Reagents for immunoturbidimetric testing

Abstract

There is a need to quantify both low and high concentrations of target substances with high sensitivity. [Solution] The present invention provides an immunoturbidimetric reagent comprising a first particle and a second particle, wherein the ratio of the volume-average particle size (Dv2) of the second particle to the volume-average particle size (Dv1) of the first particle, Dv2 / Dv1, is 0.37 or more and 0.86 or less, the volume-average particle size of the second particle is 150 nm or more, and the ratio of the refractive index (R2) of the second particle to the refractive index (R1) of the first particle, R2 / R1, is 0.87 or more and 0.97 or less.

Description

Technical Field

[0001] The present disclosure relates to a reagent for immunoturbidimetry testing.

Background Art

[0002] As a simple and rapid immunoassay method, immunoturbidimetry using particles can be mentioned. In this method, a dispersion of particles bound with a ligand having an affinity for a target substance is mixed with a sample that may contain the target substance. At this time, since an aggregation reaction of the particles occurs according to the amount of the target substance contained in the sample, the target substance can be qualitatively or quantitatively determined by optically detecting this aggregation reaction as a change amount such as scattered light intensity, transmitted light intensity, absorbance, etc. Further, it is known that the scattered light intensity of the particles used in immunoturbidimetry can be estimated from the Mie scattering theory depending on the particle size and refractive index of the particles.

[0003] In immunoassay items in clinical tests, since both the low-concentration region and the high-concentration region of the target substance have important diagnostic meanings, it is required to be able to quantify both the low-concentration target substance and the high-concentration target substance.

[0004] In response to such problems, Patent Document 1 discloses a latex agglutination method that uses two types of latex particle groups of different average particle sizes to expand the high-concentration measurement range and increase the sensitivity of measurement in the low-concentration region.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] As disclosed in Patent Document 1, it is known to use two types of latex particle groups with different average particle sizes, large and small, for highly sensitive measurement and expansion of the measurement range by the latex aggregation method. However, simply using two types of latex particle groups with different average particle sizes is not sufficient for highly sensitive measurement and the measurement range. Further improvement in highly sensitive measurement and expansion of the measurement range are required.

Means for Solving the Problems

[0008] For highly sensitive measurement and expansion of the measurement range, a method of increasing the difference in the average particle size of, for example, two types of latex particles, large and small, can be considered. However, if the particle size of the large particles is increased, the particles tend to sediment. On the other hand, if the particle size of the small particles is decreased, the amount of change in the scattered light intensity caused by the aggregation reaction itself also becomes small, which is not preferable for measurement in a high-concentration range. Therefore, in order to solve these problems, the inventors have found an immunoturbidimetric assay reagent comprising a first particle and a second particle, wherein the ratio Dv2 / Dv1 of the volume average particle size (Dv2) of the second particle to the volume average particle size (Dv1) of the first particle is 0.37 or more and 0.86 or less, Dv2 is 150 nm or more, and the ratio R2 / R1 of the refractive index (R2) of the second particle to the refractive index (R1) of the first particle is 0.87 or more and 0.97 or less.

Modes for Carrying Out the Invention

[0009] Hereinafter, embodiments of the present disclosure will be described in detail, but the technical scope of the present invention is not limited to these embodiments.

[0010] <Particle> The immunoturbidimetric reagent (test reagent) of this disclosure comprises a first particle and a second particle, wherein the ratio of the volume-average particle size (Dv2) of the second particle to the volume-average particle size (Dv1) of the first particle, Dv2 / Dv1, is 0.37 or more and 0.86 or less, Dv2 is 150 nm or more, and the ratio of the refractive index (R2) of the second particle to the refractive index (R1) of the first particle, R2 / R1, is 0.87 or more and 0.97 or less.

[0011] In agglutination testing, it is common to use light with wavelengths in the visible light range. In this wavelength range, the absorbance of particles increases with increasing particle size up to 1 μm (Non-Patent Literature 1). The volume-average particle size (Dv2) of the second particle is 150 nm or larger, and the ratio of the volume-average particle size (Dv2) of the second particle to the volume-average particle size (Dv1) of the first particle is between 0.37 and 0.86. When such first and second particles agglutinate, the increase in absorbance before and after agglutination is large, resulting in high sensitivity. With the above particle size combination, even when the concentration of the target substance is high, the sensitivity increases at a rate above a certain level as the concentration of the target substance increases, making it possible to evaluate the concentration contained in the sample. The ratio Dv2 / Dv1, which is the ratio of the volume-average particle size (Dv2) of the second particle to the volume-average particle size (Dv1) of the first particle, is 0.37 or more and 0.86 or less, more preferably 0.38 or more and 0.79 or less, and even more preferably 0.38 or more and 0.60 or less.

[0012] As described above, in the immunoturbidimetric reagent of this disclosure, the ratio of the refractive index (R2) of the second particle to the refractive index (R1) of the first particle, R2 / R1, is between 0.87 and 0.97. When the refractive index ratio is within this range, when combined with the ratio of the volume-average particle size mentioned above, highly sensitive measurements are possible from low to high concentration ranges. In reagents where R2 / R1 is less than 0.87, if both the first and second particles are organic polymer microparticles, manufacturing becomes difficult. If the first and second particles are a combination of organic polymer microparticles and inorganic microparticles, the inorganic microparticles generally have a higher specific gravity, making them prone to sedimentation. In reagents where R2 / R1 is greater than 0.97, the difference in refractive index between the first and second particles is small, and sufficient effect cannot be obtained.

[0013] One means of adjusting the ratio of the refractive indices of the particles is to adjust the content of the constituent units of the polymer that make up the particles. The particles used in this disclosure can be any conventionally known particles that have the aforementioned particle size and refractive index. For example, polystyrene, styrene-butadiene copolymer, styrene-styrene sulfonate copolymer, styrene-glycidyl methacrylate copolymer, etc. can be used.

[0014] Preferred particles as the first and second particles used in this disclosure (in this specification, the first and second particles may be simply referred to as particles) are particles comprising a polymer containing a structural unit represented by formula (1) and a structural unit represented by formula (2). [ka] (R 1 Each structural unit independently represents either a hydrogen atom or a methyl group. 2 Each structural unit independently represents a phenyl group or a naphthyl group, which may be substituted. In the case of substitution, the substituent is either a methyl group or an ethyl group.

[0015] [ka] (R 3 Each structural unit independently represents either a hydrogen atom or a methyl group. 4 (Each structural unit independently represents an arbitrary group.)

[0016] Equation (1) is R 2 The polymer contains substituted or unsubstituted phenyl and naphthyl groups. Phenyl and naphthyl groups are known to increase the refractive index. The ratio of the refractive indices of the particles can be adjusted by controlling the content of polymers having the structural unit shown in formula (1) and polymers having the structural unit shown in formula (2). In the immunoturbidimetric reagent of this disclosure, the first particle has a higher content of polymers having the structural unit shown in formula (1) relative to the content of polymers having the structural unit shown in formula (2) compared to the second particle. As a result, the refractive index (R1) of the first particle is higher than the refractive index (R2) of the second particle, and the aforementioned refractive index ratio is obtained.

[0017] The particles used in the immunoturbidimetric reagent of this disclosure preferably have a core particle and a shell on the surface of the core particle. The core particle preferably has a high refractive index, resulting in a large difference in absorbance before and after agglutination. The shell preferably covers the core particle and has high hydrophilicity to suppress nonspecific adsorption. Having such a core-shell structure makes it possible to have both a high refractive index and hydrophilicity on the particle surface, making it possible to achieve both high-sensitivity detection and suppression of nonspecific adsorption in the agglutination method.

[0018] The particles of this embodiment contain a polymer having a structural unit represented by formula (1), and it is preferable that the content ratio of the structural unit represented by formula (1) to the first particle is 60% by mass or more and 90% by mass or less. The shell preferably has a film thickness of 5 nm or more and 50 nm or less, and contains a polymer having a structural unit represented by formula (2).

[0019] The core particles may also contain polymerization initiators, copolymers derived from vinyl monomers, colorants such as pigments, dyes, and fluorescent agents, and inorganic fillers. The content of these materials in the particles is preferably 9% by mass or less.

[0020] The particles used in the immunoturbidimetric reagent of this disclosure preferably have a shell with a film thickness of 5 nm or more and 50 nm or less, and contain a polymer having the structure shown in formula (2). Formula (2) has a carbonyloxy group (-CO-O-), and therefore has high hydrophilicity. By covering the core particle surface with a shell containing a polymer having the structure shown in formula (2), the hydrophobic portion of the particle surface is covered, and the hydrophilicity is increased. As a result, hydrophobic interactions with proteins and other substances in the sample other than the target substance are reduced, and nonspecific adsorption can be suppressed.

[0021] Furthermore, by having a shell film thickness of 5 nm or more and 50 nm or less, the hydrophobic surface of the core particles can be covered without being exposed. At the same time, the particle dispersibility is moderate, particle aggregation is not inhibited, and moderate aggregation occurs, resulting in high sensitivity. As a result, both high sensitivity and suppression of nonspecific adsorption can be achieved.

[0022] In one embodiment, it is preferable that the structure of formula (1) is represented by formula (1-A). Having formula (1-A) allows for an increased refractive index of the core particles. Furthermore, because the structure of formula (1-A) is highly hydrophobic, hydrophilic shell components are less likely to be incorporated into the core portion. [ka] (R 10 (This represents a phenyl group, a tolyl group, or a naphthyl group.)

[0023] In this embodiment, the structure represented by formula (1-A) is obtained by polymerizing the monomer represented by formula (X1), which will be described later. Specific examples of monomers include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene, as will be described later, but styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferred. These monomers may be used individually or in combination.

[0024] The core particles preferably further have a crosslinked structure. The crosslinked structure is obtained by polymerization using a crosslinkable radical polymerizable monomer, which is a monomer having two or more radical polymerizable unsaturated bonds in one molecule. Examples of such crosslinkable monomers include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, and dipentaerythritol hexamethacrylate, as well as conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate. In addition, two or more types of crosslinkable radical polymerizable monomers may be used. The crosslinked structure is more preferably the structure shown in formula (3). By having a crosslinked structure in the core particles, the particles having a core and shell structure become physically strong, eliminating concerns about cracking or chipping even when repeated centrifugation operations are performed during purification. [ka] (Z represents a substituted or unsubstituted phenylene group or naphthalene group; in the case of substitution, the substituent is a methyl group or an ethyl group. Z may differ for each structural unit.)

[0025] The crosslinkable radically polymerizable monomer used to form the crosslinkable structure of formula (3) includes, for example, 1,2 - divinylbenzene, 1,3 - divinylbenzene, 1,4 - divinylbenzene, 2,6 - diethynylnaphthalene, 2,7 - diethynylnaphthalene. These may be used alone or multiple of them may be used simultaneously.

[0026] Among the exemplified crosslinkable radically polymerizable monomers, divinylbenzene is preferred. When divinylbenzene is used, it has excellent handling properties during the radical polymerization reaction, the monomer conversion rate during core particle formation is improved, and it is difficult for the core component to be incorporated into the shell, which is preferable.

[0027] In the first particle, it is preferable that the mass ratio of the core in the particle is 60% by mass or more and 95% by mass or less. By setting the mass ratio of the core in the particle to 60% by mass or more, the content of the core with a high refractive index can be made sufficiently large, improving the refractive index. As a result, the difference in absorbance before and after aggregation becomes large, and the sensitivity in the region where the concentration of the target substance is low can be improved. Also, by setting the mass ratio of the core in the particle to 95% by mass or less, a sufficient shell layer can be provided to cover the surface of the core particles with the shell layer, improving the hydrophilicity of the particle surface and improving non - specific adsorption suppression.

[0028] In this embodiment, formula (2) is preferably a structure represented by formula (2 - A) (R in formula (2) 4 is formula (2 - A’)). The structure represented by formula (2) has, as R 4 a group containing an epoxy group, a group containing a hydroxy group, a group containing a carboxy group, or any group represented by formula (2 - A’). In any case, it has at least one of a hydroxy group, a carboxy group, and an epoxy group, suppressing non - specific adsorption.

[0029]

Chemical formula

[0030] [ka] (R 31 and R 32 One of the terms represents a hydroxyl group, and the other represents a hydroxyl group or a group represented by formula (2-B). * (This indicates the bonding position.)

[0031] [ka] (R 20 R indicates a single bond or a methylene group. 22 , R 23 , R 24 Each of these represents a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group, respectively. 22 , R 23 , and R 24 At least one of them contains a hydroxyl group or a carboxyl group. 1 This represents a sulfur atom or an imino group. * (This indicates the bonding position.)

[0032] If R4 has the equation (2-A'), then equation (2) can be expressed as (2-A) above.

[0033] Examples of specific structures of formula (2-A) having the group represented by formula (2-B) are shown below in (2-A-1) to (2-A-12), but are not limited to these.

[0034] [ka]

[0035] The structure represented by formula (2-A) having the group represented by formula (2-B) according to this embodiment is obtained by reacting a polymer obtained by polymerizing the monomer represented by formula (X2), described later, with formula (X3), described later. There are no particular limitations as specific examples of the monomer represented by (X2), but glycidyl (meth)acrylate is preferred.

[0036] In the particle shell of this embodiment, the content of the structural unit represented by formula (2) is preferably 90% by mass or more and 100% by mass or less. By setting the content of the structural unit represented by formula (2) to 90% by mass or more, the hydrophilicity of the shell is increased, and the suppression of nonspecific adsorption can be improved. To further suppress nonspecific adsorption, 95% by mass or more is even more preferable. The shell may also contain polymerization initiators, copolymers derived from vinyl monomers, colorants such as pigments, dyes, and fluorescent agents, and inorganic fillers. The content is preferably 9% by mass or less.

[0037] Furthermore, the particles of this embodiment may have structures other than those of formula (1) and formula (2). Examples of structures other than those of formula (1) and formula (2) that the particles may contain include, but are not particularly limited to, structures obtained by polymerizing monomers of styrenes, acrylics, methacrylics, etc. Also, the particles may have multiple structures other than those of formula (1) and formula (2) simultaneously.

[0038] The ratio of equation (1) to equation (2) can be evaluated by measuring the particles of this embodiment using FT-IR (Fourier Transform Infrared Spectroscopy). The ATR method (Attenuated Total Reflection) is preferred as the measurement method, and an infrared absorption spectrum (FT-IR spectrum) can be obtained by utilizing the fact that infrared light penetrates to a size of approximately μm from the sample surface. In the red FT-IR spectrum of the particles of this embodiment, the 1500-1650 cm⁻¹ originates from the C=C bond of the aromatic ring. -1The peak height present is A, and it originates from the carbonyl group of the carbonyloxy group (ester bond), ranging from 1680 to 1750 cm. -1 If we let B be the peak height present, then the larger the value of A / B, the higher the proportion of the C=C bond in the aromatic ring relative to the carbonyl group, meaning that the proportion of equation (1) to the entire particle is higher than that of equation (2).

[0039] The ratio of the volume-average particle size (Dv) to the number-average particle size (Dn) (Dv / Dn) is preferably 1.25 or less. It is known that the closer the Dv / Dn value is to 1, the narrower the particle size distribution becomes. When the Dv / Dn value is 1.25 or less, there is little variation in particle size, and no large particles are included, so the size difference between the aggregated aggregate and the sample after aggregation is large, and the sensitivity is increased.

[0040] <Method for manufacturing particles> For the production of the particles, known methods for forming core and shell structures can preferably be used without particular limitation. The shell structure can be formed by adding a monomer for shell formation to a dispersion of core particles, followed by the addition of a water-soluble polymerization initiator.

[0041] As one embodiment, this disclosure provides the following method for producing particles used in the immunoturbidimetric reagent of this disclosure. A first step involves carrying out a polymerization reaction of a reaction system in the first step that includes a first monomer composition containing 90% by mass or more of the monomer represented by formula (X1) to obtain core particles; The second step involves polymerizing the core particles, a second monomer composition containing the monomer represented by formula (X2), and a reaction solution from the second step containing a water-soluble polymerization initiator to obtain particles in which a layer of 5 nm or more and 50 nm or less is formed on the outside of the core particles. However, in the reaction solution of the second step, The content ratio of the unpolymerized monomer represented by formula (X1) to the reaction solution of the second step is 1000 ppm or less; and A third step involves reacting the particles forming the aforementioned layer with the compound represented by formula (X3), A method for producing particles, including

[0042] [ka] (R 11 R represents a hydrogen atom or a methyl group. 12 (This indicates a substituted or unsubstituted phenyl group or naphthyl group; in the case of substitution, the substituent is a methyl group or an ethyl group.)

[0043] [ka] (R 13 R represents a hydrogen atom or a methyl group. 14 (This indicates an ethylene group or a carbonyl group.)

[0044] [ka] (R 15 R represents an amino group or a thiol group. 16 , R 17 R represents a group having a hydrogen atom, a methyl group, a hydroxyl group, or a carboxyl group. 16 , R 17 At least one of them represents a group having a hydroxyl group or a group having a carboxyl group.

[0045] The first step preferably involves the use of soap-free emulsion polymerization. Using soap-free emulsion polymerization results in a uniform particle size distribution, stable sensitivity, and improved detection limits in regions where the concentration of the target substance is low.

[0046] It is preferable that the amount of monomer represented by formula (X1) in the reaction solution before the start of the second step is 1000 ppm or less. By keeping the amount at 1000 ppm or less, when the shell is formed in the second step, the amount of the hydrophobic monomer represented by formula (X1) is small, making it less likely for the shell to contain a hydrophobic repeating structure, and thus suppressing nonspecific adsorption. In the reaction solution of the second step, the content ratio of the monomer represented by formula (X2) to the total amount of monomers contained in the first monomer composition and the monomers contained in the second monomer composition is preferably 9% by mass or more and less than 40% by mass in the production of the first particles, and preferably 30% by mass or more and 60% by mass or less in the production of the second particles.

[0047] Furthermore, this disclosure provides, as a further embodiment, the following method for producing particles. A first step involves carrying out a polymerization reaction of a reaction system of the first step, which includes a monomer represented by formula (X1) and a first monomer composition containing a monomer represented by formula (X2); A second step involves polymerizing the reaction solution from the second step, which contains a second monomer composition comprising a monomer represented by formula (X2) and a water-soluble polymerization initiator, with the liquid from the first step to obtain particles; However, in the reaction solution of the second step, the content ratio of the monomer represented by formula (X2) to the total amount of monomers contained in the first monomer composition and the monomers contained in the second monomer composition is 30% by mass or more and less than 80% by mass. Furthermore, the second step may be performed multiple times; and A third step involves reacting the particles forming the aforementioned layer with the compound represented by formula (X3), A manufacturing method that includes [details omitted].

[0048] The water-soluble polymerization initiator used in this embodiment is not particularly limited, but water-soluble azo compounds and water-soluble peroxides are preferably used. The water-soluble azo compound is preferably any of the following: 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]tetrahydrate, 2,2'-azobis(2-methylpropionamidine)dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, or 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate.

[0049] The water-soluble peroxide is preferably one of the following: potassium persulfate, ammonium persulfate, sodium persulfate, tert-butyl hydroperoxide, cumyl hydroperoxide, paramenthane hydroperoxide, or diisopropylbenzene hydroperoxide.

[0050] In this embodiment, the monomer represented by formula (X1) is preferred for improving sensitivity because the polymer obtained by polymerizing the monomer has a high refractive index. Examples include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene, but styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferred. These monomers may be used individually or in combination. Further specific examples of styrenes include styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, and p-phenylstyrene.

[0051] In this embodiment, since the monomer represented by formula (X2) has a glycidyl group in its side chain, the compound represented by formula (X3) can be reacted with the glycidyl group in the shell having the polymer of formula (X2), thereby introducing the carboxyl group and hydroxyl group contained in formula (X3) onto the particle surface. The monomer represented by formula (X2) is not particularly limited, but glycidyl (meth)acrylate is preferred.

[0052] In this embodiment, the monomer represented by formula (X3) is added to the shell on the particle surface in the third step by the reaction of the amino group or thiol group in formula (X3) with the glycidyl group in the shell, thereby forming formula (2). The monomer represented by formula (X3) is not particularly limited, but examples include mercaptosuccinic acid, aspartic acid, 3-mercapto-1,2-propanediol, 3-amino-1,2-propanediol, 2-amino-1,3-propanediol, ethanolamine, and trishydroxymethylaminomethane.

[0053] <First Particle> The particles of this embodiment can be used without limitation as long as Dv2 / Dv1 is 0.37 or more and 0.86 or less, and R2 / R1 is 0.87 or more and 0.97 or less. Preferably, the ratio of the number of structural units shown in formula (1) to the number of structural units shown in formula (2) is greater than the ratio of the number of structural units shown in formula (1) to the number of structural units shown in formula (2) in the second particle.

[0054] Here, the number of structural units shown in equation (1) in the first particle is denoted as P1U1, the number of structural units shown in equation (2) in the first particle is denoted as P1U2, the number of structural units shown in equation (1) in the second particle is denoted as P2U1, and the number of structural units shown in equation (2) in the second particle is denoted as P2U2. Then, the ratio of P1U1 to P1U2 is expressed as P1U1 / P1U2, the ratio of the number of P2U1 to P2U2 is expressed as P2U1 / P2U2, the ratio of P1U2 to P1U1 is expressed as P1U2 / P1U1, and the ratio of the number of P2U1 to P2U2 is expressed as P2U2 / P2U1. Therefore, it is preferable that P1U1 / P1U2 is greater than P2U1 / P2U2.

[0055] The content ratio of the structural unit shown in formula (1) to the first particle is preferably 60% by mass or more and 90% by mass or less. Having 60% by mass or more of the structural unit shown in formula (1) in the first particle increases the refractive index of the particle. When the reagent containing the particle reacts with the target substance and aggregates, the absorbance increases, but when particles with a high refractive index aggregate, the absorbance increases even more, so the difference in absorbance before and after aggregation becomes larger and sensitivity is improved. Furthermore, a content ratio of the structural unit shown in formula (1) of 90% by mass or less is preferable because it increases the liquid stability of the aqueous dispersion.

[0056] In the first particle, the content of the structure shown in formula (2) can be between 9% and 40% by mass. This range reduces the hydrophobicity of the particle surface, suppressing nonspecific adsorption. Furthermore, the particle's dispersibility is not excessively high, maintaining a moderate level, and particle aggregation is not inhibited, resulting in higher sensitivity.

[0057] In the first particle, in the particle manufacturing method described above, it is preferable that the content ratio of the monomer represented by formula (X2) to the total amount of monomers contained in the first monomer composition and the monomers contained in the second monomer composition be 9% by mass or more and less than 40% by mass.

[0058] <Second Particle> The second particle relating to the immunoturbidimetric reagent of this disclosure can be used without limitation as long as Dv2 / Dv1 is 0.37 or more and R2 / R1 is 0.87 or more and 0.97 or less, but it contains a polymer having the structural unit shown in formula (1) and a polymer having the structural unit shown in formula (2), and preferably the content of the polymer having the structural unit shown in formula (2) is higher than that of the first particle. In other words, it is preferable that P2U2 / P2U1 is greater than P1U2 / P1U1.

[0059] As mentioned above, the higher the content of the structural unit represented by formula (1) contained in the particle, the higher the refractive index. In other words, if P2U2 / P2U1 is greater than P1U2 / P1U1, the second particle will have a lower refractive index compared to the first particle.

[0060] The second type of particle can take two forms: The first form of the second particle is a particle having a core particle and a shell on the surface of the core particle, wherein the shell contains a polymer having the structural unit shown in formula (2), and the content of the polymer is high. The second form of the second particle is a form in which the polymer having the structural unit shown in formula (2) is contained throughout the particle, and the content of the polymer is high. In the second particle, in the particle manufacturing method described above, it is preferable that the content ratio of the monomer represented by formula (X2) to the total amount of monomers contained in the first monomer composition and the monomers contained in the second monomer composition be 30% by mass or more and 60% by mass or less.

[0061] In the immunoturbidimetric reagent of this disclosure, as described above, the content of the structural unit represented by formula (2) in the second particle is higher than the content in the first particle, resulting in the second particle having a relatively lower refractive index compared to the first particle.

[0062] A second embodiment of the second particle of the present disclosure is a particle containing a copolymer having structural units represented by formula (1) and structural units represented by formula (2). The surface of the copolymer-containing particle may further have a shell containing a polymer having structural units represented by formula (2).

[0063] The following are possible methods for manufacturing the second form of the second particle: A first step involves carrying out a polymerization reaction of a reaction system of the first step, which includes a monomer represented by formula (X1) and a first monomer composition containing a monomer represented by formula (X2); A second step involves polymerizing the reaction solution from the second step, which contains a second monomer composition comprising a monomer represented by formula (X2) and a water-soluble polymerization initiator, with the liquid from the first step to obtain particles; However, in the reaction solution of the second step, the content ratio of the monomer represented by formula (X2) to the total amount of monomers contained in the first monomer composition and the monomers contained in the second monomer composition is 30% by mass or more and less than 80% by mass. Furthermore, the second step may be performed multiple times; and A third step involves reacting the particles forming the aforementioned layer with the compound represented by formula (X3), Includes.

[0064] <Substances that specifically bind to a target substance (ligands)> A ligand is a compound that specifically binds to a receptor on a particular target substance. Ligands bind to a specific site on the target substance, and ligands and target substances have a selective or specific high affinity. Examples of target substance-ligand combinations include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc., as long as the objectives of this disclosure can be achieved. Specifically, ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, etc.

[0065] <Affinity Particles> Particles on which the ligand is immobilized will hereafter be referred to as affinity particles, and particles before the ligand is immobilized will be referred to as pre-sensitization particles. The first and second particles may be affinity particles, and their volume-average particle size and refractive index may be the volume-average particle size and refractive index of the affinity particles. In this embodiment, the ligand is preferably an antibody or antigen, and more preferably an antibody with an isoelectric point between 5.0 and 8.0. Affinity particles using antibodies with an isoelectric point in this range have a reduced charge on the surface of the affinity particles due to the influence of the antibody. As a result, the interaction between the affinity particles and the cationic components contained in the sample is reduced, and excellent measurement accuracy can be obtained even when the sample is large. The isoelectric point can be measured by isoelectric focusing electrophoresis. In this disclosure, ligand immobilization on pre-sensitized particles can be carried out by any known method, and the ligand can be immobilized by physically or chemically binding it to the pre-sensitized particles. For example, the ligand can be immobilized on the pre-sensitized particles by covalent bonds, hydrogen bonds, ionic bonds, electrostatic attraction, or van der Waals forces. Examples of chemical binding methods include methods utilizing carbodiimide-mediated reactions or NHS ester activation reactions, or methods in which avidin is bound to a carboxyl group and then a biotin-modified ligand is bound to it.

[0066] The amount of ligand per 1 mg of pre-sensitized particles is preferably 1.0 μg or more and 150.0 μg or less, and more preferably 2.0 μg or more and 100.0 μg or less. Furthermore, the affinity particles in this disclosure are affinity particles obtained by mixing two affinity particles, and it is even more preferable that the amount of ligand bound to the two affinity particles is different. That is, preferably, it includes a first affinity particle and a second affinity particle, and it is preferable that the amount of ligand bound to the first affinity particle and the amount of ligand bound to the second affinity particle are different.

[0067] The zeta potential of the affinity particles in this disclosure is preferably between -50mV and -15mV. A zeta potential of -50mV or higher ensures an appropriate balance between the electrostatic repulsion and electrostatic attraction of the affinity particles. This reduces the influence of ionic components in the sample, resulting in superior measurement accuracy. Furthermore, a zeta potential of -15mV or lower appropriately suppresses the particle aggregation rate. Excellent measurement accuracy can also be obtained even when the concentration of the target substance in the sample is high. The zeta potential of the affinity particles is more preferably between -40mV and -15mV.

[0068] In the immunoturbidimetric reagent of the present invention, it is preferable that the amount of ligand bound to the second particle is 0.1 or more and 1.5 or less by mass ratio to the amount of ligand bound to the first particle. A large amount of ligand bound to the first particle results in high binding affinity to the target substance, making it suitable for highly sensitive detection of low concentrations of the target substance. Furthermore, if the second particle also has a ligand amount of 0.1 times or more the amount of ligand bound to the first particle, it can be suitable for highly sensitive detection of high concentrations of the target substance.

[0069] In the immunoturbidimetric reagent disclosed herein, it is preferable that the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle is 0.1 or more and 1.5 or less. By having the concentration of the first particle 1.5 times or less than the concentration of the second particle, the reagent exhibits high binding affinity to the target substance and can be suitably used for highly sensitive detection of low concentrations of the target substance. Furthermore, if the second particle is present at a concentration of 0.1 times or more than the first particle, it can be suitably used for highly sensitive detection of high concentrations of the target substance.

[0070] <Detection method, measurement method> This disclosure provides, as a further embodiment, a measurement method using immunoturbidimetric methods with particles. The immunoturbidimetric method using particles provides a method for optically detecting interparticle aggregation that occurs when affinity particles of the above embodiment are mixed with a sample. In this embodiment, absorbance is used as the method for detecting this optical change. There are no restrictions on the equipment used for measurement; any optical instrument capable of detecting absorbance is acceptable. In particular, it is preferable to use a general-purpose automated analyzer that allows for easy control of sample and reagent dispensing volume, mixing time, measurement wavelength, etc.

[0071] The following describes an example of a measurement method using an automated analyzer. First, a predetermined amount of the sample is dispensed into the reaction vessel. Next, as mixing step 1, a predetermined amount of the first reagent is dispensed into the same reaction vessel, stirred and mixed, and then the temperature is adjusted to a predetermined level and maintained for a predetermined time. The temperature at this time is preferably in the range of 20°C to 50°C.

[0072] Subsequently, as mixing step 2, a predetermined amount of the second reagent is dispensed into the same reaction vessel, mixed by stirring, and then the reaction is carried out for a predetermined time. At this time, it is preferable to start mixing step 2 at least 280 seconds after the start of mixing step 1. By waiting 280 seconds after the start of mixing step 1, the sample and the first reagent are uniformly mixed, improving the measurement accuracy. Furthermore, it is preferable that the reaction time for mixing step 2 be between 200 seconds and 600 seconds. The start of the mixing step may refer to the time when the predetermined solution is dispensed and added into the same reaction vessel, or it may refer to the time when stirring and mixing is performed. However, it is preferable that stirring and mixing be started within 30 seconds of dispensing and adding.

[0073] Furthermore, the absorbance in mixing step 2 is measured, and the desired absorbance change is calculated. Preferably, the absorbance 42 seconds after the start of mixing step 2 is between 0.90 and 2.00. The absorbance 42 seconds after the start of mixing step 2 represents the initial absorbance before the agglutination reaction progresses, and having this value within the above range ensures sufficient measurement accuracy even at low concentrations of the target substance. Additionally, the measurement wavelength for absorbance preferably includes 500 nm to 750 nm. Including wavelengths within this range is preferable because it makes it easier to obtain a sufficient absorbance change and achieve good measurement accuracy even at low concentrations of the target substance.

[0074] The concentration of affinity particles in mixing step 2 is preferably 0.02% by mass or more and 0.20% by mass or less. This ensures an appropriate ratio of aggregated particles, thereby obtaining sufficient measurement accuracy. Furthermore, the pH in mixing step 2 is preferably 5.0 or more and 11.0 or less.

[0075] The viscosity of the mixed liquid in mixing step 2 is preferably 0.95 to 1.40. The viscosity within this range controls the particle aggregation rate, resulting in a more accurate measurement of the optimal absorbance change. Furthermore, excellent measurement accuracy is achieved even at high concentrations of the target substance.

[0076] <Specimen and target substance> The specimens that can be used in this disclosure are not particularly limited as long as they contain the target substance, but examples include blood, serum, plasma, etc. Examples of target substances in this disclosure include, but are not limited to, proteins such as ferritin, sialylated glycan antigen KL-6, CEA (carcinoembryonic antigen), glycan antigen 15-3, hepatitis C virus (HCV) antigen, hepatitis C virus antibody, hepatitis B virus antigen, and hepatitis B virus antibody.

[0077] <First reagent and second reagent> The reagent for the immunoturbidimetric test using particles used in the measurement method of this embodiment may include a first reagent and a second reagent containing affinity particles.

[0078] The surface tension of the first reagent is preferably between 20 mN / m and 50 mN / m. This range ensures uniform mixing of the affinity particles and the sample. As a result, the effect of the sample components on the affinity particles is homogenized, leading to a higher accuracy in measuring the change in absorbance.

[0079] The electrical conductivity of the first reagent is preferably between 0.5 mS / cm and 70.0 mS / cm. This range maintains the electrostatic repulsion of affinity particles, resulting in more uniform interaction between affinity particles, particularly with anionic sample components, leading to improved measurement accuracy. More preferably, the electrical conductivity of the first reagent is between 0.5 mS / cm and 65.0 mS / cm.

[0080] The pH of the first and second reagents is preferably between 5.0 and 11.0. Being within this range allows for a uniform dispersion state of affinity particles and a uniform mixing state when the samples are mixed, resulting in superior measurement accuracy. The pH values ​​of the first and second reagents may be different.

[0081] The first and second reagents preferably contain a buffer. The type of buffer is not particularly limited and any substance that provides buffering capacity is acceptable. For example, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPSO, EPPS, tricine, bicine, TAPS, CHES, and CAPS are preferably used as acetic acid, citrate, phosphoric acid, Tris, glycine, boric acid, and Good's buffers. One type of buffer may be used alone, or two or more types may be used in combination. Furthermore, the buffers used in the first and second reagents may be the same or different.

[0082] The first and second reagents preferably further contain sugars or sugar alcohols. The inclusion of sugars promotes hydration of the affinity particle surface and sample components, reducing the interaction between affinity particles and sample components, thereby improving measurement accuracy. Furthermore, the increased viscosity of the liquid controls the particle migration speed, resulting in high measurement accuracy when the mixing step 2 time is 300 seconds or less. Examples of such sugars and sugar alcohols include, but are not limited to, monosaccharides such as glucose and fructose, disaccharides such as sucrose, lactose, maltose, cellobiose, and trehalose, or oligosaccharides such as maltotriose and dextran, and sugar alcohols such as erythritol, mannitol, sorbitol, and xylitol. One type of sugar or sugar alcohol may be used alone, or two or more types may be used in combination.

[0083] The first and second reagents preferably further contain a surfactant. Known nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants can be used. Among these, it is preferable to contain at least one of sorbitan fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene phenyl ether, or polyoxyethylene cyclohexyl ether. These surfactants have high hydrophilicity and a high solubilizing and stabilizing effect on sample components. Therefore, the interaction between affinity particles and sample components is reduced, and excellent measurement accuracy can be obtained even when the concentration of the target substance is high. The concentration of the surfactant in mixing step 2 is preferably 0.001% by mass or more and 0.200% by mass or less.

[0084] The first reagent may further contain a sensitizer. Examples of sensitizers include water-soluble polymers such as polyethylene glycol, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, and polyglycosylethyl methacrylate.

[0085] In addition, the first and second reagents of this embodiment may contain chelating agents such as EDTA, CyDTA, DTPA, EGTA, NTA, and NTP; non-specific inhibitors such as proteins, protein hydrolysates, amino acids, animal serum, antibodies, antibody fragments, and reducing agents such as bovine serum albumin, casein, and gelatin; stabilizers such as proteins and preservatives; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0086] The method for measuring physical properties in this disclosure is described below. <Method for measuring the zeta potential of particles> The zeta potential of particles can be measured using a zetasizing device, the Nano-ZS (manufactured by Malvern Panalytic Corporation). The zeta potential is measured when the particles are dispersed in a 0.01N potassium hydroxide aqueous solution at pH 7.8 at a concentration of 0.003% by mass. The measurement conditions are 25°C, latex (n ≈ 1.59) is selected as the refractive index of the particles, and pure water is selected as the solvent. Ten measurements are taken, and the average value of the ten measurements is used as the zeta potential.

[0087] <Method for measuring the volume-average particle size> The volume-average particle size can be measured using a zetasizing device, the Nano-ZS (manufactured by Malvern Panalytic). The volume-average particle size can be measured when the particles are dispersed in ion-exchanged water at a concentration of 0.003% by mass. Measurement conditions are 25°C, with latex (n≈1.59) selected as the particle refractive index, and pure water selected as the solvent. Ten measurements are taken, and the zeta potential can be calculated using the average of these ten measurements.

[0088] Furthermore, the ratio of volume-average particle sizes when two or more particles with different particle sizes are mixed can be determined by observation using a scanning electron microscope or similar device. Specifically, more than 300 particles are photographed at a magnification of 50,000x, and the particle sizes of these 300 particles are measured using a known image processing method. The volume of each particle is determined from the measured particle sizes, and a volume distribution is created. The particle size that causes a peak in the created volume distribution is taken as the volume-average particle size of each particle, and the ratio of the particle sizes of the two particles can be determined by calculating the difference.

[0089] Another method for measuring the ratio of volume-average particle sizes when two or more types of particles are mixed is to fractionate the mixed particles using methods such as density gradient centrifugation, and then measure them using the method described above.

[0090] <Method for measuring the amount of antibody against particles (antibody sensitization amount of affinity particles)> This disclosure describes a method for measuring the antibody sensitization level of affinity particles. The antibody sensitization level of affinity particles can be determined by protein quantification. Here, the antibody sensitization level of particles refers to the amount of antibody bound or adsorbed per 1 mg of particle. In the following examples, the measurement was performed as follows.

[0091] First, mix 7 mL of solution A and 140 μL of solution B from the Protein Assay BCA Kit (manufactured by Wako Pure Chemical Industries) to prepare a solution called solution AB. Next, add 25 μL of affinity particle dispersion (particle concentration 0.1%) to 200 μL of solution AB and incubate at 60°C for 30 minutes. Next, the solution is centrifuged at 20400g at 4°C for 5 minutes, and 200 μL of the supernatant is pipetted into a 96-well microplate. 200 μL of standard samples, each containing antibody mixed in 10 mM HEPES buffer at a desired concentration (5 points within the concentration range of 0 μg / mL to 200 μg / mL), is added to a separate microplate. The absorbance at 562 nm is measured using a microplate reader, and the antibody amount is calculated from the calibration curve of the standard samples. The antibody amount per particle (μg / mg) is determined by dividing the calculated antibody amount by the particle weight.

[0092] <Method for measuring the refractive index of particles> The refractive index of particles can be measured using Abbemat (manufactured by Anton Paar). In the following examples, the refractive index was specifically measured when the particle dispersion was dispersed to a concentration of 5% by mass. The measurement conditions selected were 25°C and a measurement wavelength of 589.3 nm. The particle refractive index was calculated from the Lorentz-Lorentz equation using the measured refractive index value, the specific gravity of the dispersion medium, the refractive index, and the specific gravity of the particles. [Examples]

[0093] The present disclosure will be described in detail below with reference to examples, but the present invention is not limited to these examples. Furthermore, although the following examples use ferritin and HCV antibody as target substances, the effects of this disclosure are due to particle design and can be obtained regardless of the target substance; therefore, they are not limited to ferritin and HCV antibody.

[0094] (Synthesis of particle 1) (Process 1) In a 2L four-neck separable flask, 93.1g of styrene (St: manufactured by Kishida Chemical Co., Ltd.), 1.7g of divinylbenzene (DVB: manufactured by Kishida Chemical Co., Ltd.), and 1190.7g of deionized water were weighed out and mixed. This mixture was then maintained at 70°C while stirring at 200 rpm, and the inside of the four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Next, a solution prepared separately by dissolving 4.0g of V-50 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in 50g of deionized water was added to the mixture, and a polymerization reaction was carried out for 48 hours to obtain a copolymer particle dispersion of St and DVB.

[0095] (Process 2) Next, 148.3 g of the above dispersion, diluted with deionized water to a solid content concentration of 2.0% by weight, was weighed into another four-necked separable flask. Then, 0.39 g of glycidyl methacrylate (GMA: manufactured by Kishida Chemical Co., Ltd.) was added, and the mixture was maintained at 70°C while stirring at 100 rpm, and the inside of the four-necked separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Then, a solution prepared separately by dissolving 0.018 g of V-50 in 1 g of deionized water was added to the aforementioned mixture, and the mixture was stirred continuously for 17 hours to obtain a dispersion containing St / DVB / GMA composite particles.

[0096] (Step 3) An aqueous solution of mercaptosuccinic acid (MSA: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 3-mercapto-1,2-propanediol (MPD: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), prepared in advance, was added to a dispersion containing St / DVB / GMA composite particles (MSA and MPD were in a 6:4 (mol fraction) ratio, and the total number of moles of MSA and MPD was equivalent to the number of moles of the aforementioned glycidyl methacrylate). Triethylamine (manufactured by Kishida Chemical Co., Ltd.) was then added to adjust the pH to 10. Next, the dispersion was heated to 70°C while stirring at 200 rpm, and maintained at this temperature for 18 hours to obtain a dispersion of particle 1. Subsequently, particle 1 was separated from the dispersion using a centrifuge, and the particle 1 was further purified by repeating the operation of redispersing particle 1 in ion-exchanged water eight times to finally obtain a particle 1 dispersion with a solid content of 5.0% by mass. The volume-average particle size of the obtained particle 1 was 400 nm.

[0097] (Synthesis of particle 4 from particle 2) In the synthesis method for particle 1, particle 4 was synthesized from particle 2 using the same experimental procedure as for particle 1, except that the amounts of St, DVB, and V-50 used in step 1 and the amount of GMA in step 2 were changed as shown in Table 1. The physical properties of the obtained particles 1 to 4 are summarized in Table 1.

[0098] In all of particles 1 to 4, the core particle contains a styrene-divinylbenzene copolymer, that is, it has a structural unit represented by formula (1), R 1 R is a hydrogen atom. 2 It was a phenyl group. Also, the shell was R 4 However, it included a structural unit represented by formula (2) having a structural unit represented by any of the following formulas (31), (32), (33), or (34). [ka] [ka] [ka] [ka]

[0099] [Table 1]

[0100] (Synthesis of particle 5) 22.7 g of St, 33.9 g of GMA, 0.86 g of DVB, and 2168.6 g of deionized water were weighed into a 2 L four-neck separable flask to form a mixture. This mixture was then kept at 70°C while stirring at 200 rpm, and the flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Next, a solution of 1.1 g of V-50 dissolved in 30 g of deionized water, which had been prepared separately, was added to the mixture to initiate soap-free emulsion polymerization. Two hours after the start of polymerization, 5.8 g of GMA was added to the aforementioned four-neck separable flask, and the mixture was kept at 70°C for another 22 hours while stirring at 200 rpm to obtain a copolymer particle dispersion of St and DVB. Next, an aqueous solution of MSA and MPD prepared in advance (MSA and MPD in a 6:4 (mol fraction) ratio, with the total number of moles of MSA and MPD being equal to the number of moles of GMA mentioned above) was added, and triethylamine was added to adjust the pH to 10. Then, the dispersion was heated to 70°C while stirring at 200 rpm, and maintained in this state for 18 hours to obtain a dispersion of particle 5. After that, particle 5 was separated from the dispersion using a centrifuge, and the particle 5 was further purified by repeating the operation of redispersing particle 5 in ion-exchanged water eight times, finally obtaining a particle 5 dispersion with a solid content of 5.0% by mass. The volume-average particle size of the obtained particle 5 was 250 nm.

[0101] (Synthesis of particle 6) Particle 6 was synthesized using the same experimental procedure as for particle 5, except that the amounts of St, DVB, V-50, and GMA used were changed as shown in Table 2. The physical properties of the obtained particles 5 and 6 are summarized in Table 2.

[0102] In both particle 5 and 6, the particle is R 1 R is a hydrogen atom. 2 R is a structural unit represented by formula (1) which is a phenyl group, and 4 However, it included a structural unit represented by formula (2) having a structural unit represented by any of the above formulas (31), (32), (33), or (34).

[0103] [Table 2]

[0104] (Production of affinity particle 1) For the dispersion of particle 1, 300 μL of the dispersion (3 mg as particle solids), diluted with deionized water to a solid content concentration of 1.0% by mass, was taken into a 1.5 mL microtube. 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (hereinafter EDC) (manufactured by Tokyo Chemical Industry Co., Ltd.) and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium (hereinafter NHS) (manufactured by Tokyo Chemical Industry Co., Ltd.) were added, and the mixture was stirred at room temperature for 30 minutes to obtain an activated particle dispersion containing carboxyl groups (activated particle dispersion). After centrifugal washing, 270 μL of pH 5.5 phosphate buffer-physiological saline (PBS) was added, and the particles with activated carboxyl groups were dispersed using ultrasound. To this, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.12 mg of antibody) was added and stirred at room temperature for 3 hours to obtain test particles sensitized with the antibody. After centrifugation washing of these test particles, 500 μL of PBS was added to obtain affinity particle 1.

[0105] (Production of affinity particle 13 from affinity particle 2) In the method for producing affinity particle 1, affinity particle 12 was produced from affinity particle 2 using the same experimental procedure as for the production of affinity particle 1, except that the particle type and the amount of antibody dispersion added were changed as shown in Table 3. Furthermore, affinity particle 13 was produced using the same experimental procedure as for the production of affinity particle 1, except that Immutex-Carboxyl® P0307 (hereinafter referred to as P0307, ​​manufactured by JSR Life Science Co., Ltd.) was used as the particle type and the amount of antibody dispersion added was changed as shown in Table 3.

[0106] [Table 3]

[0107] (Preparation of Reagent 1) Second reagent 1 was prepared using the prepared affinity particles 1 and 4. Specifically, affinity particles 1 and 4 were centrifuged and redispersed in 500 μL of a buffer (HEPES buffer) containing 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (TritonX-100, Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​dissolved in deionized water. Then, the mixture and dilution with HEPES buffer were performed so that affinity particle 1 was 0.08% by mass, affinity particle 4 was 0.1% by mass, and the total was 0.2% by mass, to obtain second reagent 1.

[0108] (Preparation of Reagent 2 to Reagent 12) Second reagents 2 through 12 were prepared using the same experimental procedure as for second reagent 1, except that the type of affinity particles and the concentration of affinity particles were changed as shown in Table 4. The physical properties of the obtained second reagents 1 through 12 are summarized in Table 4. In all cases of second reagents 1 through 10, the condition P1U1 / P1U2 > P2U1 / P2U2 was satisfied.

[0109] [Table 4]

[0110] (Preparation of Reagent 1) First reagent 1 was prepared by dissolving 50 mM HEPES, 0.05% by mass Triton X-100, and 1.0% by mass sodium chloride (manufactured by Kishida Chemical Co., Ltd.) in deionized water.

[0111] (Example 1: Measurement of absorbance change) A mixture was prepared by mixing 15 μL of the specified sample with 60 μL of the first reagent 1, and the mixture was incubated at 37°C for 290 seconds. Next, 30 μL of the second reagent 1 was mixed into the mixture, and the absorbance was measured after stirring for 42 seconds. Furthermore, this mixture was allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance measured after 30 seconds was defined as the change in absorbance. Absorbance measurements were performed using an Eppendorf BIOSPECTROMETER spectrophotometer, with a measurement wavelength of 572 nm. The optical path length was 1 cm. The types of samples used are described in the explanation of each evaluation item below.

[0112] (Method for measuring the change in absorbance per 1 ng / mL of ferritin) In measuring the change in absorbance, the change in absorbance was calculated when each sample was prepared to have ferritin concentrations of 0.0 ng / mL (physiological saline), 25.0 ng / mL, 1000.0 ng / mL, and 2000.0 ng / mL.

[0113] (Examples 2 to 9, Comparative Examples 1 to 3) In the measurement of the absorbance change in Example 1, the absorbance change was measured using the same experimental procedure as in Example 1, except that the type of the second reagent was changed as shown in Table 5. Furthermore, the difference between the absorbance change at a ferritin concentration of 2000.0 ng / mL and the absorbance change at a ferritin concentration of 1000.0 ng / mL was calculated and is shown in Table 5. [Table 5]

[0114] These results show that the second reagent, having a volume-average particle size ratio and refractive index ratio that satisfy the provisions of this disclosure, exhibits a large change in absorbance at a target substance concentration of 25.0 ng / mL, demonstrating excellent minimum detection sensitivity. Furthermore, the difference in absorbance change between the target substance concentrations of 2000.0 ng / mL and 1000.0 ng / mL is positive, indicating that it has the effect of increasing the absorbance change even in the high-concentration measurement range.

[0115] On the other hand, in the second reagents with volume-average particle size ratios of 0.96 (Comparative Example 1) and 0.36 (Comparative Example 2), and a refractive index ratio of 0.99 (Comparative Example 3), the difference in absorbance change was negative, indicating that the high-concentration measurement range was inferior.

[0116] (Synthesis of particle 7 and particle 8) Particles 7 and 8 were synthesized using the same experimental procedure as for the synthesis of particles 1 and 4, except that the ratio of MSA to MPD in the aqueous solution used in step 3 was changed to 10:0 (mol fraction). The volume-average particle sizes of the obtained particles 7 and 8 were 400 nm and 250 nm, respectively. The zeta potentials of the obtained particles 7 and 8 were -41 mV and -36 mV, respectively.

[0117] (Preparation of affinity particles 14 and 15) The HCV antigen solution (TRINA BIOREACTIVES) was solvent-replaced using an ultrafiltration device (Amicon Ultra 10K: Merck). The solution was diluted to a protein concentration of 0.14 mg / mL or 0.07 mg / mL to obtain "HCV antigen solution R1 (0.14 mg / mL)" or "HCV antigen solution R2 (0.07 mg / mL)". For dilution, 10 mM HEPES buffer with 20 mM (±) dithiothreitol (DTT: Fujifilm Wako Pure Chemical Industries, Ltd.) added was used.

[0118] 60 μL of a 1.7% by mass suspension of particles 7 and 8 was placed in a microcentrifuge tube. 30 μL of a 0.1% aqueous solution of EDC and 30 μL of a 0.1% aqueous solution of NHS were added to it. The above was stirred at room temperature for 30 minutes to obtain a dispersion of particles with activated carboxyl groups (activated particle dispersion).

[0119] After centrifugation, 50 μL of 10 mM HEPES buffer was added, and the particles with activated carboxyl groups were dispersed by ultrasound. 50 μL of "HCV antigen solution R1" was added to the dispersion of particles 7 with activated carboxyl groups, and 50 μL of "HCV antigen solution R2" was added to the dispersion of particles 8 with activated carboxyl groups. The mixture was stirred at room temperature for 1 hour to bind the antigen to the carboxyl groups of the particles.

[0120] After centrifugation washing, 240 μL of masking buffer (0.3 M glycine solution containing 0.1% Tween 20, pH 8.0) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was then left to stand overnight at 4°C to allow glycine to bind to the remaining activated carboxyl groups.

[0121] After centrifugation washing, affinity particles 14 and 15 were obtained by adding 0.5 mL of storage buffer (10 mM HEPES, pH 7.9 containing 0.01% Tween20) and dispersing the particles using ultrasound. The volume-average particle size, refractive index, and amount of protein supported on the particle surface of the obtained affinity particles are summarized in Table 6.

[0122] [Table 6]

[0123] (Preparation of the second reagent 13) Second reagent 13 was prepared using the prepared affinity particles 14 and 15. Specifically, affinity particles 14 and 15 were centrifuged and redispersed in 500 μL of buffer containing 10 mM HEPES and 0.01% by mass Tween 20 dissolved in deionized water. Then, the mixture and dilution with HEPES buffer were performed so that affinity particles 14 accounted for 0.09% by mass and affinity particles 15 accounted for 0.2% by mass, thereby obtaining second reagent 13.

[0124] The ratio of the volume-average particle sizes (Dv2 / Dv1) of affinity particles 14 and 15 was 0.60, and the ratio of their refractive indices (R2 / R1) was 0.97. Furthermore, the second reagent 13 satisfied the condition P1U1 / P1U2 > P2U1 / P2U2.

[0125] (Preparation of Reagent 2) The following materials were mixed and dissolved in ultrapure water to the concentrations described below, and the pH was adjusted to 7.5 to obtain the first reagent 2. 100mM HEPES 500 mM sodium chloride ·0.1 mass% Tween20 • 0.5% by mass CHAPS (3-[(3-colamidopropyl)dimethylammonio]-1-propanesulfonic acid) • 0.2% by mass of polyethylene glycol (molecular weight approximately 500,000)

[0126] (Example 10: Evaluation of HCV antibody detection) The absorbance change was measured using the same experimental procedure as in Example 1, except that 10 μL of the specified sample was used, the first reagent was changed to 65 μL of first reagent 2, and the second reagent was changed to 25 μL of second reagent 13. Negative control serum (ACCURUN810 Multimarker Negative Control: Minaris Medical Co., Ltd.) and HCV positive control serum (ACCURUN Series Infectrol D: Minaris Medical Co., Ltd.) were used as samples. The HCV positive control serum used had a cutoff index (COI) value of 34.9, as measured by Lumipulse Presto Ortho HCV (PrestoII / L2400). HCV positive solutions were prepared by serially diluting the HCV positive control serum with negative control serum, and an HCV positive solution with a COI value of 1 was prepared.

[0127] Reagent 13, an HCV-positive solution with a COI value of 1, showed a significantly higher change in absorbance compared to the negative control serum. This indicates superior minimum detection sensitivity in HCV antibody detection.

[0128] Furthermore, in HCV-positive solutions diluted stepwise, the change in absorbance increased in proportion to the HCV-positive control serum up to a COI value of 34.9. This indicates that the HCV antibody detection method has the effect of significantly increasing the change in absorbance even in the high-concentration measurement range.

[0129] This embodiment includes the following configurations and methods. (Composition 1) It contains the first particle and the second particle, The ratio of the volume-average particle size (Dv2) of the second particle to the volume-average particle size (Dv1) of the first particle, Dv2 / Dv1, is 0.37 or more and 0.86 or less. Dv2 is 150nm or more, A reagent for immunoturbidimetric testing, characterized in that the ratio R2 / R1, which is the ratio of the refractive index (R2) of the second particle to the refractive index (R1) of the first particle, is 0.87 or more and 0.97 or less. (Configuration 2) The immunoturbidimetric reagent according to configuration 1, characterized in that both the first particle and the second particle each contain a structural unit represented by formula (1) and a structural unit represented by formula (2). [ka] (R 1 Each structural unit independently represents either a hydrogen atom or a methyl group. 2 (Each structural unit independently represents a phenyl group or a naphthyl group, which may be substituted.) [ka] (R 3 Each structural unit independently represents either a hydrogen atom or a methyl group. 4 (Each structural unit independently represents an arbitrary group.) (Composition 3) The immunoturbidimetric reagent according to configuration 2, characterized in that the ratio P1U1 / P1U2, which is the ratio of the number of structural units (P1U1) represented by formula (1) to the number of structural units (P1U2) represented by formula (2) in the first particle, is greater than the ratio P2U1 / P2U2, which is the ratio of the number of structural units (P2U1) represented by formula (1) to the number of structural units (P2U2) represented by formula (2) in the second particle. (Composition 4) R in equation (2) above 4 The immunoturbidimetric reagent according to configuration 2 or 3, characterized in that each structural unit independently comprises a group containing an epoxy group, a group containing a hydroxyl group, a group containing a carboxyl group, or any of the groups represented by formula (2-A'). [ka] (R 31 and R 32 One of the terms represents a hydroxyl group, and the other represents a hydroxyl group or a group represented by formula (2-B). * (This indicates the bonding position.) [ka] (R 20R indicates a single bond or a methylene group. 22 , R 23 , R 24 Each of these represents a hydrogen atom, a methyl group, a hydroxyl group, a carboxyl group, a hydroxymethyl group, or a carboxymethyl group, respectively. 22 , R 23 , and R 24 At least one of them contains a hydroxyl group or a carboxyl group. 1 This represents a sulfur atom or an imino group. * (This indicates the bonding position.) (Composition 5) The immunoturbidimetric reagent according to any one of configurations 2 to 4, characterized in that both the first particle and the second particle have a core containing a structural unit represented by formula (1) and a shell containing a structural unit represented by formula (2). (Composition 6) The immunoturbidimetric reagent according to any one of configurations 1 to 5, characterized in that both the first particle and the second particle have a zeta potential of -50 mV or more and -15 mV or less. (Composition 7) The immunoturbidimetric reagent according to any one of configurations 1 to 6, characterized in that both the first particle and the second particle have ligands, and the mass ratio of the amount of ligand in the second particle to the amount of ligand in the first particle is 0.1 or more and 1.5 or less. (Composition 8) The immunoturbidimetric reagent according to configuration 7, characterized in that the ligand is an antibody having an isoelectric point of 5.0 to 8.0. (Composition 9) An immunoturbidimetric reagent according to any one of configurations 1 to 8, characterized in that the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle contained in the reagent is 0.1 or more and 1.5 or less. (Composition 10) A reagent for immunoturbidimetric testing according to any one of the configurations 1 to 9, characterized by comprising at least one of sugars and sugar alcohols. (Composition 11) A reagent for immunoturbidimetric testing according to any one of the configurations 1 to 10, characterized by comprising at least one surfactant selected from the group consisting of sorbitan fatty acid esters, polyoxyethylene alkyl ethers, and polyoxyethylene phenyl ethers.

Claims

1. It contains the first particle and the second particle, The ratio of the volume-average particle size (Dv2) of the second particle to the volume-average particle size (Dv1) of the first particle, Dv2 / Dv1, is 0.37 or more and 0.86 or less. Dv2 is 150 nm or greater, A reagent for immunoturbidimetric testing, characterized in that the ratio R2 / R1, which is the ratio of the refractive index (R2) of the second particle to the refractive index (R1) of the first particle, is 0.87 or more and 0.97 or less.

2. The immunoturbidimetric reagent according to claim 1, characterized in that both the first particle and the second particle contain a structural unit represented by formula (1) and a structural unit represented by formula (2). 【Chemistry 1】 (R １ Each structural unit independently represents either a hydrogen atom or a methyl group. ２ (Each structural unit independently represents a phenyl group or a naphthyl group, which may be substituted.) 【Chemistry 2】 (R ３ Each structural unit independently represents either a hydrogen atom or a methyl group. ４ (Each structural unit independently represents an arbitrary group.)

3. The immunoturbidimetric reagent according to claim 2, characterized in that the ratio P1U1 / P1U2, which is the ratio of the number of structural units represented by formula (1) to the number of structural units represented by formula (2) (P1U2) in the first particle, is greater than the ratio P2U1 / P2U2, which is the ratio of the number of structural units represented by formula (1) to the number of structural units represented by formula (2) (P2U2) in the second particle.

4. R in formula (2) above ４ The immunoturbidimetric reagent according to claim 2, characterized in that each structural unit is independently a group containing an epoxy group, a group containing a hydroxyl group, a group containing a carboxyl group, or any of the groups represented by formula (2-A'). 【Transformation 3】 (R ３１ and R ３２ One of the terms represents a hydroxyl group, and the other represents a hydroxyl group or a group represented by formula (2-B). ＊ (This indicates the bonding position.) 【Chemistry 4】 (R ２０ represents a single bond or a methylene group. R ２２ , R ２３ , R ２４ each represents a hydrogen atom, a methyl group, a hydroxy group, a carboxy group, a hydroxymethyl group or a carboxymethyl group. At least one of R ２２ , R ２３ , and R ２４ contains a hydroxy group or a carboxy group. Y １ represents a sulfur atom or an imino group. ＊ indicates the bonding position. )

5. The immunoturbidimetric reagent according to claim 2, characterized in that both the first particle and the second particle each have a core containing a structural unit represented by formula (1) and a shell containing a structural unit represented by formula (2).

6. The immunoturbidimetric reagent according to claim 1, characterized in that both the first particle and the second particle have a zeta potential of -50 mV or more and -15 mV or less.

7. The immunoturbidimetric reagent according to claim 1, characterized in that both the first particle and the second particle have ligands, and the mass ratio of the amount of ligand in the second particle to the amount of ligand in the first particle is 0.1 or more and 1.5 or less.

8. The immunoturbidimetric reagent according to claim 7, characterized in that the ligand is an antibody having an isoelectric point of 5.0 or higher and 8.0 or lower.

9. The immunoturbidimetric reagent according to claim 1, characterized in that the ratio of the concentration (by mass) of the second particle to the concentration (by mass) of the first particle contained in the reagent is 0.1 or more and 1.5 or less.

10. The immunoturbidimetric reagent according to claim 1, characterized by comprising at least one of sugars and sugar alcohols.

11. The immunoturbidimetric reagent according to claim 1, characterized by comprising at least one surfactant selected from the group consisting of sorbitan fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene phenyl ether.

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