Latex particle dispersion, reagent kit, detection method, precipitation suppressing agent, and method for suppressing precipitation

Abstract

The present invention relates to a latex particle dispersion containing latex particles and an aqueous medium, the latex particle dispersion being characterized by further containing at least one type of compound selected from the group consisting of hexametaphosphoric acid and salts thereof.

Description

Latex particle dispersion, reagent kit, detection method, sedimentation inhibitor, and sedimentation inhibitor method

[0001] The present invention relates to a latex particle dispersion, a reagent kit, a detection method, a sedimentation inhibitor, and a sedimentation inhibition method. This application claims priority to Japanese Patent Application No. 2023-165930, filed on September 27, 2023, the contents of which are incorporated herein by reference.

[0002] Reagents or reagent kits (hereinafter also referred to as "detection reagents, etc.") for detecting target substances using latex tubidimetric immunoassay (LTIA; also referred to as "latex immunoagglutination") can be installed in general-purpose automatic analyzers and are excellent in speed, accuracy, and cost-effectiveness. Furthermore, the detection reagents, etc. have made a dramatic contribution to clinical testing due to improved detection sensitivity.

[0003] Detection reagents and the like generally consist of two types of reagents: a first reagent (buffer solution) and a second reagent (latex particle dispersion). The first reagent serves to uniformly dilute the specimen (hereinafter also referred to as "sample") to be analyzed, ensuring a uniform and unbiased agglutination reaction by latex particles. The second reagent contains, as its main reaction component, monoclonal antibody-sensitized latex particles, which combine a monoclonal antibody that exhibits high specificity for a target substance such as an antigen with latex particles that maintain the properties of the sensitizing antibody. The second reagent further contains an aqueous medium for dispersing the latex particles, a preservative, and the like. The second reagent is also referred to as a latex reagent.

[0004] Applying detection reagents to an automated analyzer enables simple and rapid measurement of a large number of samples. The detection reagents are contained in containers and stored in the reagent storage of the automated analyzer. From the viewpoint of usability, it is desirable for the reagent composition to remain uniform over long periods of time when the containers are left stationary in the reagent storage, i.e., for the reagents to have good on-board stability.

[0005] However, when the particle size of the latex particles contained in the detection reagent used in the LTIA method is increased to increase sensitivity, the latex particles may settle over time after filling the latex reagent into a container, resulting in an inhomogeneous latex reagent. Measuring a sample using such an inhomogeneous latex reagent may result in an inaccurate measurement. Therefore, to perform an accurate measurement using a reagent containing latex particles, it is necessary to redisperse the latex particles in the aqueous medium by inverting the latex reagent container or stirring the latex reagent before measurement.

[0006] Patent Document 1 discloses that its object is to "provide a method for inhibiting the sedimentation of reactive substance-bound microparticles in a dispersion of the microparticles," and that this object is solved by "a method for inhibiting the sedimentation of microparticles to which a reactive substance is bound, the method comprising the step of causing at least one selected from the group consisting of polyanions and salts thereof, dextran, cyclodextrin, polyethylene glycol, and glycerol to coexist in a dispersion of the microparticles."

[0007] Patent Document 2 discloses that its objective is to "improve particle sedimentation properties while maintaining high sensitivity in an immunological latex turbidimetric reagent," and that this objective is achieved by "an immunological latex turbidimetric reagent comprising a suspension of latex particles carrying an antigen or antibody, the reagent comprising 7.5 to 15 wt / vol % of saccharides."

[0008] Patent Document 3 discloses an improved method for regulating an assay involving the interaction of two binding partners: "A method for determining an analyte in a sample in an interaction assay, the method comprising contacting the sample with an interaction modulator, wherein the interaction modulator is an enhancer of the interaction assay at low analyte concentrations and a retarder of the interaction assay at high analyte concentrations, and the interaction modulator is a copolymer of 4-styrenesulfonic acid and maleic acid (poly-(4-styrenesulfonic acid-co-maleic acid, PSSM) and / or polyacrylic acid (PAA), and the interaction assay is a homogeneous agglutination assay."

[0009] JP 2007-114129, JP 11-051938, Special publication 2020-507752

[0010] In the method for inhibiting sedimentation of fine particles disclosed in Patent Document 1, the sedimentation inhibition effect was confirmed only for gold colloid particles of 5 to 100 nm, and the method does not suggest anything about inhibiting sedimentation of latex particles in the latex reagent used in the LTIA method.

[0011] The method for suppressing the sedimentation of microparticles disclosed in Patent Document 2 is a technology that aims to improve dispersibility by increasing the specific gravity of the suspension by adding sugars to the particle suspension, such as monosaccharides such as glucose and fructose, disaccharides such as sucrose and lactose, or oligosaccharides such as maltotriose. However, the present inventors have found that adding these sugars to the latex reagent used in the LTIA method may reduce the sensitivity of the detection reagent (Test Example 3).

[0012] In the method described in Patent Document 3, an enhancer such as polyacrylic acid is not added to the solution containing latex particles, and therefore, the enhancer such as polyacrylic acid does not improve the on-board stability of the latex reagent.

[0013] Until now, there has been no technology that can suppress the sedimentation of latex particles in a latex particle dispersion used in the LTIA method without adversely affecting the performance of the detection reagent, such as reducing the sensitivity of the reagent.

[0014] An object of the present invention is to provide a latex particle dispersion, a reagent kit, a detection method, a sedimentation inhibitor, and a sedimentation inhibition method that have improved on-board stability.

[0015] The present inventors have conducted extensive research to solve the above problems and have found that in a detection reagent for detecting a target substance by latex immunoturbidimetry, which is composed of a buffer solution and a latex particle dispersion, the latex particle dispersion containing at least one compound consisting of hexametaphosphate and its salts suppresses the sedimentation of latex particles in the latex particle dispersion for a long period of time, and when applied to the detection of a target substance by latex immunoturbidimetry, the measurement sensitivity is at least maintained compared to a reagent not containing these components, thereby completing the present invention. The present invention includes the following aspects.

[0016] [1] A latex particle dispersion comprising latex particles and an aqueous medium, further comprising at least one compound selected from the group consisting of hexametaphosphoric acid and salts thereof. [2] The latex particle dispersion according to [1], which is a latex particle dispersion in a detection reagent used in detecting a target substance by latex immunoturbidimetry. [3] The latex particle dispersion according to [2], wherein the detection reagent comprises a buffer solution and a latex particle dispersion. [4] The latex particle dispersion according to any one of [1] to [3], wherein the salt is an alkali metal salt. [5] The latex particle dispersion according to any one of [1] to [4], wherein the aqueous medium comprises water. [6] The latex particle dispersion according to any one of [1] to [5], wherein the latex particles are polymer particles derived from a monomer component comprising a monomer having a styrene or vinylnaphthalene structure. [7] The latex particle dispersion according to any one of [1] to [6], wherein the latex particles are latex particles carrying a substance capable of binding to a target substance. [8] The latex particle dispersion according to [7], wherein the substance capable of binding to a target substance is an antigen or antibody against the target substance. [9] The latex particle dispersion according to any one of [1] to [8], wherein the average particle size of the latex particles is 50 to 1000 nm.

[10] The latex particle dispersion according to any one of [1] to [9], wherein the latex particles are contained in an amount of 0.001 to 0.20 mass% with respect to the total mass of the latex particle dispersion.

[11] A reagent kit used for detecting a target substance in a sample by latex immunoturbidimetry, comprising the latex particle dispersion according to any one of [1] to

[10] .

[12] A method for detecting a target substance in a specimen by latex immunoturbidimetry, the detection method comprising the step of contacting the specimen with the latex particle dispersion according to any one of [1] to

[10] .

[13] A sedimentation inhibitor for latex particles in a latex particle dispersion in a detection reagent containing a buffer solution and a latex particle dispersion used in the detection of a target substance by latex immunoturbidimetry, the sedimentation inhibitor comprising at least one compound selected from the group consisting of hexametaphosphate and its salts as an active ingredient.

[14] The sedimentation inhibitor according to

[13] , wherein the salt is an alkali metal salt.

[15] A method for inhibiting the sedimentation of latex particles carrying a substance capable of binding to a target substance in latex immunoturbidimetry, the method comprising the step of contacting the latex particles with at least one compound selected from the group consisting of hexametaphosphate and its salts in an aqueous medium.

[16] The method according to

[15] , wherein the salt is an alkali metal salt.

[17] A method for preserving latex particles in a latex immunoturbidimetric assay, comprising the step of contacting the latex particles with at least one compound selected from the group consisting of hexametaphosphoric acid and its salts in an aqueous medium.

[18] A method for preserving a latex particle dispersion in a latex immunoturbidimetric assay, comprising the step of contacting the latex particles with at least one compound selected from the group consisting of hexametaphosphoric acid and its salts in an aqueous medium, wherein the content of latex particles in the supernatant of the latex particle dispersion after storage for 7 days is 80% or more of the content before storage.

[0017] The present invention provides latex particle dispersions, reagent kits, detection methods, sedimentation inhibitors, and sedimentation inhibitor methods with improved on-board stability.

[0018] Fig. 1 is a graph showing the content ratio of latex particles before and after inversion mixing in Test Example 1. Fig. 2 is a graph showing the content ratio of latex particles before and after inversion mixing in Test Example 2.

[0019] In this specification and claims, when a numerical range is expressed using "to", the numerical range includes the numerical values ​​on both sides of "to".

[0020] [Particle Dispersion] The latex particle dispersion of the present invention is a latex particle dispersion containing latex particles and an aqueous medium, and further containing at least one compound selected from the group consisting of hexametaphosphoric acid and salts thereof. By allowing the compound to coexist with the latex particles, the dispersibility of the latex particles in the aqueous medium can be improved, and as a result, on-board stability can be improved.

[0021] Hexametaphosphoric acid and its salts used in the latex particle dispersion of the present invention are preferably water-soluble salts. The salts are not particularly limited, but include, for example, ammonium salts and alkali metal salts, with alkali metal salts being preferred. Examples of alkali metal salts include lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr). The alkali metal salt is preferably at least one selected from the group consisting of sodium salts and potassium salts. Furthermore, sodium salts are most preferred, i.e., sodium hexametaphosphate (CAS RN [10124-56-8] is most preferred. The alkali metal salts can be used alone or in combination of two or more.

[0022] The content of the compound in the latex particle dispersion of the present invention is not particularly limited, but is preferably 0.001 to 10.0 mass %, more preferably 0.001 to 5.0 mass %, even more preferably 0.005 to 2.0 mass %, and even more preferably 0.01 to 1.0 mass %, relative to the total mass of the latex particle dispersion. When the content of the compound in the latex particle dispersion of the present invention is within the range of 0.001 to 10.0 mass %, relative to the total mass of the latex particle dispersion, a decrease in measurement sensitivity is not caused, and therefore the dispersibility of the latex particle dispersion can be improved without affecting the detection accuracy of the target substance.

[0023] The pH of the latex particle dispersion of the present invention is not particularly limited, but is preferably 4.0 to 10.0, more preferably 5.0 to 9.0, and even more preferably 6.0 to 8.0. Adjustment and measurement of the pH can be performed by well-known and conventional techniques by those skilled in the art. For example, the pH of the latex particle dispersion of the present invention can be adjusted by adding a base such as sodium hydroxide or an acid such as hydrochloric acid.

[0024] The latex particle dispersion of the present invention is used as a latex particle dispersion in a detection reagent used in detecting a target substance by latex immunoturbidimetry. The detection reagent generally consists of two types of reagents, but may consist of three or more types. In the present application, any solution to which latex particles have been added is referred to as a latex particle dispersion. When the detection reagent consists of two types of reagents, it generally consists of a buffer solution (first reagent) and a latex particle dispersion solution (second reagent). The detection reagent may be configured such that latex particles are added to the first reagent, or such that latex particles are added to both the first and second reagents. The latex particle dispersion of the present invention can be any of the first, second, or third or more reagents used in latex immunoturbidimetry. The latex particle dispersion of the present invention is preferably a latex particle dispersion in a detection reagent consisting of a buffer solution and a latex particle dispersion solution.

[0025] The latex particle dispersion of the present invention can be used as a reagent for detecting a target substance by latex immunoturbidimetry, and can also be combined with other reagent components to form a kit. Typical examples of the reagent include in vitro diagnostic reagents. The components of the reagent kit will be described later. The reagent used in latex immunoagglutination may also be referred to as an immunoagglutination reagent.

[0026] (Aqueous Medium) The aqueous medium used in the latex particle dispersion of the present invention may be any medium capable of dispersing latex particles, and among these, those containing water are preferred. The water content is not particularly limited as long as it is an amount capable of dispersing latex particles, and may be, for example, in the range of 40 to 100 mass %, 60 to 100 mass %, 80 to 100 mass %, or 90 to 100 mass % relative to the total amount of the aqueous medium.

[0027] The aqueous medium may also be a buffer solution such as Good's buffer, Tris buffer, phosphate buffer, glycine buffer, ammonia buffer, citrate buffer, acetate buffer, or succinate buffer. In addition to the water or buffer solution, the aqueous medium may also contain a lower alcohol such as methanol, ethanol, or isopropanol, or a water-miscible organic solvent such as dimethyl sulfoxide or dimethylformamide. In addition to the water or buffer solution, the aqueous medium may also contain a protein such as bovine serum albumin or casein, an amino acid, a sugar, a surfactant, an antifoaming agent, or a preservative.

[0028] The content of the aqueous medium may be, for example, 50.00 to 99.99% by mass, or 70.00 to 99.99% by mass, based on the total amount of the latex particle dispersion.

[0029] (Latex Particles) In the latex particle dispersion of the present invention, the latex particles are not particularly limited as long as they are latex particles commonly used as reagents for immunoassays, and may contain a fluorescent dye or a magnetic material. Latex particles can be obtained by polymerizing or copolymerizing various monomers. Examples of the monomer include polymerizable monomers having a phenyl group, such as styrene, α-methylstyrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, 4-vinylbenzoic acid, divinylbenzene, and vinyltoluene; polymerizable monomers having a phenyl group and a sulfonate, such as styrene sulfonate, divinylbenzene sulfonate, o-methylstyrene sulfonate, and p-methylstyrene sulfonate; polymerizable unsaturated aromatic compounds such as 1-vinylnaphthalene, 2-vinylnaphthalene, α-naphthyl (meth)acrylate, and β-naphthyl (meth)acrylate; polymerizable unsaturated carboxylic acids, such as (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; and polymerizable monomers having a naphthyl group, such as 1-vinylnaphthalene, 2-vinylnaphthalene, α-naphthyl (meth)acrylate, and β-naphthyl (meth)acrylate. Examples of suitable polymerizable unsaturated carboxylic acid esters include methyl acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, glycidyl (meth)acrylate, ethylene glycol-di-(meth)acrylic acid ester, and tribromophenyl (meth)acrylate; unsaturated carboxylic acid amides include (meth)acrylonitrile, (meth)acrolein, (meth)acrylamide, N-methylol-(meth)acrylamide, methylenebis(meth)acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide; polymerizable unsaturated nitriles; vinyl halides; and conjugated dienes. Among these, preferred are polymer particles derived from a monomer component containing a monomer having a styrene or vinylnaphthalene structure. Here, unless otherwise specified, "polymer particles derived from a monomer component" refers to polymer particles obtained by polymerizing or copolymerizing a monomer component. These monomers are appropriately selected depending on the required surface properties, specific gravity, etc., and can be used alone or in combination of two or more.

[0030] The synthetic polymer (also referred to as synthetic polymer) constituting the latex particles of the present invention is not particularly limited, and examples thereof include polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, itaconic acid polymer, styrene-hydrophilic carboxy monomer copolymer such as styrene-(meth)acrylic acid copolymer, styrene-acrylic acid copolymer, styrene-itaconic acid copolymer, etc. Among these, preferred are styrene-methacrylic acid copolymer, styrene-itaconic acid copolymer, styrene and styrene-styrene sulfonate copolymer.

[0031] The content of the latex particles is preferably 0.001 to 0.20% by mass based on the total mass of the latex particle dispersion.

[0032] In the present invention, the average particle size of the latex particles may be any size that allows for a difference in sensitivity depending on the measurement target and measurement range, and is preferably, for example, 50 nm to 1000 nm. Although a larger average particle size of latex particles results in higher sensitivity, the particles tend to sink more easily. This has made their application to measurement reagents difficult. However, the latex particle dispersion of the present invention makes particles less likely to sink, even if they have a large particle size. Furthermore, by adding the components of the present invention, even latex particles with a medium or small average particle size can be made to remain less likely to sink for a longer period of time. Therefore, the effects of the present invention can be enjoyed regardless of the average particle size of the latex particles.

[0033] The average particle size of the latex particles is not particularly limited, but is preferably 50 nm or more, more preferably 80 nm or more, and even more preferably 100 nm or more.

[0034] It is also possible to further expand the range of measurable concentrations by using latex particles having two or more different average particle sizes, large and small.

[0035] The average particle size of latex particles can be analyzed by laser diffraction / scattering (LS), Coulter principle, dynamic light scattering photon correlation spectroscopy, electron microscope, etc. In this specification, the average particle size refers to the volume-average particle size, and may be, for example, a value measured using a laser diffraction / scattering particle size distribution analyzer based on the laser diffraction / scattering (LS) method. As the laser diffraction / scattering particle size distribution analyzer, for example, the "LS 13 320" manufactured by Beckman Coulter can be used, and the measurement parameters are appropriately set according to the solvent used and the composition of the latex particles to be measured. The validity of the measurement parameters can be confirmed by checking that the particle size measured using standard polystyrene particles (certified by a third-party organization) commercially available from Beckman Coulter, etc., is within the manufacturer's specifications.

[0036] (Substance capable of binding to a target substance) In the present invention, substances capable of binding to a target substance include proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, haptens, etc., and are not particularly limited in terms of molecular weight or origin (natural or synthetic), but examples include antibodies or antigens that can be used in immunological assays that utilize immune reactions. The antibodies may be polyclonal or monoclonal antibodies. Monoclonal antibodies are more preferred.

[0037] The antibody used in the present invention may be not only the whole antibody molecule but also a functional fragment of an antibody having antigen-antibody reaction activity. It may be an antibody obtained through an immunization process of a general animal (mouse, goat, sheep, etc.), or an antibody (chimeric antibody, humanized antibody, fully humanized antibody, etc.) whose amino acid sequence has been altered by genetic recombination techniques to that of an animal species different from that of the animal immunized with an immunogen (target substance). The functional fragment of an antibody may be F(ab'), a fragment having antigen-antibody reaction activity. ２Examples of such antibodies include Fab', single-chain antibodies (scFv), variable domain of heavy chain of heavy chain antibodies (VHH), and new antigen receptor (IgNAR) antibodies. Functional fragments of these antibodies can be produced by treating the antibodies obtained as described above with a protease (e.g., pepsin or papain).

[0038] In the present invention, a substance capable of binding to a target substance to be supported on latex particles can be immobilized and supported on the latex particles by known methods such as physical adsorption, chemical bonding, or a combination of these.

[0039] In the case of physical adsorption, this can be carried out according to known methods, such as by mixing and contacting a substance capable of binding to a target substance with latex particles in a solution such as a buffer solution, or by contacting a substance capable of binding to a target substance dissolved in a buffer solution with a carrier.

[0040] When the chemical binding method is used, it can be carried out according to known methods described in, for example, "Clinical Pathology Extra Special Issue No. 53, Immunoassay for Clinical Testing - Techniques and Applications -" edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publishing Society, published in 1983; and "New Biochemical Experiment Course 1, Protein IV" edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, published in 1991, by mixing and contacting a substance capable of binding to a target substance and latex particles with a bivalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, and then reacting the amino groups, carboxyl groups, thiol groups, aldehyde groups, hydroxyl groups, or the like of the substance capable of binding to the target substance and the latex particles with the bivalent crosslinking reagent.

[0041] If treatment is necessary to suppress spontaneous aggregation or nonspecific reactions of latex particles, the surface of the latex particles may be treated by known methods, such as contacting and coating with proteins such as bovine serum albumin (BSA), casein, gelatin, egg albumin or a salt thereof, surfactants, or skim milk powder, to perform a carrier blocking treatment (masking treatment). Furthermore, as described in, for example, Japanese Patent Application Laid-Open No. 11-337551, latex particles carrying antigens or antibodies that do not immunologically react with the target substance may be further added to the detection reagent. Such particles are generally added to the first reagent for the purpose of suppressing nonspecific reactions. One aspect of the present invention is the use of a latex particle dispersion in a detection reagent used for detecting a target substance by latex immunoturbidimetry. Another aspect is the use of a latex particle dispersion for detecting a target substance by latex immunoturbidimetry. Yet another aspect is the use of a latex particle dispersion for producing a detection reagent used for detecting a target substance by latex immunoturbidimetry. Yet another aspect is the use of latex particles, an aqueous medium, and at least one compound selected from the group consisting of hexametaphosphoric acid and salts thereof to prepare a latex particle dispersion.

[0042] [Reagent Kit] A reagent kit for use in detecting a target substance in a sample by latex immunoturbidimetry of the present invention includes at least the latex particle dispersion of the present invention. The kit typically includes a two-reagent system including a particle dispersion (second reagent) containing particles carrying a substance that binds to the target substance and a first reagent containing a buffer solution. Examples of the buffer solution used in the first reagent include Good's buffer, Tris buffer, phosphate buffer, glycine buffer, ammonia buffer, citrate buffer, acetate buffer, and succinate buffer. The first reagent may also contain, in addition to the buffer solution, a component that suppresses nonspecific reactions, an agglutination promoter, an antifoaming agent, a preservative, and the like. In addition to the first and second reagents, the kit may also include, for example, a sample diluent, a sample extract, instructions for use, and sample collection tools (collection pipettes, syringes, cotton swabs, filters, etc.).

[0043] [Detection Method] The method for detecting a target substance in a specimen by the latex turbidimetric immunoassay (LTIA) of the present invention is carried out by contacting the specimen with the above-mentioned latex particle dispersion. Methods for measuring a target substance by the LTIA can be broadly divided into two types.

[0044] The first method involves reacting the target substance with latex particles carrying a substance capable of binding to the target substance to form a sandwich-type immune complex, and measuring the target substance based on the degree of agglutination of the latex particles associated with the formation of the immune complex.

[0045] The second method involves adding multiple target substances or their analogs (including fragments thereof), or proteins to which they are immobilized, to a reagent, causing them to compete with the target substances in the sample, thereby inhibiting the formation of an immune complex between the target substances contained in the reagent and latex particles carrying a substance capable of binding to the target substances, and measuring the target substances (antigens) from the degree of inhibition of agglutination of the latex particles associated with the inhibition of immune complex formation.

[0046] The target substance and the substance capable of binding to the target substance can be selected arbitrarily depending on the purpose. For example, if the target substance is an antigen, antibodies such as polyclonal antibodies and monoclonal antibodies (including recombinant antibodies and functional fragments of each antibody) can be selected as the substance capable of binding to the target substance. If the target substance is an antibody, antigens such as natural and recombinant antigens can be selected as the substance capable of binding to the target substance.

[0047] The present invention can be used in any of the above methods, and specifically includes the following steps: (1) mixing a target substance in a sample with a buffer solution or the like; (2) adding a latex particle dispersion containing latex particles carrying a substance capable of binding to the target substance to the resulting mixture after step (1), and contacting the mixture with the sample; and (3) optically detecting the degree of aggregation of the latex particles in the liquid. Here, the latex particles carrying a substance capable of binding to the target substance used in step (2) may be of one type, two types, or three or more types. Furthermore, some of the latex particles may be contained in the buffer solution used in step (1). Step (3) may be started at the same time as step (2) or step (1).

[0048] In the present invention, methods for optically observing the degree of aggregation of latex particles include methods (endpoint method, rate method, etc.) that measure scattered light intensity, absorbance, or transmitted light intensity using an optical instrument. The concentration (quantitative value) of the target substance contained in the sample can be calculated by comparing the measured value of absorbance, etc. obtained by measuring a sample with the measured value of absorbance, etc. obtained by measuring a standard substance (a sample with a known concentration of the target substance). The measurement of absorbance, etc. of transmitted light or scattered light may be performed using a single wavelength or a dual wavelength (the difference or ratio between two wavelengths). The measurement wavelength is generally selected from the range of 500 nm to 900 nm.

[0049] The measurement of a target substance in a sample according to the present invention may be performed manually or using an apparatus such as a measuring device, but is preferably performed using an apparatus. The measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (dedicated machine).

[0050] [Sedimentation Inhibitor] The sedimentation inhibitor for latex particles in a latex particle dispersion in a detection reagent containing a buffer solution and a latex particle dispersion used in the detection of a target substance by latex immunoturbidimetry of the present invention contains, as an active ingredient, at least one compound selected from the group consisting of hexametaphosphoric acid and its salts. One aspect of the present invention is the use of at least one compound selected from the group consisting of hexametaphosphoric acid and its salts for inhibiting the sedimentation of latex particles in a detection reagent containing a buffer solution and a latex particle dispersion used in the detection of a target substance by latex immunoturbidimetry. Another aspect is the use of at least one compound selected from the group consisting of hexametaphosphoric acid and its salts for the manufacture of the sedimentation inhibitor for latex particles in a detection reagent containing a buffer solution and a latex particle dispersion used in the detection of a target substance by latex immunoturbidimetry. Another aspect is the use of at least one compound selected from the group consisting of hexametaphosphoric acid and its salts for inhibiting sedimentation of latex particles in a latex particle dispersion. Yet another aspect is the use of at least one compound selected from the group consisting of hexametaphosphoric acid and its salts for producing an agent for inhibiting sedimentation of latex particles in a latex particle dispersion. The type and content of the active ingredient in the latex particle dispersion are the same as those described for the latex particle dispersion of the present invention.

[0051] [Method for inhibiting sedimentation] The method for inhibiting sedimentation of latex particles in a latex immunoturbidimetric assay of the present invention comprises the step of contacting the latex particles with at least one compound selected from the group consisting of hexametaphosphoric acid and its salts in an aqueous medium. It is believed that the contact of the compound with the latex particles in the aqueous medium inhibits sedimentation of the latex particles, although the details of the mechanism are unclear. One aspect of the present invention is a method for inhibiting sedimentation of latex particles, comprising the step of mixing a latex particle dispersion with at least one compound selected from the group consisting of hexametaphosphoric acid and its salts. Another aspect is a method for inhibiting sedimentation of latex particles, in which the mixing is performed in an aqueous medium.

[0052] In the present invention, "latex particles settle" includes both the case where the particles settle as they are and the case where the particles adsorb to each other or agglomerate to increase in size before settling. Furthermore, in the present invention, "settling of latex particles is inhibited" includes the case where the particles are inhibited from settling as they are, the case where the particles are inhibited from adsorbing to each other or agglomerating to increase in size before settling, and the case where both of these are inhibited.

[0053] In the present invention, the presence or absence of a sedimentation inhibitory effect on latex particles is evaluated by adding a test additive to a latex particle dispersion and comparing the degree of particle sedimentation with that without the additive, as shown in the examples described below. The degree of particle sedimentation can be evaluated by comparing the absorbance at 600 nm or the like before mixing with the absorbance at the same wavelength after mixing. For example, after leaving the container containing the latex particle dispersion stationary for 7 or 15 days, a solution is sampled from the top of the dispersion and the absorbance is measured. The sedimentation inhibitory effect of the additive is evaluated when the absorbance after mixing is, for example, 80.0% or more of the absorbance before mixing.

[0054] [Storage Method] The method for storing a latex particle dispersion for use in latex immunoturbidimetry of the present invention comprises a step of storing the latex particle dispersion in a state in which the latex particle dispersion contains at least one compound selected from the group consisting of hexametaphosphoric acid and its salts. Although the details of the mechanism are unclear, contact between the compound and the latex particles in an aqueous medium inhibits sedimentation of the latex particles. As a result, it is believed that the latex particles in the latex particle dispersion can be stored (preserved) for a long period of time without sedimentation. One aspect of the present invention is a method for storing a latex particle dispersion for use in latex immunoturbidimetry, comprising a step of mixing the latex particle dispersion with at least one compound selected from the group consisting of hexametaphosphoric acid and its salts.

[0055] In the present invention, whether or not latex particles in a latex particle dispersion can be stably stored without settling is evaluated as being stable when a test additive is added to the latex particle dispersion and the degree of particle settling is improved compared to when no additive is added, as shown in the examples described below. The degree of particle settling can be evaluated by comparing the absorbance at 600 nm or the like before mixing with the absorbance at the same wavelength after mixing. For example, after leaving the container containing the latex particle dispersion stationary for 7 days, a solution is sampled from the top of the dispersion and the absorbance is measured. A stable storage effect is evaluated when the absorbance after mixing is, for example, 80.0% or more of the absorbance before mixing.

[0056] [Specimen] Examples of specimens containing a target substance in the present invention include samples obtained from living organisms, such as human or animal blood, serum, plasma, culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, or cell or tissue extracts. Furthermore, specimens in the present invention include not only specimens obtained directly from living organisms, but also specimens that have been pretreated by dilution, purification, extraction, filtration, or the like. Examples of blood specimens include whole blood, red blood cells, plasma, serum, and the like, and also include plasma specimens collected using blood collection tubes containing an anticoagulant.

[0057] [Target Substance] In the present invention, examples of target substances include proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, and haptens, but are not particularly limited as long as they are theoretically measurable. Examples include CRP (C-reactive protein), Lp(a), MMP3 (matrix metalloproteinase 3), antiphospholipid antibodies, type IV collagen, PSA, BNP (brain natriuretic peptide), insulin, albumin, cystatin C, RF (rheumatoid factor), KL-6, procalcitonin (PCT), FDP, D-dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), PAI-1, phenytoin, phenobarbital, carbamazepine, valproic acid, theophylline, TARC (thymus and activation-regulated chemokine), and sIL-2R (soluble interleukin-2 receptor).

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below.

[0059] Test Example 1: Evaluation of sedimentation of latex particles: procalcitonin measurement reagent Various additives were added to a latex particle dispersion (second LTIA measurement reagent) and stored, and the sedimentation of latex particles in the solution was evaluated.

[0060] Comparative Example 1 The test method and evaluation method are as follows. (1) Preparation of the second LTIA measurement reagent: 5 mM MOPS-NaOH (pH 7.0) Anti-human procalcitonin monoclonal antibody-sensitized latex (two types) The anti-human procalcitonin monoclonal antibody was obtained using a commercially available procalcitonin antigen (*) by a method known to those skilled in the art. Furthermore, a combination of monoclonal antibodies that allows sandwich measurement of the procalcitonin antigen was selected by a method known to those skilled in the art. The anti-human procalcitonin monoclonal antibody-sensitized latex was prepared with reference to the method described in JP 2017-181377 A. That is, an anti-human procalcitonin monoclonal antibody solution diluted to 0.36 mg / mL with an equivalent amount of 5 mM Tris-HCl (pH 8.5) was added to a 1.0% latex solution (5 mM Tris buffer solution (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) with an average particle size of 0.3 μm, and the mixture was stirred at 4° C. for 2 hours. Thereafter, an equal amount of 5 mM Tris-HCl (pH 8.5) containing 0.5% BSA was added, and the mixture was stirred at 4° C. for 1 hour to prepare an anti-human procalcitonin monoclonal antibody-sensitized latex particle solution. The latex particle solution was diluted with 5 mM MOPS-NaOH (pH 7.0) so that the absorbance at 600 nm became approximately 5.5 OD, and this was used as the second reagent of the LTIA measurement reagent. (*) Commercially available antigen; Recombinant human Procalcitonin: Hytest

[0061] (2) Storage Conditions 2 mL of the second reagent was dispensed into two glass test tubes, and then the tubes were left to stand in a refrigerator (2 to 8° C.) for 7 days.

[0062] (3) Evaluation Method: One of two glass test tubes was mixed by inversion, and the other was left standing. 100 μL of the sample was taken from the liquid surface. This was mixed with 900 μL of 5 mM MOPS-NaOH (pH 7.0) (10-fold dilution), and the absorbance at 600 nm and 800 nm was measured. The ratio of the absorbance at 600 nm to the absorbance at 800 nm (600 / 800 ratio) was calculated as an index of particle dispersibility in the solution. The absorbance ratio at 600 nm (before mixing by inversion) / (after mixing by inversion) was calculated as the latex particle content ratio (%) at the liquid surface. The measurement results are shown in Table 1. The latex particle content ratios before and after mixing by inversion are shown in the graph in Figure 1. The absorbance values ​​at 600 nm and 800 nm were multiplied by 10, and the absorbance of the original solution is listed in Table 1.

[0063] Comparative Examples 2 to 8, Examples 1 to 3 Measurements were performed in the same manner as in Comparative Example 1, except that the components listed in the "Additive Name" column of Table 1 were added to the second LTIA measurement reagent to the final concentrations listed in the "Final Concentration" column. The measurement results are shown in Table 1. The following additives were used: Sucrose, polyacrylic acid 5000, polyacrylic acid 25000, polyacrylic acid 1000000, sodium polyacrylate 22000-70000, sodium polyphosphate, sodium hexametaphosphate: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. The numbers attached to the polyacrylic acids indicate the degree of polymerization. PEO-3: manufactured by Sumitomo Seika Chemicals Co., Ltd. Molecular weight: 600,000-1,100,000.

[0064]

[0065] (4) Results and Discussion The ratio of the absorbance at 600 nm to the absorbance at 800 nm (600 / 800 ratio) represents the dispersibility of latex particles in the reagent. The 600 / 800 ratio after inversion mixing in Comparative Example 1 (without additive) (2.17 in this case) was used as the standard, and dispersibility was evaluated as good if it did not decrease by 0.10 or more. This evaluation method was also used in Test Example 2. The dispersibility was good in all of Comparative Examples 2 to 8 and Examples 1 to 3. The absorbance ratio at 600 nm (before inversion mixing) / (after inversion mixing) was calculated as the content ratio (%) of latex particles at the liquid surface. The smaller this value, the more easily the particles settle, and the closer this value is to 100%, the greater the additive's effect of suppressing particle settling. In this study, when the content ratio was 80% or more, the particle sedimentation inhibitory ability was evaluated as being present, and this was recorded as "A" in the "Sedimentation Inhibitory Ability" column in Table 1. When the content ratio was less than 80%, the particle sedimentation inhibitory ability was evaluated as not being present, and this was recorded as "B" in the "Sedimentation Inhibitory Ability" column in Table 1. However, the sedimentation inhibitory ability of Comparative Example 1 was recorded as "-". In addition, the degree of foaming of the second LTIA measurement reagent was evaluated, and in the "Degree of Reagent Foaming" column in Table 1, a "B" was recorded if the reagent was clearly more foamy than Comparative Example 1, and an "A" was recorded if the degree of foaming was the same as Comparative Example 1. In addition, a sample for which the degree of foaming was not evaluated was recorded as "Not performed". A sample with foaming to the same degree as Comparative Example 1 is easy to handle and is suitable because weighing errors are unlikely to occur when measuring the required amount using a pipette or the reagent probe of an automatic analyzer. In Comparative Example 2, sucrose had anti-sedimentation ability, but was not a preferred component because it reduced measurement sensitivity (see Test Example 3, described below). Comparative Examples 3 to 8 were not suitable because they had low anti-sedimentation ability and / or, even if they had anti-sedimentation ability, the reagents to which they were added tended to foam. Only sodium hexametaphosphate shown in Examples 1 to 3 had high anti-sedimentation ability, and the degree of foaming of the reagents to which this component was added was similar to that of Comparative Example 1, indicating the most excellent properties.

[0066] Test Example 2: Evaluation of Sedimentation of Latex Particles: TARC Measurement Reagent Test Example 1 revealed that sodium hexametaphosphate has a high sedimentation suppression ability, and the degree of foaming of the added reagent is similar to that of Comparative Example 1, making it easy to handle. Next, a compound having a structure similar to sodium hexametaphosphate was added to a latex particle dispersion (second LTIA measurement reagent) and stored, and the sedimentation of latex particles in the solution was evaluated.

[0067] Comparative Example 9: No Additive The test and evaluation methods are as follows. The composition of the reagent is shown below. (1) Preparation of the second LTIA measurement reagent: 5 mM MOPS-NaOH (pH 7.0) Anti-human TARC monoclonal antibody-sensitized latex (two types) The anti-human TARC monoclonal antibody was obtained using a commercially available TARC antigen (**) by a method known to those skilled in the art. Furthermore, a combination of monoclonal antibodies capable of sandwich assay with the TARC antigen was selected by a method known to those skilled in the art. The anti-human TARC monoclonal antibody-sensitized latex was prepared with reference to the method described in JP 2017-181377 A. Specifically, an anti-TARC antibody solution diluted to 0.36 mg / mL with an equal volume of 5 mM Tris-HCl (pH 8.5) was added to a 1.0% latex solution (5 mM Tris buffer solution (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5)) with an average particle size of 0.3 μm, and the mixture was stirred at 4° C. for 2 hours. An equal volume of 5 mM Tris-HCl (pH 8.5) containing 0.5% BSA was then added, and the mixture was stirred at 4° C. for 1 hour to prepare an anti-human TARC monoclonal antibody-sensitized latex particle solution. The latex particle solution was diluted with 5 mM MOPS-NaOH (pH 7.0) to an absorbance of approximately 5.5 OD at 600 nm, and used as the second reagent in the LTIA measurement reagent. (**) Commercially available antigen: CCL17, thymus and activation regulated chemokine (Shenandoah) Biotechnology, Inc.), CCL17 / TARC, Human (LifeSpan Biosscience, Inc.), Human TARC (CCL17) (Abeomics, Inc.), etc.

[0068] (2) Storage Conditions 2 mL of the second reagent was dispensed into two glass test tubes, and then the tubes were left to stand in a refrigerator (2 to 8° C.) for 15 days.

[0069] (3) Evaluation Method: One of two glass test tubes was mixed by inversion, and the other was left standing. 100 μL of the sample was taken from the liquid surface. This was mixed with 900 μL of 5 mM MOPS-NaOH (pH 7.0) (10-fold dilution), and the absorbance at 600 nm and 800 nm was measured. The ratio of the absorbance at 600 nm to the absorbance at 800 nm (600 / 800 ratio) was calculated as an index of particle dispersibility in the solution. The absorbance ratio at 600 nm (before mixing by inversion) / (after mixing by inversion) was calculated as the latex particle content ratio (%) at the liquid surface. The measurement results are shown in Table 2. The latex particle content ratios before and after mixing by inversion are shown in Figure 2. The absorbance values ​​at 600 nm and 800 nm were multiplied by 10, and the absorbance of the original solution is shown in Table 2.

[0070] <Comparative Examples 10 to 15, Examples 4 and 5> Measurements were carried out in the same manner as in Comparative Example 9, except that the components listed in the "Additive Name" column of Table 2 were added to the second reagent to the final concentrations listed in the "Final Concentration" column. The measurement results are shown in Table 2. The following additives were used: Sodium metaphosphate, pentasodium triphosphate, sodium diphosphate decahydrate: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Sodium phosphate: manufactured by Tokyo Chemical Industry Co., Ltd.

[0071]

[0072] (4) Results and Discussion In Test Example 2, the dispersibility of latex particles was good under all conditions. The content ratio of latex particles was 78.1% in Comparative Example 9, which contained no additive, while the value decreased in all Comparative Examples 11 to 15, indicating that the particles were more likely to settle. The components that exhibited the ability to inhibit particle settling were sucrose in Comparative Example 10 and sodium hexametaphosphate in Examples 4 and 5. Although sucrose in Comparative Example 10 exhibited the ability to inhibit settling, it was not a preferred component because it reduced measurement sensitivity (see Test Example 3, described below). Sodium hexametaphosphate was found to exhibit the ability to inhibit particle settling over a wide concentration range, from 0.01 to 1.0% final concentration. Phosphoric acid condensates are generally known as dispersants, but surprisingly, only sodium hexametaphosphate exhibited the ability to inhibit the settling of antibody-bound particles. That is, the phosphoric acid condensates used in the comparative examples, such as sodium polyphosphate, sodium metaphosphate, pentasodium triphosphate, and sodium diphosphate decahydrate, did not exhibit the ability to inhibit the precipitation of antibody-bound particles, and only sodium hexametaphosphate exhibited the ability to inhibit the precipitation of antibody-bound particles.

[0073] [Test Example 3: Absorbance measurement by LTIA method: TARC measurement] The second reagent of the LTIA measurement reagent was combined with the first reagent described below, and a standard solution containing a known concentration of TARC antigen was measured as a sample. The effect of the additives contained in the second reagent on detection sensitivity was evaluated by verifying the measurement sensitivity.

[0074] Comparative Example 16: No Additive The test and evaluation methods are as follows. The composition of the reagent is shown below. (1) Preparation of LTIA measurement reagent An LTIA measurement reagent consisting of the following first and second reagents was prepared. i) First reagent: 100 mM MOPS-NaOH (pH 7.5), 500 mM NaCl, 0.5% BSA ii) Second reagent: The second reagent used in Comparative Example 9 was used as is.

[0075] (2) Samples TARC standard solution Sample 1: Physiological saline (TARC 0 pg / mL) Sample 2: TARC 661 pg / mL Sample 3: TARC 1856 pg / mL Sample 4: TARC 4746 pg / mL Sample 5: TARC 9760 pg / mL Sample 6: TARC 20421 pg / mL

[0076] (3) Measurement Procedure: The first and second reagents were combined, and the TARC concentration in the sample was measured using a Hitachi Automated Analyzer 3500. Specifically, 120 μL of the first reagent was added to 2.4 μL of sample, and the mixture was incubated at 37°C for 5 minutes. Then, 40 μL of the second reagent was added and stirred. The change in absorbance associated with aggregation formation was measured over the next 5 minutes at a dominant wavelength of 570 nm and a secondary wavelength of 800 nm.

[0077] <Comparative Example 17, Examples 6 and 7> In Comparative Example 17, the measurement was carried out in the same manner as in Comparative Example 10, except that the second reagent (containing sucrose at a final concentration of 5.0%) was used. In Example 6, the measurement was carried out in the same manner as in Example 4, and in Example 7, the measurement was carried out in the same manner as in Example 5, except that the second reagent (containing sodium hexametaphosphate at final concentrations of 0.01% and 1.0%, respectively) was used.

[0078] (4) Evaluation method The ratio of the absorbance value obtained by measuring the standard substance solution to the absorbance value of Comparative Example 16 (without additive) (each absorbance value ÷ absorbance value of Comparative Example 16 (%)) was calculated, and this was defined as the "measurement sensitivity (relative value)" and is shown in Table 3.

[0079]

[0080] (5) Results and Discussion The sucrose added in Comparative Example 17 was a component that had shown high particle sedimentation suppression ability in Test Examples 1 and 2, but it reduced measurement sensitivity as shown in Table 3. In particular, the sensitivity of samples with low TARC concentrations (Sample 2 and Sample 3) was reduced by half, to 50% and 57%, compared to Comparative Example 16. In clinical testing, detection of extremely small amounts of target substances contained in serum, etc., which are the subject of analysis, is required, so it can be said that such a reduction in measurement sensitivity for samples with low concentrations of target substances is extremely undesirable. In contrast, sodium hexametaphosphate of the present invention can be said to be an extremely practical component that suppresses particle sedimentation without causing a reduction in measurement sensitivity, regardless of the TARC concentration.

[0081] The latex particle dispersion of the present invention suppresses sedimentation of latex particles, and therefore, when applied to a latex particle dispersion (second reagent) used in latex immunoturbidimetry, measurement becomes possible without redispersing the latex particle dispersion (second reagent) by stirring, etc. Furthermore, the on-board stability of the latex particle dispersion (second reagent) used in latex immunoturbidimetry can be improved.

Claims

1. A latex particle dispersion comprising latex particles and an aqueous medium, the latex particle dispersion further comprising at least one compound selected from the group consisting of hexametaphosphoric acid and salts thereof.

2. The latex particle dispersion according to claim 1, which is a latex particle dispersion in a detection reagent used in detecting a target substance by latex immunoturbidimetry.

3. The latex particle dispersion according to claim 1, which is a latex particle dispersion in a detection reagent containing a buffer and a latex particle dispersion used in the detection of a target substance by latex immunoturbidimetry.

4. The latex particle dispersion of claim 1, wherein the salt is an alkali metal salt.

5. The latex particle dispersion according to any one of claims 1 to 4, wherein the aqueous medium comprises water.

6. The latex particle dispersion according to any one of claims 1 to 4, wherein the latex particles are polymer particles derived from a monomer component containing a monomer having a styrene or vinylnaphthalene structure.

7. The latex particle dispersion according to any one of claims 1 to 4, wherein the latex particles are latex particles carrying a substance capable of binding to a target substance.

8. The latex particle dispersion according to claim 7, wherein the substance capable of binding to the target substance is an antigen or an antibody to the target substance.

9. The latex particle dispersion according to any one of claims 1 to 4, wherein the average particle size of the latex particles is 50 to 1,000 nm.

10. The latex particle dispersion according to any one of claims 1 to 4, wherein the latex particles are contained in an amount of 0.001 to 0.20% by mass based on the total mass of the latex particle dispersion.

11. A reagent kit for use in detecting a target substance in a specimen by latex immunoturbidimetry, comprising the latex particle dispersion liquid according to any one of claims 1 to 4.

12. A method for detecting a target substance in a specimen by latex immunoturbidimetry, comprising the step of contacting the specimen with the latex particle dispersion liquid according to any one of claims 1 to 4.

13. A sedimentation inhibitor for latex particles in a latex particle dispersion in a detection reagent containing a buffer solution and a latex particle dispersion used in the detection of a target substance by latex immunoturbidimetry, the sedimentation inhibitor comprising, as an active ingredient, at least one compound selected from the group consisting of hexametaphosphoric acid and its salts.

14. The settling inhibitor of claim 13, wherein said salt is an alkali metal salt.

15. A method for inhibiting sedimentation of latex particles carrying a substance capable of binding to a target substance in a latex immunoturbidimetric assay, the method comprising the step of contacting the latex particles with at least one compound selected from the group consisting of hexametaphosphoric acid and its salts in an aqueous medium.

16. The method of inhibiting settling according to claim 15, wherein the salt is an alkali metal salt.

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