described in claim 11.

13. Sensitized particles obtained by binding a substance with specific affinity for the substance to be measured or a substance similar to the substance to be measured to a colored particle according to any one of claims 11 to 12.

14. A measurement reagent comprising sensitized particles as described in claim 13 dispersed in a buffer solution.

15. A method for measuring the target substance in a sample by an immunoassay using the sensitized particles described in claim 13.

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