Latex particle for supporting physiologically active substance

Abstract

Provided are novel latex particles for supporting a physiologically active substance, the latex particles having high analysis sensitivity and accuracy and being stably producible. The latex particles for supporting a physiologically active substance comprise a polymer chain represented by general formula (wherein, at least one of R2-R6 is a halogen atom).

Description

Latex particles for supporting physiologically active substances

[0001] The present invention relates to latex particles for supporting physiologically active substances, a method for producing the same, and use thereof. In the present specification, the term "analysis" includes both "measurement" for quantitatively or semi-quantitatively determining the amount of an analyte and "detection" for determining the presence or absence of an analyte.

[0002] Currently, in the field of clinical diagnostic testing, there is a demand for measuring a wide variety of substances serving as indicators for disease diagnosis in a large number of specimens in a short time with high accuracy, and feeding the results back rapidly and accurately to the treatment site. For example, accurate quantification of trace amounts of analytes is widely carried out by analytical systems in which physiologically active substances (proteins such as antibodies, enzymes, and receptors, antigens, nucleic acid substances such as DNA and RNA, or sugar chains, etc.) are bound to particle surfaces, and by immunological measurement utilizing antigen-antibody reactions. As a method for particularly improving detection sensitivity and accuracy, a method using particles in which an antigen or antibody against an analyte is supported on the surface of fine particles made of a polymer such as polystyrene (so-called latex particles) is known.

[0003] As a method using latex particles, for example, latex agglutination is widely used. The latex agglutination method is a method for measuring an analyte in a short time by visually or optically detecting the degree of aggregation of latex particles caused by the reaction between the analyte and an antigen or antibody bound to the latex particles.

[0004] Many of the analytes quantified by antigen-antibody reactions are generally trace components present in biological samples, and quantitative accuracy in the low-concentration range is crucial. However, depending on the progression of a disease, the concentration of the target substance may show abnormally high values. Therefore, in clinical testing settings, reagents capable of accurately measuring from low to high values ​​are required. In latex agglutination, increasing the particle size of latex particles is known as a way to improve sensitivity in the low-concentration range. However, larger latex particles have high absorbance, limiting the concentration that can be formulated as a reagent, and their small specific surface area limits the amount of antibody modification, resulting in a narrower measurement range.

[0005] As a method to solve these problems, latex particles using high refractive index materials have been reported. Patent Document 1 reports an immunoaggregation reaction using particles made of titanium dioxide, an inorganic material. However, because it has a higher specific gravity than conventional materials such as polystyrene and tends to settle easily, there are problems such as limitations on the upper limit of particle size when manufacturing it as a diagnostic agent. Patent Document 2 reports hollow-shaped particles containing compounds having at least one skeleton selected from the group consisting of a fluorene skeleton, a dinaphthothiophene skeleton, a naphthalene skeleton, anthracene skeleton, and a phenanthrene skeleton, as compounds with a refractive index of 1.60 or higher. However, hollow-shaped particles are difficult to synthesize and quality control is also difficult, making them impractical, and there have been no reports of them being put into practical use in clinical settings. Therefore, latex particles used in clinical diagnostic agents are required to improve the stability of manufacturing clinical diagnostic agents and the sensitivity and accuracy of clinical diagnostic agents, but these have not yet been achieved.

[0006] Furthermore, it has been reported that immunoassay reagents characterized by latex particles containing halogen atoms can reduce the adhesion of contaminants in reaction cells (Patent Document 3). For example, Examples 1, 3, and 4 of Patent Document 3 describe the production of latex particles using styrene and 4-fluorostyrene as monomers, and Example 2 describes the production of latex particles using styrene and 2,3,4,5,6-pentafluorostyrene as monomers. Since halogen atoms are negatively charged, they are generally thought to suppress the adsorption of biological samples to hydrophobic resins. On the other hand, they are thought to affect the interaction between antibodies and antigens and to hinder the aggregation characteristics of latex particles, and there are no reports that they contribute to improving the reactivity of antibodies and antigens. For this reason, there are no reports of including halogen atoms or halogenated polymers in high concentrations in the reaction field of antibodies and antigens, and in Patent Document 3, the ratio of halogen-containing monomers to the total monomers used in the production of latex particles is in the range of 5 to 20% by weight in all examples (the particles produced in Examples 1 to 4). Furthermore, in the manufacturing method described in Patent Document 3, it is unlikely that halogen atoms are unevenly distributed on the particle surface.

[0007] Japanese Patent Publication No. 6683907, Japanese Patent Publication No. 7161213, International Publication No. 2020 / 203755

[0008] The object of the present invention is to provide novel latex particles for carrying physiologically active substances, a method for producing the same, and an analytical reagent for latex agglutination using the said latex particles for carrying physiologically active substances, which have high analytical sensitivity and accuracy and can be manufactured stably.

[0009] The present invention relates to the following: [1] General formula (10): R15-Bm-An-R1 (10) or general formula (1): In general formula (10) and general formula (1), R2 to R6 and R8 to R14 are each independently: (i) a monovalent functional group selected from the group consisting of hydrogen atoms, halogen atoms, methyl groups, ethyl groups, propyl groups, cyclopropyl groups, hydroxyl groups, amino groups, methoxy groups, thiol groups, cyano groups, vinyl groups, isocyanate groups, aldehyde groups, carboxyl groups, nitro groups, allyl groups, aryl groups, boronic acids, isocyanide groups, ketone groups, phenyl groups, sulfo groups, epoxy groups, glycidyl groups, and pyridine groups; or (ii) a monovalent functional group in which one or more carbon atoms are substituted with oxygen atoms, sulfur atoms, or nitrogen atoms. (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, at least one of R2 to R6 is a halogen atom, provided that none of R11 to R14 are phenyl groups substituted with halogen atoms, R7 is a single bond, ester bond, thioether bond, amide bond, or ether bond, and R1 and R15 are the functional groups specified in (i) to (iv) above, or functional groups derived from polymerization initiators. The order of the repeating units A and B can be random or in blocks.[1] A latex particle for carrying a physiologically active substance, comprising a polymer chain represented by

[10] or (1) in general formula (1), where m and n represent the total number of repeating units B and A contained in one polymer chain, and m is an integer of 0 or more, and n is an integer of 1 or more. [2] A latex particle for carrying a physiologically active substance according to [1], wherein m is an integer of 1 or more in general formula (1). [3] A latex particle for carrying a physiologically active substance according to [1], wherein the latex particle for carrying a physiologically active substance comprises a core portion located inside the particle and a shell portion located around it. [4] A latex particle for carrying a physiologically active substance according to [3], wherein the mole percentage of repeating units A in the polymer chain represented by general formula (10) or (1) constituting the shell portion [{n / (n+m)} × 100] is greater than the mole percentage of repeating units A in the polymer chain represented by general formula (10) or (1) constituting the core portion [{n / (n+m)} × 100]. [5] Latex particles for carrying physiologically active substances according to [3], wherein the core portion is mainly composed of a polymer chain represented by general formula (10) or (1) where m is 1 or more and n is 0 or more, and the shell portion is mainly composed of a polymer chain represented by general formula (10) or (1) where m is 0 or more and n is 1 or more. [6] Latex particles for carrying physiologically active substances according to [5], wherein the core portion is mainly composed of a polymer chain represented by general formula (10) or (1) where m is 1 or more and n is 0, and the shell portion is mainly composed of a polymer chain represented by general formula (10) or (1) where m is 1 or more and n is 1 or more. [7] Latex particles for carrying physiologically active substances according to any of [1] to [6], wherein the halogen atom is an atom selected from the group consisting of fluorine, chlorine, bromine, and iodine. [8] Latex particles for carrying physiologically active substances according to any one of [1] to [7], obtained by polymerization using a first monomer, a second monomer, a polymerization initiator, and a surfactant, wherein the first monomer is of the following general formula (X): [In the formula, R2 to R6 and R8 to R10 are each independently (i) a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (ii) a monovalent functional group in which one or more carbon atoms are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom.] (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, wherein at least one of R2 to R6 is a halogen atom, and R7 is a single bond, ester bond, thioether bond, amide bond, or ether bond, and the second monomer is represented by the following general formula (Y): [In the formula, R11 to R14 are each independently (i) a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (ii) a monovalent functional group in which one or more carbon atoms are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom.] (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) wherein the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, and none of R11 to R14 are phenyl groups substituted with halogen atoms. The latex particle for carrying a physiologically active substance according to [8], wherein the first monomer and / or the second monomer further contain a functional group for carrying a physiologically active substance. A method for producing latex particles for carrying physiologically active substances according to any of [1] to [8], comprising polymerizing the first monomer and the second monomer described in

[10] and [8].

[11] A method for producing

[10] , wherein the first monomer and / or the second monomer further contains a functional group for supporting a physiologically active substance.

[12] An analytical reagent for latex agglutination, comprising any of the physiologically active substance-supporting latex particles of [1] to [9].

[0010] According to the present invention, it is possible to provide latex particles for carrying physiologically active substances that have high analytical sensitivity and accuracy and can be manufactured stably, in particular latex particles for carrying physiologically active substances with improved sensitivity in the low-concentration range. Furthermore, according to one preferred embodiment of the present invention, latex particles for carrying physiologically active substances having a core-shell structure, the amount of functional groups for carrying physiologically active substances in the shell portion and / or the amount of halogenated phenyl groups in repeating unit A derived from the first monomer can be easily increased, thereby further improving analytical sensitivity and accuracy (especially, for example, sensitivity in the low-concentration range). In addition, by adopting a core-shell structure, it becomes easier to control the amount of functional groups for carrying physiologically active substances in the shell portion and / or the amount of halogenated phenyl groups, thus enabling stable manufacturing.

[0011] This is a photograph, instead of a drawing, showing a scanning electron microscope image of the latex particles produced in Example 1. This is a graph showing the results of the reactivity test of the D-dimer measurement reagent produced in Example 8. This is a graph showing the results of the sensitivity test of the D-dimer measurement reagent (Example). This is a graph showing the results of the sensitivity test of the D-dimer measurement reagent (Comparative Example). This is a graph showing the results of the reactivity test of the TAT measurement reagent produced in Example 12. This is a graph showing the results of the sensitivity test of the TAT measurement reagent (Example). This is a graph showing the results of the sensitivity test of the TAT measurement reagent (Comparative Example).

[0012] The latex particles for carrying physiologically active substances of the present invention (hereinafter sometimes simply referred to as the latex particles of the present invention) have a general formula (10): R15-Bm-An-R1 (10) or general formula (1): In general formula (10) and general formula (1), R2 to R6 and R8 to R14 are each independently: (i) a monovalent functional group selected from the group consisting of hydrogen atoms, halogen atoms, methyl groups, ethyl groups, propyl groups, cyclopropyl groups, hydroxyl groups, amino groups, methoxy groups, thiol groups, cyano groups, vinyl groups, isocyanate groups, aldehyde groups, carboxyl groups, nitro groups, allyl groups, aryl groups, boronic acids, isocyanide groups, ketone groups, phenyl groups, sulfo groups, epoxy groups, glycidyl groups, and pyridine groups; or (ii) a monovalent functional group in which one or more carbon atoms are substituted with oxygen atoms, sulfur atoms, or nitrogen atoms. (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, at least one of R2 to R6 is a halogen atom, provided that none of R11 to R14 are phenyl groups substituted with halogen atoms, R7 is a single bond, ester bond, thioether bond, amide bond, or ether bond, and R1 and R15 are the functional groups specified in (i) to (iv) above, or functional groups derived from polymerization initiators. The order of the repeating units A and B can be random or in blocks.The polymer chain includes a polymer chain represented by [m and n, where m and n represent the total number of repeating units B and A contained in one polymer chain, and m is an integer greater than or equal to 0, and n is an integer greater than or equal to 1].

[0013] The latex particles of the present invention comprise a polymer chain represented by general formula (10) or general formula (1), wherein the polymer chain comprises repeating unit A containing a halogenated phenyl group, and optionally comprises repeating unit B without a halogenated phenyl group. The latex particles of the present invention are not limited to these, but can be produced by a polymerization reaction between a first monomer represented by general formula (X) and a second monomer represented by general formula (Y), which can be used optionally, as described in detail below. In this case, repeating unit A is derived from the first monomer, and repeating unit B is derived from the second monomer.

[0014] The halogenated phenyl group contained in repeating unit A is bonded directly to the main chain of the polymer chain represented by general formula (10) or general formula (1), or via group R7, and a halogen atom is bonded to at least one of the remaining five carbon atoms, excluding the carbon atom bonded to the main chain of the polymer chain. Examples of halogen atoms include fluorine, chlorine, bromine, iodine, and astatine, and preferably fluorine, chlorine, bromine, or iodine can be used.

[0015] The sequence of repeating units A and B constituting the polymer chain represented by general formula (10) or general formula (1) can be random or blocky, for example, by polymerization in the presence of the first monomer and the second monomer, or by polymerizing the first monomer and the second monomer separately to form blocks, and then reacting the products thereof.

[0016] The number of repeating units A (n) and B (m) contained in a single polymer chain can be appropriately determined depending on the target latex particles. For example, the number and weight of each can be determined according to the target particle size and polymerization conditions (including the type of monomer and its function / effect). Furthermore, the composition ratio can be determined by adjusting the polymerization conditions, for example, the ratio of the first monomer to the second monomer used.

[0017] Specifically, for example, the total number of repeating units A (n) and B (m) contained in one polymer chain can be 1 or more, 4 or more, 8 or more, 30 or more, or 50 or more, and can also be 50,000 or less, 40,000 or less, 30,000 or less, or 25,000 or less. Also, for example, the number of repeating units A (n) contained in one polymer chain can be 1 or more, 2 or more, 4 or more, 8 or more, 20 or more, 100 or more, 500 or more, or 2,500 or more, and can also be 50,000 or less, 40,000 or less, 30,000 or less, 25,000 or less, 20,000 or less, or 15,000 or less. Furthermore, for example, the number of repeating units B (m) contained in a single polymer chain can be 0 or more, 1 or more, 2 or more, 4 or more, 8 or more, 20 or more, 100 or more, 500 or more, or 2500 or more, and can also be 50,000 or less, 40,000 or less, 30,000 or less, 25,000 or less, 20,000 or less, or 15,000 or less. Latex particles can be manufactured by combining polymer chains configured as described above as appropriate.

[0018] Furthermore, the total mole percentage of the first monomer and the second monomer is [{(n+m) / (n+m)}×100] = 100 (%). As a specific example of the composition ratio, the mole percentage per monomer unit of the first monomer [{n / (n+m)}×100] when the sum of the first monomer and the second monomer in the latex particles is considered the total monomer is 2% or more, preferably 4% or more, more preferably 10% or more, even more preferably 15% or more, and also 100% or less, preferably 90% or less, more preferably 80% or less, even more preferably 60% or less, and particularly preferably 50% or less. Here, the total mole percentage and mole percentage are calculated as the sum of each target monomer in the latex particles.

[0019] The latex particles of the present invention can be manufactured as whole particles by the polymerization method used (for example, the first polymerization method (one-step method), the second polymerization method (two-step method), and the third polymerization method described below), or they can be particles having a core-shell structure consisting of a core portion inside the particle and a shell portion outside it (hereinafter sometimes referred to as core-shell particles). In the aforementioned core-shell structure, the shell portion may be a single layer or two or more layers. Particles manufactured as whole particles can be manufactured, for example, by the first polymerization method described above, and particles having a core-shell structure can be manufactured, for example, by the second polymerization method or the third polymerization method described above.

[0020] The latex particles of the present invention can be produced, for example, by a polymerization reaction using a first monomer, a second monomer different from the first monomer, a surfactant (emulsifier), and a polymerization initiator.

[0021] The first monomer usable in this invention is of the following general formula (X): [In the formula, R2 to R6 and R8 to R10 are each independently (i) a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (ii) a monovalent functional group in which one or more carbon atoms are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom.] (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, and at least one of R2 to R6 is a halogen atom, and R7 is a single bond, ester bond, thioether bond, amide bond, or ether bond.

[0022] Examples of the first monomer include 2,4,6-triiodophenyl acrylate, 4,4'-diiodo-trans-stilbene, 2,4,6-tribromophenyl acrylate, 2-fluorostyrene, 3-fluorostyrene, 4-fluorostyrene, 4-fluoro-α-methylstyrene, 4-fluorocinnamaldehyde, 4-fluorocinnamic acid, 1-fluoro-4-(2-nitroethenyl)benzene, and trans-3 ,4-difluorocinnamic acid, 4-ethyl fluorocinnamate, 4-fluorochalcone, 4-fluoro-4'-methyl chalcone, trans,trans-1,5-bis(4-fluorophenyl)-1,4-pentadien-3-one, 2,3,4,5,6-pentafluorostyrene, 2-bromostyrene, 3-bromostyrene, 4-bromostyrene, trans-4-bromosinnamaldehyde, 4-bromo-β,β-difluoro Rosstyrene, 4-bromocinnamic acid, (E)-3-(4-bromophenyl) ethyl acrylate, trans-4-bromostilbene, 3-(4-bromophenyl)-1-phenylpropane-2-en-1-one, 4,4'-dibromo-trans-stilbene, 1-bromo-4-(1,2,2-triphenylethenyl)benzene, 4-bromo-4'-[di(p-tolyl)amino]stilbene, tetrakis(4-bromophenyl) Examples include ethylene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 4-chlorocinnamic acid, methyl 4-chlorocinnamate, 1-chloro-4-(2-nitrovinyl)benzene, ethyl (E)-3-(4-chlorophenyl)acrylate, trans-2,4-dichlorocinnamic acid, 4-chloro-3-nitrocinnamic acid, 4,4'-dichloro-trans-stilbene, and 4,4'-dichlorochalcone. Furthermore, two or more of these monomers can be used in mixture form.

[0023] A second monomer usable in this invention is of the following general formula (Y): [In the formula, R11 to R14 are each independently (i) a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (ii) a monovalent functional group in which one or more carbon atoms are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom.] (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, and none of R11 to R14 are phenyl groups substituted with halogen atoms.

[0024] Examples of second monomers include styrene, styrene derivatives (e.g., chloromethylstyrene, sodium styrenesulfonate), divinylbenzene, acrylic acid or methacrylic acid, itaconic acid, maleic anhydride, maleic acid, phthalic acid, acrylic acid esters or methacrylic acid esters [e.g., methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, hexadecyl (meth)acrylate], vinyl acetate, dimethacrylate, diacrylate, and butadiene. Furthermore, two or more of these monomers can be used in combination.

[0025] The latex particles of the present invention can be synthesized, for example, by emulsion polymerization, soap-free emulsion polymerization, miniemulsion polymerization, dispersion polymerization, and suspension polymerization.

[0026] Emulsion polymerization is a polymerization method that uses a medium such as water, monomers that are poorly soluble in the medium, an emulsifier, and a polymerization initiator. Monomers are dispersed in water and form micelles stabilized by the emulsifier. A polymerization initiator added to the aqueous system polymerizes the monomers in the water one after another. As polymerization progresses, the monomers in the micelles are consumed and latex particles are formed.

[0027] Miniemulsion polymerization is a polymerization method in which monomer droplets are miniaturized to submicron size by applying strong mechanical energy, such as using an ultrasonic oscillator, to an aqueous medium in which monomers and surfactants are dissolved, and then the polymerization reaction proceeds within the oil droplets using an oil-soluble initiator.

[0028] Surfactants usable in the present invention can be appropriately selected from known methods, but examples include sodium dodecylbenzenesulfonate, linear alkylbenzenesulfonic acid or its sodium salt, sodium lauryl sulfate, polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (80) sorbitan monooleate (Tween 80), octylphenol ethoxylate (Triton X-100), polyvinyl alcohol, lauryl alcohol ethoxylate, alkyl polyglycoside, polyoxyethylene alkyl ether, cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium chloride, sorbitan monolaurate, sodium stearate, polyoxypropylene polyoxyethylene block copolymer (Pluronic), calcium stearate, amine stearate, and sodium lauryl ether sulfate.

[0029] Polymerization initiators usable in the present invention include known radical polymerization initiators usable in emulsion polymerization and miniemulsion polymerization, such as peroxide initiators, persulfate initiators, azo initiators, or redox initiators.

[0030] Examples of the peroxidation initiators include benzoyl peroxide (BPO), di-t-butyl peroxide (DBPO), and ammonium peroxide.

[0031] Examples of persulfate initiators include potassium persulfate (KPS), ammonium persulfate (APS), and sodium persulfate (NPS).

[0032] Examples of the azo initiators include azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobisisobutyrate (MAIB), 4,4'-azobis(4-cyanovaleric acid), and 2,2'-azobis(2,4-dimethylvaleronitrile). Among the azo initiators, examples of low-temperature azo initiators that can be implemented at low temperatures include the water-soluble azo polymerization initiator VA-044 (Wako Pure Chemical Industries) or the oil-soluble azo polymerization initiator V-70 (Wako Pure Chemical Industries).

[0033] Examples of the redox initiators include N,N,N',N'-tetramethylethylenediamine (TMEDA) / potassium persulfate (KPS), FeSO4. ４ / KPS, FeSO ４ / H ２ O ２ Ascorbic acid (vitamin C) / H ２ O ２ Examples include:

[0034] The method for producing the bioactive substance-supporting latex particles of the present invention is not particularly limited as long as it can produce the bioactive substance-supporting latex particles of the present invention as described above. It can be carried out in the same manner as conventionally known emulsion polymerization and miniemulsion polymerization (e.g., M. Antonietti, K. Landfester, Prog. Polym. Sci., 2002, 27, 689-757, JM Asua, Prog. Polym. Sci., 2002, 27, 1283-1346), except that the first monomer usable in the present invention is used during the polymerization reaction.

[0035] The reaction conditions during the polymerization of latex particles, such as the solvent, mixing ratio, temperature, and reaction time, can be appropriately determined, for example, by conducting pilot tests, depending on the monomer used, the type of functional group for supporting the physiologically active substance, the surfactant, the polymerization initiator, the average particle size of the synthesized latex particles, and the amount of physiologically active substance supported on the surface of the latex particles.

[0036] As a first polymerization method for latex particles for supporting physiologically active substances that can be used in the present invention, for example, a method of reacting a first monomer, a second monomer, a surfactant, and a polymerization initiator in one step can be mentioned (one-step method). Thereby, latex particles containing a polymer (polymer chain) of the first monomer and the second monomer as a main component can be obtained. In the present specification, the phrase "latex particles containing a target polymer as a main component" means latex particles containing a small amount of a by-product polymer other than the target polymer. Examples of the by-product polymer include polymers in which the target polymerization reaction was not completed, and polymers that did not reach the target polymer due to deviating from the desired polymerization reaction. In addition, the term "small amount" means an amount that does not affect the various effects of the latex particles of the present invention. A person skilled in the art can easily obtain latex particles having the structure within the scope of design.

[0037] The physiologically active substance can be immobilized on the latex particles, for example, by physical adsorption. In the present invention, by irreversibly immobilizing the physiologically active substance via a functional group for supporting a physiologically active substance that can be introduced into the latex particles as desired, it becomes possible to stably produce an analytical reagent for latex agglutination that has high reactivity with an analyte. Examples of the functional group for supporting a physiologically active substance include a carboxyl group, an amino group, an epoxy group, an aldehyde group, a sulfo group, an imide group, a cyano group, a urethane group, an allyl group, a nitro group, a maleimide group, another amino group, a mercapto group, a hydroxyl group, a glycidyl group, and a sulfhydryl group. As can be understood from the fact that some of the functional groups for supporting a physiologically active substance exemplified herein overlap with the monovalent functional groups listed as R2 to R6 and R11 to R14 in general formula (1), some of said monovalent functional groups may also function as functional groups for supporting a physiologically active substance. When using the first polymerization method, it is preferable that the functional group for supporting a physiologically active substance is included in the first monomer and / or the second monomer. It is also preferable that the content of the first monomer and / or the functional group for supporting a physiologically active substance is higher closer to the surface layer of the particle.

[0038] When the first polymerization method (one-step method) described above is used to produce the latex particles of the present invention, the content of the first monomer in the total monomer (sum of the first monomer and the second monomer) used as the starting material is not particularly limited, but the polymerization can be carried out in the range of 0.01 to 99.5% by weight, preferably 0.5 to 50% by weight, more preferably 1.0 to 40% by weight, and even more preferably 5 to 30% by weight, based on the total monomer weight. The content of the second monomer in the total monomer used as the starting material is not particularly limited, but the polymerization can be carried out in the range of 0.5 to 99.9% by weight, preferably 50 to 99.5% by weight, more preferably 60 to 99% by weight, and even more preferably 70 to 95% by weight, based on the total monomer weight in the latex particles.

[0039] Furthermore, when using the first polymerization method (one-step method), if only the first monomer is used and the second monomer is not used as the monomer, latex particles with m=0, that is, latex particles that do not contain repeating units B, can be obtained. On the other hand, by appropriately selecting the type of monomer and polymerization conditions, the content of the first monomer on the surface of the latex particles can be increased, and the content of the first monomer on the inside can be decreased. For example, the content of the first monomer on the surface of the manufactured latex particles is not particularly limited, but it can be said to be 2% or more, preferably 4% or more, more preferably 10% or more, even more preferably 15% or more, and 90% or less, preferably 80% or less, even more preferably 60% or less, and even more preferably 50% or less, as the molar percentage [{n / (n+m)} × 100] of repeating units A in the polymer chain represented by general formula (10) or general formula (1).

[0040] As a second polymerization method for latex particles for supporting physiologically active substances that can be used in the present invention, for example, there may be mentioned a method (two-step method) in which a second monomer, a surfactant and a polymerization initiator are reacted (first step), and then the first monomer, the second monomer, a surfactant and a polymerization initiator are reacted in the presence of the polymer (polymer chain) obtained in the first step (second step). This makes it possible to obtain latex particles having a core-shell structure composed of a core portion mainly containing a polymer of the second monomer, and a shell portion mainly containing a copolymer of the first monomer and the second monomer that coats the core portion. A person skilled in the art can easily obtain the latex particles having the core-shell structure within the scope of design.

[0041] In the present specification, the core portion refers to the central structure of the latex particle, and the shell portion is a structure that coats the core portion, and particularly refers to the surface layer structure of the latex particle. The core portion and the shell portion may be continuous or discontinuous. Analysis of latex particles and the polymers contained therein can be appropriately performed by a person skilled in the art by appropriately selecting known methods. For example, when Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) is performed on a cross section passing through the center of a latex particle, the element distribution from the outside to the center of the particle can be analyzed. Alternatively, by gradually melting the particles, the polymer of the shell portion and the polymer of the core portion can be fractionated, and when gas chromatography / mass spectrometry is performed on each fraction, the type and content of the monomers can be analyzed. Furthermore, when X-ray Photoelectron Spectroscopy (XPS) is performed on the surface of the particles or the like, the content of elements in the shell portion can be analyzed.

[0042] While physiologically active substances can be immobilized on latex particles by means of physical adsorption, for example, in this invention, they are immobilized covalently via a physiologically active substance-supporting functional group, making it possible to produce analytical reagents for latex agglutination that are stable and highly reactive with the analyte. When using the second polymerization method, it is preferable that the physiologically active substance-supporting functional group is contained in at least the first monomer and / or the second monomer used in the second step. This makes it easier to control the second monomer and the physiologically active substance-supporting functional group present on the surface, and makes it possible to minimize the total content of the second monomer in the latex particles while increasing the surface content of the second monomer. As a result, for example, it is possible to suppress the specific gravity of the latex particles while achieving high reactivity between antibodies and antigens, making it possible to produce analytical reagents for latex agglutination that are highly reactive with the analyte.

[0043] When the second polymerization method (two-step method) described above is used to produce latex particles having a core-shell structure, the content of the first monomer (hereinafter sometimes referred to as halogenated monomer) in the second step is not particularly limited, but the polymerization can be carried out in the range of 10 to 99.5% by weight, preferably 20 to 80% by weight, more preferably 30 to 70% by weight, and even more preferably 40 to 60% by weight, based on the total weight of monomers (sum of the first monomer and the second monomer) used as starting materials in the second step. The lower and upper limits of each numerical range can be arbitrarily combined.

[0044] In latex particles having a core-shell structure, configuring the ratio of halogenated monomer in the shell portion to fall within the aforementioned numerical range provides the following advantages compared to, for example, a particle with 100% by weight of halogenated monomer, resulting in highly accurate measurements. Firstly, it is possible to produce latex particles with a relatively low specific gravity, thus ensuring high dispersibility. Therefore, the latex particles do not settle even after prolonged standing, resulting in high measurement accuracy over a long period.

[0045] Secondly, by copolymerizing with styrene monomer, a variety of halogenated monomers can be introduced into the particles. While some, like bromostyrene, can be shelled as 100% halogenated monomer, it can be difficult to introduce common halogenated monomers into the latex particle shell on their own. By copolymerizing with styrene monomer, halogenated monomers can be introduced stably.

[0046] This makes it easier to immobilize physiologically active substances, especially antibodies, on the particle surface. Since polystyrene is excellent at immobilizing antibodies, copolymerizing it with styrene can impart both antibody immobilization and high sensitivity through halogenated monomers.

[0047] In the latex particles of the present invention, the inclusion of halogenated phenyl groups can improve analytical sensitivity and accuracy (particularly sensitivity in the low-concentration range). Therefore, in the latex particles of the present invention, latex particles having a core-shell structure are preferred, and it is more preferable that the shell portion contains a sufficient amount of halogenated phenyl groups. For example, when the core-shell type particles of the present invention, which is one of the preferred embodiments, are produced using the second polymerization method (two-step method) described above, the molar percentage [{n / (n+m)} × 100] of the repeating unit A in the polymer chain represented by general formula (10) or general formula (1) that constitutes the produced shell portion is not particularly limited, but is 2% or more, preferably 4% or more, more preferably 10% or more, even more preferably 15% or more, and also 90% or less, preferably 80% or less, more preferably 60% or less, and even more preferably 50% or less. The lower and upper limits of each numerical range can be arbitrarily combined.

[0048] A third polymerization method for latex particles carrying physiologically active substances that can be implemented in the present invention is a modification of the second polymerization method for latex particles. As long as the particle has the core-shell structure described above, the polymerization steps are not limited to two steps, but may be three or more steps, and may also contain other substructures or subcomponents.

[0049] Regardless of the polymerization method used, for example, using any of the first to third polymerization methods described above, the latex particles of the present invention allow for the analysis of the proportion of halogen elements in the particle surface layer, when carbon atoms, oxygen atoms, and halogen atoms are total atoms, by appropriately using TOF-SIMS, XPS, etc. The proportion is not particularly limited, but it can be at least 0.5 atm%, preferably 1 atm%, more preferably 3 atm%, even more preferably 4 atm%, and at least 50 atm%, preferably 40 atm%, more preferably 35 atm%, and even more preferably 20 atm%.

[0050] The size of the latex particles for carrying physiologically active substances in the present invention can be, for example, a particle diameter of 15 to 3000 nm, preferably 30 to 2000 nm, more preferably 40 to 1000 nm, even more preferably 50 to 800 nm, and particularly preferably 60 to 500 nm. Those skilled in the art can appropriately select, design, and manufacture these particles according to known methods.

[0051] Apart from using the latex particles for supporting bioactive substances of the present invention, analytical reagents for latex agglutination can be manufactured according to known methods. The analytical reagent of the present invention is a reagent for analyzing an analyte, wherein a bioactive substance capable of reacting with the analyte in a biological sample is immobilized on bioactive substance-supporting latex particles, preferably via a bioactive substance-supporting functional group. The method for supporting the bioactive substance on the bioactive substance-supporting latex particles can be appropriately selected from known methods, such as a method of physically supporting the bioactive substance on the bioactive substance-supporting latex particles, or a method of chemically supporting the bioactive substance on the bioactive substance-supporting functional group of the bioactive substance-supporting latex particles.

[0052] Furthermore, the combination of physiologically active substances and analyte substances can be appropriately selected and used from known methods.

[0053] Examples of physiologically active substances usable in the present invention include substances that can react with the analyte in a biological sample, such as antigens, antibodies, enzymes, receptors, DNA, RNA, and glycans.

[0054] The analytes in biological samples usable in this invention include IgG, C-reactive protein (CRP), ferritin, β-2 microglobulin, α-fetoprotein (AFP), IgE, hepatitis B virus (HBS antibody or HBc antibody), D-dimer, fibrin / fibrinogen degradation products (FDP), soluble fibrin (SF), plasmin / α2-plasmin inhibitor complex (PPI), prostate-specific antigen (PSA), elastase 1, elastase XDP, thrombomodulin (TM), albumin (preferably serum albumin), thrombin / antithrombin complex (TAT), presepsin, myoglobin, CEA, AFP, ferritin, β2M, PIVKA2, PSA, PAP, CA19-9, CA125, CA15-3, CA72-4, ST-439, SCC antigen, γ-SM, NSE, BCA225, pepsinogen (I, II), proGRP, CYFRA, HER2 / neu, thyroid-stimulating hormone (TSH), thyroid hormones (T3, T4, FT3, FT4), T3UPTAKE, cortisol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), growth hormone (HGH), ACTH, insulin, C peptide, DHEA-S, estradiol (E2), estriol (E3), te Progesterone, progesterone, human chorionic gonadotropin (HCG, HCGpreg, β-HCG), PTH, intact PTH, whole PTH, hANP, osteocalcin, calcitonin, procalcitonin, thyroglobulin, human brain natriuretic peptide (BNP), human brain natriuretic peptide precursor N-terminal fragment (NTproBNP), PINP, urinary deoxypyridinoline, BAP, β-crossplus (urine), HCV antibody, HCV IgE antibody, HBs antigen, HBs antibody, HBe antigen / antibody, HBc antibody, IgM-type HBc antibody, hepatitis B virus core-associated antigen, HAV antibody, IgM-type HAV antibody, IgG-type HAV antibody, HIV antigen / antibody, HIV antibody 1 / 2, HIV p24 antigen, HTLV-1 antibody, IgG-type cytomegalovirus antibody, syphilis TP antibody, anti-CCP antibody, total IgE, specific IgE, antinuclear antibody, anti-DNA antibody, anti-ssDNA antibody, anti-ds antibody, anti-TSH receptor antibody, anti-thyroid peroxidase antibody,Examples of antibodies that can be used include anti-thyroglobulin antibodies, anti-mitochondrial antibodies, anti-SSA / Ro antibodies, anti-SSB / La antibodies, anti-Sm antibodies, anti-RNP antibodies, anti-galactose-deficient IgG antibodies, P-ANCA, MPO-ANCA, IgG, IgA, IgM, IgD, myoglobin, C3 / C4, troponin T, troponin I, α1-M, KL-6, eosinophil basic protein (ECP), phenytoin, phenobarbital, carbamazepine, valproic acid, tobramycin, gentamicin, vancomycin, theophylline, digoxin, cyclosporine, tacrolimus, folic acid, CK-MB, VB12, sIL-2R, EPO, etc.

[0055] For example, when using an antibody as the physiologically active substance, monoclonal antibodies or polyclonal antibodies can be used. Furthermore, in addition to the immunoglobulin molecule itself, antibody fragments such as Fab, Fab', and F(ab') can also be used. ２ Alternatively, Fv, or peptide aptamers can also be used.

[0056] For example, when using DNA as the physiologically active substance, a DNA probe of approximately 5 to 100 base pairs complementary to the substance to be analyzed can be used.

[0057] The analyzable test samples usable in embodiments of the present invention are not particularly limited, as long as they are samples that may contain the analyte, and particularly include biological samples such as blood, serum, plasma, urine, cerebrospinal fluid, or cell or tissue lysates.

[0058] The analytical reagent of the present invention can be used in known latex agglutination methods. In latex agglutination methods, the amount of the analyte in a sample can be analyzed (particularly measured) by optically analyzing (particularly measuring) the degree of agglutination that occurs when the analytical reagent and the sample are brought into contact in a liquid. In a specific method for optically detecting the degree of agglutination of latex particles, for example, the measurement can be performed using an optical instrument that measures scattered light intensity, absorbance, or transmitted light intensity. The preferred measurement wavelength is 300 to 800 nm. The measurement method can be performed by measuring the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity by selecting the size (average particle size) or concentration of the latex particles used, or by setting the reaction time, according to known methods. It is also possible to use these methods in combination.

[0059] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0060] 《Example 1: Synthesis of Latex Particles》 50 g of deionized water, 50 g of styrene, and 0.5 g of 0.025% by mass aqueous solution of linear alkylbenzene sulfonate were added to a reaction vessel and dispersed in a homogenizer to obtain an emulsion. Then, 450 g of deionized water was added and the mixture was heated while stirring at 200 rpm. When the temperature reached 80°C, 10 g of 0.05% by mass aqueous solution of potassium peroxodisulfate was added dropwise, and the polymerization reaction was carried out for 4 hours. (End of first step)

[0061] Next, 10 g of 2,4,6-triiodophenyl acrylate was dissolved in 10 g of styrene. To this solution, 0.56 g of methacrylic acid, 0.25 g of 0.025% by mass aqueous solution of linear alkylbenzene sulfonate sodium, and 50 g of ion-exchanged water were added and dispersed in a homogenizer to obtain an emulsion. This emulsion was added to the above reaction vessel and the reaction was stopped after 3 hours. (End of second step) After the reaction, the dispersion was neutralized and washed with aqueous potassium bicarbonate to obtain an aqueous dispersion of latex particles containing iodine on the surface (hereinafter referred to as iodratex particles).

[0062] Examples 2-5: Latex particles were produced in the same manner as in Example 1, except that the monomers shown in Table 1 were used instead of 2,4,6-triiodophenyl acrylate.

[0063] Comparative Examples 1-2: Latex particles were produced in the same manner as in Example 1, except that the monomers shown in Table 1 were used instead of 2,4,6-triiodophenyl acrylate.

[0064] 《Example 6: Particle Size Measurement and Surface Elemental Analysis of Latex Particles》 Dried latex particles from Examples 1-5 and Comparative Examples 1-2 were observed using a scanning electron microscope (JEOL Ltd., JCM-7000 NeoScope) and the average particle size was measured (N=20). The results are shown in Table 1. Figure 1 shows an image of the iodratex particles produced in Example 1 observed using a field emission scanning electron microscope (Hitachi High-Tech Corporation, Regulus 8220). Furthermore, the surface of the iodratex particles was analyzed using an energy-dispersive X-ray analyzer (Oxford Instruments, ULTIM MAX), and it was found that they contained iodine atoms at an atomic concentration of 0.9%.

[0065]

[0066] Example 7: Binding of Anti-D-Dimer Antibody to the Surface of Latex Particles Using reaction buffer (2 mg / L MES pH 6.2), 1 mL of the latex particle dispersion (solid content 4% by mass) obtained in Example 1, 36.6 μL of 20 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution, and 800 μL of 2.5 mg / mL anti-D-dimer antibody were mixed and stirred at room temperature for 60 minutes. Then, 400 μL of 20% Lysine solution was added, and 440 μL was taken, and 360 μL of 2.4% dn-BSA solution was added and stirred at room temperature for 30 minutes. After the reaction, the solution was centrifuged and washed with 0.05% sodium azide aqueous solution, and then redispersed to obtain a dispersion of iodratex particles in which the anti-D-dimer antibody (Patent No. 5860394) was chemically bound to the carboxyl groups on the surface of the latex particles. In Example 3 and Comparative Examples 1 and 2, the latex particle dispersions obtained were also prepared in the same manner as described above, to obtain dispersions of latex particles to which anti-D-dimer antibodies were chemically bound.

[0067] Example 8: Preparation of D-dimer measurement reagent. A 2 mg / L MOPS pH 7.2 solution was used as reagent R1. For reagent R2, a solution was prepared by mixing 1.6 mL of the latex particle dispersion on which the anti-D-dimer antibody was immobilized in Example 7 with 8.4 mL of the 2 mg / L MOPS pH 7.2 solution.

[0068] 《Example 9: D-dimer Reactivity Evaluation》 The D-dimer measurement reagent prepared in Example 8 was measured using an automated analyzer (PHC Corporation, STACIA fully automated clinical laboratory system) with D-dimer antigen (PHC Corporation, D-D-dimer calibrator, ST-756XS) as the measurement sample and the measurement protocol for Elpia Ace D-D-dimer II reagent (PHC Corporation, RM75-751LK). The measurement samples were prepared at six concentrations: 0 μg / mL, 2 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 46 μg / mL. The measurement results are shown in Figure 2. The latex particles containing halogenated phenyl polymer (Example) all showed high reactivity. Furthermore, the iodratex particles (Example 1) showed remarkably high reactivity compared to latex particles containing an allyl iodide polymer (Comparative Example 2), being approximately 35 times more reactive at a low concentration of 2 μg / mL and approximately 15 times more reactive at a high concentration of 46 μg / mL. This demonstrated that the presence of a phenyl group in addition to a halogen increases reactivity.

[0069] 《Example 10: D-Dimer Sensitivity Evaluation》 To evaluate the sensitivity of the measurement sample to low concentration ranges, the measurement sample, prepared by diluting D-dimer antigen (D-D-dimer calibrator, ST-756XS, manufactured by PHC Corporation) with common diluent V (manufactured by PHC Corporation), was measured using an automated analyzer (STACIA, a fully automated clinical laboratory system, manufactured by PHC Corporation) in the same manner as in Example 9. The measurement sample was prepared at five concentrations: 0 μg / mL, 0.06 μg / mL, 0.12 μg / mL, 0.23 μg / mL, and 0.47 μg / mL. The measurement results are shown in Figure 3 (Example) and Figure 4 (Comparative Example). In all cases, the latex particles in the Example showed a concentration-dependent increase in reactivity, whereas in the Comparative Example, no increase in reactivity was observed in the latex particles. It was shown that latex particles containing halogenated phenyl polymers have high sensitivity and can measure accurately down to low concentration ranges.

[0070] Example 11: Binding of anti-TAT antibody to latex particle surface. For Examples 2-5 and Comparative Examples 1-2, antibodies were bound to the latex particle surface in the same manner as in Example 7, except that anti-antithrombin antibody and anti-thrombin antibody (Patent No. 6594641) were used.

[0071] 《Example 12: Preparation of TAT Measurement Reagent》 The R1 reagent of the LPIA Genesis TAT ​​reagent (manufactured by PHC Corporation, RM75-671LK) was used as the R1 reagent. For the R2 reagent, 0.15 mL each of the latex particle dispersion containing the anti-antithrombin antibody and anti-thrombin antibody immobilized in Example 11 was added, along with 0.05% NaN ３ A solution prepared by mixing 1.6 mL of aqueous solution was used.

[0072] 《Example 13: TAT Reactivity Evaluation》 The TAT measurement reagent prepared in Example 12 was measured using an automated analyzer (PHC Corporation, STACIA fully automated clinical laboratory system) with TAT antigen (PHC Corporation, LPIA Genesis TAT ​​Calibrator, ST671XS) as the measurement sample and the measurement protocol for LPIA Genesis TAT ​​reagent (PHC Corporation, RM75-671LK). The measurement samples were prepared at four concentrations: 0 ng / mL, 23 ng / mL, 46 ng / mL, and 103 ng / mL. The measurement results are shown in Figure 5. The latex particles containing halogenated phenyl polymer (Example) all showed high reactivity. Furthermore, the latex particles of the Example showed remarkably high reactivity, more than 20 times higher at the low concentration range of 23 ng / mL compared to the latex particles of the Comparative Example, demonstrating that reactivity is high regardless of the target of measurement or the type of bound antibody. Furthermore, the 103 ng / mL concentration in the example is thought to indicate an excess reaction.

[0073] 《Example 14: TAT Sensitivity Evaluation》 To evaluate the sensitivity of the measurement sample to low concentration ranges, the measurement sample, prepared by diluting TAT antigen (LPIA Genesis TAT ​​Calibrator, ST671XS, manufactured by PHC Corporation) with common diluent V (manufactured by PHC Corporation) in the same manner as in Example 13, was measured using an automated analyzer (STACIA, a fully automated clinical laboratory system, manufactured by PHC Corporation). The measurement sample was prepared at five concentrations: 0 ng / mL, 1.3 ng / mL, 2.6 ng / mL, 5.2 ng / mL, and 10.3 ng / mL. The measurement results are shown in Figure 6 (Example) and Figure 7 (Comparative Example). In all cases, the latex particles in the Example showed a concentration-dependent increase in reactivity, whereas in the Comparative Example, no increase in reactivity was observed in the latex particles. It was shown that latex particles containing halogenated phenyl polymers have high sensitivity and can accurately measure down to low concentration ranges, regardless of the target substance or the type of bound antibody.

[0074] The novel latex particles for carrying physiologically active substances according to the present invention can be used in the manufacture of analytical reagents for clinical diagnostic tests.

Claims

1. General formula (10): R15-Bm-An-R1 (10) or general formula (1): In general formula (10) and general formula (1), R2 to R6 and R8 to R14 are each independently: (i) a monovalent functional group selected from the group consisting of hydrogen atoms, halogen atoms, methyl groups, ethyl groups, propyl groups, cyclopropyl groups, hydroxyl groups, amino groups, methoxy groups, thiol groups, cyano groups, vinyl groups, isocyanate groups, aldehyde groups, carboxyl groups, nitro groups, allyl groups, aryl groups, boronic acids, isocyanide groups, ketone groups, phenyl groups, sulfo groups, epoxy groups, glycidyl groups, and pyridine groups; or (ii) a monovalent functional group in which one or more carbon atoms are substituted with oxygen atoms, sulfur atoms, or nitrogen atoms. (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, at least one of R2 to R6 is a halogen atom, provided that none of R11 to R14 are phenyl groups substituted with halogen atoms, R7 is a single bond, ester bond, thioether bond, amide bond, or ether bond, and R1 and R15 are the functional groups specified in (i) to (iv) above, or functional groups derived from polymerization initiators. The order of the repeating units A and B can be random or in blocks.Latex particles for carrying physiologically active substances, containing polymer chains represented by [m and n represent the total number of repeating units B and A contained in one polymer chain, where m is an integer greater than or equal to 0 and n is an integer greater than or equal to 1].

2. The latex particle for carrying a physiologically active substance according to claim 1, wherein in general formula (1), m is an integer of 1 or more.

3. The latex particle for carrying a physiologically active substance according to claim 1, wherein the latex particle for carrying a physiologically active substance comprises a core portion located inside the particle and a shell portion located around it.

4. The latex particle for carrying a physiologically active substance according to claim 3, wherein the mole percentage [{n / (n+m)} × 100] of repeating units A in the polymer chain represented by general formula (10) or (1) constituting the shell portion is greater than the mole percentage [{n / (n+m)} × 100] of repeating units A in the polymer chain represented by general formula (10) or (1) constituting the core portion.

5. The latex particle for carrying a physiologically active substance according to claim 3, wherein the core portion mainly consists of a polymer chain represented by general formula (10) or (1) where m is 1 or more and n is 0 or more, and the shell portion mainly consists of a polymer chain represented by general formula (10) or (1) where m is 0 or more and n is 1 or more.

6. The latex particle for carrying a physiologically active substance according to claim 5, wherein the core portion mainly consists of a polymer chain represented by general formula (10) or (1) where m is 1 or more and n is 0, and the shell portion mainly consists of a polymer chain represented by general formula (10) or (1) where m is 1 or more and n is 1 or more.

7. Latex particles for carrying physiologically active substances according to any one of claims 1 to 6, wherein the halogen atom is an atom selected from the group consisting of fluorine, chlorine, bromine, and iodine.

8. A latex particle for carrying a physiologically active substance according to any one of claims 1 to 7, obtained by polymerization using a first monomer, a second monomer, a polymerization initiator, and a surfactant, wherein the first monomer is of the following general formula (X): [In the formula, R2 to R6 and R8 to R10 are each independently (i) a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (ii) a monovalent functional group in which one or more carbon atoms are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom.] (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, wherein at least one of R2 to R6 is a halogen atom, and R7 is a single bond, ester bond, thioether bond, amide bond, or ether bond, and the second monomer is represented by the following general formula (Y): [In the formula, R11 to R14 are each independently (i) a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (ii) a monovalent functional group in which one or more carbon atoms are substituted with an oxygen atom, a sulfur atom, or a nitrogen atom.] (iii) The monovalent functional group (i) described above is a monovalent functional group in which the terminal atom is substituted with a monovalent functional group selected from the group consisting of a hydrogen atom, a halogen atom, a methyl group, an ethyl group, a propyl group, a cyclopropyl group, a hydroxyl group, an amino group, a methoxy group, a thiol group, a cyano group, a vinyl group, an isocyanate group, an aldehyde group, a carboxyl group, a nitro group, an allyl group, an aryl group, a boronic acid, an isocyanide group, a ketone group, a phenyl group, a sulfo group, an epoxy group, a glycidyl group, and a pyridine group, or (iv) The monovalent functional group (i) is a monovalent functional group in which the terminal atom is substituted via a carbon atom, oxygen atom, sulfur atom, or nitrogen atom with a monovalent functional group selected from the group consisting of a hydrogen atom, halogen atom, methyl group, ethyl group, propyl group, cyclopropyl group, hydroxyl group, amino group, methoxy group, thiol group, cyano group, vinyl group, isocyanate group, aldehyde group, carboxyl group, nitro group, allyl group, aryl group, boronic acid, isocyanide group, ketone group, phenyl group, sulfo group, epoxy group, glycidyl group, and pyridine group, and none of R11 to R14 are phenyl groups substituted with halogen atoms.

9. The latex particle for carrying a physiologically active substance according to claim 8, wherein the first monomer and / or the second monomer further contains a functional group for carrying a physiologically active substance.

10. A method for producing latex particles for carrying physiologically active substances according to any one of claims 1 to 8, comprising polymerization using the first monomer and the second monomer described in claim 8.

11. The manufacturing method according to claim 10, wherein the first monomer and / or the second monomer further contains a functional group for supporting a physiologically active substance.

12. An analytical reagent for latex agglutination, comprising latex particles for carrying physiologically active substances as described in any one of claims 1 to 9.

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