Method, reagent and kit for latex agglutination immunoassay of NT-probnp

Abstract

To provide a method, a reagent and a kit for accurately measuring N-terminal pro-brain natriuretic peptide (NT-proBNP) in a sample with high sensitivity.SOLUTION: In a latex agglutination immunoassay method for NT-proBNP in a sample, N-terminal pro-brain natriuretic peptide (NT-proBNP) in the sample is reacted with latex particles to which an antibody recognizing NT-proBNP or an antibody fragment thereof is bonded in an aqueous medium in the presence of a pyridinium derivative.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a latex agglutination immunoassay method for NT-proBNP, a measurement reagent, and a measurement kit. [Background technology]

[0002] Brain natriuretic peptide (hereinafter also referred to as BNP), also known as B-type natriuretic peptide, is derived from the BNP gene. After the BNP gene is transcribed and translated, a BNP precursor (hereinafter also referred to as proBNP) consisting of 108 amino acid residues is generated, which is then cleaved into N-terminal pro-brain natriuretic peptide (hereinafter also referred to as NT-proBNP), which is physiologically inactive and consists of the 76th amino acid sequence from the N-terminus of proBNP, and physiologically active BNP, which consists of the 77th to 108th amino acid sequence of proBNP.

[0003] BNP and NT-proBNP are produced and secreted rapidly, primarily in the ventricles, where gene expression is increased in response to wall stress (stretch stress). Therefore, in heart failure, where wall stress increases, blood concentrations increase according to the severity of the condition, making measurement of BNP or NT-proBNP useful in diagnosing and monitoring heart failure.

[0004] As methods for measuring NT-proBNP, there have been reported a labeling method (Patent Document 1) in which NT-proBNP in a sample is subjected to an antigen-antibody reaction with an antibody that binds to NT-proBNP to form an immune complex, and the amount of label in the immune complex is measured; and a latex agglutination immunoassay method (Patent Document 2) in which latex particles bound to an antibody that binds to NT-proBNP in a sample are used to cause an antigen-antibody reaction with NT-proBNP in the sample, and the change in turbidity due to agglutination of the latex particles is measured as absorbance.

[0005] The above-mentioned labeling method generally requires washing (B / F separation) using a washing solution to remove substances other than NT-proBNP contained in the sample before measuring the amount of label in the immune complex, which makes the measurement time-consuming.

[0006] On the other hand, latex agglutination immunoassays, which do not require labeling and do not require B / F separation, are also used in measurements using general-purpose automated analyzers for biochemical testing. However, latex agglutination immunoassays have the problem of poor measurement sensitivity, making them difficult to apply to measurements of target components that require high sensitivity. Furthermore, because latex agglutination immunoassays do not include a B / F separation step, they are susceptible to nonspecific reactions caused by impurities contained in the sample, making accurate measurements difficult.

[0007] In latex agglutination immunoassays, anti-IgM antibodies have been reported as substances that suppress the above-mentioned non-specific reactions (Patent Document 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 3987284 [Patent Document 2] Special Publication No. 2022-544394 [Patent Document 3] Japanese Patent Application Laid-Open No. 2000-346844 Summary of the Invention [Problem to be solved by the invention]

[0009] Because NT-proBNP is used for diagnosing and monitoring heart failure, there is a demand for measurement reagents and kits that are more sensitive and capable of accurate measurement. An object of the present invention is to provide a highly sensitive and accurate method for measuring NT-proBNP in a sample, a measuring reagent and a measuring kit. [Means for solving the problem]

[0010] As a result of intensive research to solve the above problems, the present inventors have found that a highly sensitive and accurate measurement of NT-proBNP is possible by a latex agglutination immunoassay method in which NT-proBNP in a sample is reacted with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium in the presence of a pyridinium derivative, and have thus completed the present invention.

[0011] That is, the present invention includes the following aspects. [1] A latex agglutination immunoassay method for NT-proBNP in a sample, in which NT-proBNP in the sample is reacted with latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP in an aqueous medium in the presence of a pyridinium derivative. [2] A latex agglutination immunoassay method for NT-proBNP in a sample, comprising: (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting, in the aqueous medium, NT-proBNP in the sample with latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound, wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative. [3] A latex agglutination immunoassay method for NT-proBNP in a sample, comprising: (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting, in the aqueous medium, NT-proBNP in the sample with latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP, and latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP, wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative. [4] The method according to any one of [1] to [3], wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v). [5] The method according to any one of [1] to [4], wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v). [6] The method according to any one of [1] to [5], wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I):

[0012] [ka]

[0013] (In the formula, R 1 represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X - represents a monovalent anion) [7] The method according to any one of [1] to [6], wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group with 1 to 4 carbon atoms or a salt thereof. [8] The method according to any one of [1] to [7], wherein the pyridinium derivative is methylpyridinium or a salt thereof. [9] The method according to any one of [1] to [8], wherein the aqueous medium contains a sensitizer.

[10] The method according to [9], wherein the sensitizer is a hydrophilic polymer.

[11] A method for suppressing non-specific reactions caused by contaminants in a biological sample in a latex agglutination immunoassay of NT-proBNP in a sample, in which NT-proBNP in a sample is reacted with latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP in an aqueous medium in the presence of a pyridinium derivative.

[12] A method for suppressing non-specific reactions caused by contaminants in a biological sample in a latex agglutination immunoassay of NT-proBNP in a sample, comprising: (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting, in the aqueous medium, NT-proBNP in the sample with latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound, wherein steps (1) and / or (2) are performed in the presence of a pyridinium derivative.

[13] A method for suppressing non-specific reactions caused by contaminants in a biological sample in a latex agglutination immunoassay of NT-proBNP in a sample, comprising: (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting, in the aqueous medium, NT-proBNP in the sample with latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP, and latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP, wherein steps (1) and / or (2) are performed in the presence of a pyridinium derivative.

[14] The method according to any one of

[11] to

[13] , wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

[15] The method according to any one of

[11] to

[14] , wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

[16] The method according to any one of

[11] to

[15] , wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I):

[0014] [ka]

[0015] (In the formula, R 1 represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X - represents a monovalent anion)

[17] The method according to any one of

[11] to

[16] , wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group with 1 to 4 carbon atoms or a salt thereof.

[18] The method according to any one of

[11] to

[17] , wherein the pyridinium derivative is methylpyridinium or a salt thereof.

[19] The method according to any one of

[11] to

[18] , wherein the aqueous medium contains a sensitizer.

[20] The method according to

[19] , wherein the sensitizer is a hydrophilic polymer.

[21] A latex agglutination immunoassay reagent for NT-proBNP in a sample, comprising latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP, and a pyridinium derivative.

[22] A latex agglutination immunoassay reagent for NT-proBNP in a sample, comprising latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP, latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP, and a pyridinium derivative.

[23] The reagent according to

[21] or

[22] , wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

[24] The reagent according to any one of

[21] to

[23] , wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

[25] The reagent according to any one of

[21] to

[24] , wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I):

[0016] [ka]

[0017] (In the formula, R 1 represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X - represents a monovalent anion)

[26] The reagent according to any one of

[21] to

[25] , wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group with 1 to 4 carbon atoms or a salt thereof.

[27] The reagent according to any one of

[21] to

[26] , wherein the pyridinium derivative is methylpyridinium or a salt thereof.

[28] The reagent according to any one of

[21] to

[27] , wherein the reagent contains a sensitizer.

[29] The reagent according to

[28] , wherein the sensitizer is a hydrophilic polymer.

[30] A latex agglutination immunoassay kit for NT-proBNP in a sample, comprising a first reagent containing an aqueous medium, latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP, and a second reagent containing latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP, wherein the first reagent and / or the second reagent contain a pyridinium derivative.

[31] The kit according to

[30] , wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

[32] The kit according to

[30] or

[31] , wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

[33] The kit according to any one of

[30] to

[32] , wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I):

[0018] [ka]

[0019] (In the formula, R 1 represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X - represents a monovalent anion)

[34] The kit according to any one of

[30] to

[33] , wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group with 1 to 4 carbon atoms or a salt thereof.

[35] The kit according to any one of

[30] to

[34] , wherein the pyridinium derivative is methylpyridinium or a salt thereof.

[36] The kit according to any one of

[30] to

[35] , wherein the first reagent further contains a sensitizer.

[37] The kit according to any one of

[30] to

[36] , wherein the second reagent further contains a sensitizer.

[38] The kit according to

[36] or

[37] , wherein the sensitizer is a hydrophilic polymer. [Effects of the Invention]

[0020] A highly sensitive and accurate method for measuring NT-proBNP in a sample, a measuring reagent and a measuring kit are provided. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, embodiments for carrying out the present invention will be described in detail. Note that the embodiment described below shows an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow. Note that numerical ranges indicated using "to" indicate ranges that include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. Furthermore, when multiple substances corresponding to each component are present in the solution, the amount of each component in a solution refers to the total amount of the multiple substances present in the reaction or reagent, unless otherwise specified.

[0022] In this embodiment, "% (w / v)" means the percentage of mass (g) based on volume (100 mL).

[0023] In this embodiment, when an antibody is expressed as "binding" to or "reacting" with NT-proBNP, or as "recognizing" NT-proBNP, the terms include the meanings commonly used in the field of the present invention, and are all used synonymously. Methods for confirming the "binding" of an antibody to NT-proBNP include methods utilizing the principles of antigen-immobilized ELISA, competitive ELISA, sandwich ELISA, surface plasmon resonance, immunochromatography, quartz crystal microbalance, and the like, which are well known to those skilled in the art.

[0024] [Aqueous medium] In this embodiment, the aqueous medium may be deionized water, distilled water, a buffer solution, or the like, with a buffer solution being preferred. Examples of buffering agents used to prepare the buffer solution include acetate buffer and Good's buffer. A single buffering agent may be used alone, or two or more buffering agents may be used in combination. The aqueous medium may contain salts, metal ions, sugars, proteins, surfactants, and the like. Examples of salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc. Examples of metal ions include sodium ions, magnesium ions, manganese ions, zinc ions, etc. Examples of sugars include mannitol and sorbitol. Examples of proteins include bovine serum albumin (hereinafter referred to as BSA) and the like. Examples of surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, etc. The aqueous medium may also contain a sensitizer. The sensitizer can improve sensitivity by promoting the aggregation of NT-proBNP, the component to be measured, with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP. Examples of sensitizers include hydrophilic polymers, salts, etc. Examples of hydrophilic polymers include surfactants, polyethylene glycol, 2-methacryloyloxyethyl phosphorylcholine (2-MPC) polymer, dextran, etc., and examples of salts and surfactants include those described above. A specific example (product) of polyethylene glycol is polyethylene glycol with a molecular weight of 20,000 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). A specific example (product) of 2-methacryloyloxyethyl phosphorylcholine (2-MPC) polymer is Lipidure-BL103 (manufactured by NOF Corporation). A specific example (product) of dextran is dextran derived from Leuconostoc spp. with a molecular weight of 450,000 to 650,000 (manufactured by Sigma-Aldrich). The sensitizer not only improves the agglutination reaction (sensitivity) of the target component, NT-proBNP, but also enhances the agglutination reaction (non-specific reaction) of substances in the sample other than the target component. The concentration of the sensitizer in the aqueous medium is preferably 0.1 to 5% (w / v), more preferably 0.4 to 3% (w / v). The aqueous medium may also contain an anti-IgM antibody. The anti-IgM antibody can suppress, for example, nonspecific agglutination reactions (hereinafter also referred to as nonspecific reactions) caused by components in the sample other than the component to be measured. The anti-IgM antibody may be derived from any animal species, including rodents such as mice and rats, and mammals such as humans, monkeys, sheep, goats, rabbits, pigs, and cows. The anti-IgM antibody may be a polyclonal antibody or a monoclonal antibody. The concentration of the anti-IgM antibody in the aqueous medium is preferably 3 μg / ml to 10 mg / ml. A concentration of the anti-IgM antibody in the aqueous medium within the above range is preferred because it reduces the possibility of nonspecific reactions and also reduces the possibility of reactions with various immunoglobulins contained in the sample, which can lead to variations in measured values.

[0025] [sample] The sample in this embodiment is not particularly limited as long as it is a sample that may contain NT-proBNP, and examples thereof include any one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, ascites, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice, and preferred examples include whole blood, plasma, serum, ascites, etc. The sample in this embodiment may be an aqueous medium containing NT-proBNP, for example, NT-proBNP diluted with phosphate-buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2, hereinafter referred to as PBS).

[0026] [Latex agglutination immunoassay method] In this embodiment, the latex agglutination immunoassay method is a method in which a sample containing NT-proBNP is reacted in an aqueous medium with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP, thereby forming a complex containing NT-proBNP and the latex particles bound to the antibody or antibody fragment thereof that recognizes NT-proBNP, and selectively agglutinating the latex particles. The agglutination can be detected by measuring absorbance, scattered light, etc.

[0027] [Latex particles] In this embodiment, the latex particles are not particularly limited as long as they enable the NT-proBNP measurement method of this embodiment or can be used in the measurement reagent or measurement kit of this embodiment. Examples include fine particles of organic polymeric substances, fine particles of inorganic oxides, and fine particles whose surfaces as core fine particles are surface-treated with organic substances, etc. Specific examples include synthetic resins such as polystyrene, copolymers mainly composed of styrene, polyvinyl chloride, polypropylene, (meth)acrylic resin, and polymethyl methacrylate. One type of latex particle may be used alone, or two or more types may be used in combination. Among these, polystyrene-based synthetic polymers and polystyrene-based synthetic polymers copolymerized with acrylic acid-based monomers, sulfonic acid-containing monomers, etc. as components for imparting electric charge are particularly preferred.

[0028] Polystyrene latex particles are particularly preferred as the latex particles. The use of latex particles with a highly hydrophobic surface, such as polystyrene latex particles, allows for smooth adsorption of proteins and peptides. Polystyrene latex particles obtained by soap-free polymerization, which does not use a surfactant as an emulsifier, are particularly preferred because they can exist stably even without a surfactant due to the repulsion between negative charges on the surface. Additionally, various modified latexes (e.g., carboxylic acid-modified latex), magnetic latex (latex containing magnetic particles), and the like can also be used as needed.

[0029] The shape of the latex particles is not particularly limited, and examples thereof include spherical, elliptical, and irregular shapes. The average particle size (D50) may be, for example, 0.03 to 0.8 μm, 0.05 to 0.4 μm, or 0.1 to 0.35 μm. The average particle size (D50) can be measured, for example, with a laser diffraction particle size distribution analyzer. The average particle size (D50) is defined as the particle size at an integrated value of 50% (volume basis) in the particle size distribution.

[0030] [NT-proBNP] NT-proBNP in this embodiment is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. NT-proBNP is produced by cleaving a BNP precursor (proBNP) consisting of the amino acid sequence shown in SEQ ID NO: 2 into BNP consisting of the amino acid sequence shown in SEQ ID NO: 3 and NT-proBNP. The amino acid sequences of NT-proBNP, proBNP, and BNP are shown in Table 1.

[0031] [Table 1]

[0032] NT-proBNP can be prepared according to known methods, for example, by expressing it in Escherichia coli or the like using genetic recombination techniques.

[0033] [Antibody or antibody fragment thereof that recognizes NT-proBNP] The antibody recognizing NT-proBNP may be a mouse antibody, rat antibody, rabbit antibody, human antibody, humanized antibody, or chimeric antibody, or may be an antibody derived from another species. Furthermore, the antibody recognizing NT-proBNP may be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0034] Antibodies that recognize NT-proBNP include monoclonal antibodies, polyclonal antibodies, dimers, multimers, etc., with monoclonal antibodies being preferred from the standpoints of homogeneity and stability.

[0035] In this embodiment, the antibody fragment refers to a portion of the antibody that recognizes NT-proBNP, and includes the variable domain of the antibody or at least the antigen-binding region. Examples of antibody fragments in this embodiment include Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies (scFv), sc(Fv)2, Fab3, domain antibodies (dAb), diabodies, triabodies, tetrabodies, and minibodies. An "Fv fragment" is the smallest antibody fragment and includes the complete antigen-recognition region and antigen-binding region.

[0036] In this embodiment, the first antibody or antibody fragment thereof that recognizes NT-proBNP and the second antibody or antibody fragment thereof that recognizes NT-proBNP may bind to the same or different sites (epitopes) of NT-proBNP. Furthermore, the first antibody or antibody fragment thereof that recognizes NT-proBNP and the second antibody or antibody fragment thereof that recognizes NT-proBNP may be the same antibody or different antibodies.

[0037] In this embodiment, the antibody or antibody fragment thereof recognizing NT-proBNP can be obtained by a conventional antibody production method using NT-proBNP or a microorganism producing NT-proBNP as an immunogen. For example, the antibody or antibody fragment can be obtained by fusing myeloma cells with antibody-producing cells obtained by immunizing an animal with the immunogen to produce cells (hybridoma), and then obtaining a monoclonal antibody produced by the hybridoma. Alternatively, a commercially available antibody can be used as the antibody recognizing NT-proBNP. Examples of commercially available monoclonal antibodies recognizing NT-proBNP include BRJNBNPS102 anti-NT-proBNP antibody (manufactured by Fapon Biotech) and BECBNPS108 anti-NT-proBNP antibody (manufactured by Fapon Biotech). In this embodiment, the monoclonal antibody recognizing NT-proBNP is also referred to as an "anti-NT-proBNP antibody."

[0038] [Latex particles bound to antibodies or antibody fragments that recognize NT-proBNP] In this embodiment, latex particles having an antibody or antibody fragment thereof that recognizes NT-proBNP bound thereto refer to latex particles having an antibody or antibody fragment thereof that recognizes NT-proBNP bound thereto (including a first antibody or antibody fragment thereof that recognizes NT-proBNP, and a second antibody or antibody fragment thereof that recognizes NT-proBNP, as described below).

[0039] The method of binding an antibody or antibody fragment thereof that recognizes NT-proBNP to latex particles is not particularly limited as long as it enables the NT-proBNP measurement method of this embodiment or is usable for the measurement reagent or measurement kit of this embodiment, and examples include binding by physical adsorption and binding by chemical bond. Examples of physical adsorption include electrostatic bond, hydrogen bond, hydrophobic bond, etc. Examples of chemical bond include covalent bond, coordinate bond, etc.

[0040] The antibody or antibody fragment thereof that recognizes NT-proBNP may be bound to latex particles directly or indirectly using the aforementioned physical adsorption and / or chemical binding. Examples of indirect binding methods include a method in which the antibody or antibody fragment thereof that recognizes NT-proBNP is bound to latex particles using the specific binding between a pair of affinity substances such as biotin and avidin (avidin, streptavidin, neutravidin, etc.), and a method in which the antibody or antibody fragment thereof is bound to latex particles by a covalent bond via a linker.

[0041] When a set of affinity substances is used, latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound can be produced by binding an antibody or antibody fragment thereof that recognizes NT-proBNP bound to one of the affinity substances (A) of the set with latex particles to which the other affinity substance (a) of the set is bound.

[0042] Examples of combinations of Aa include the following combinations. Combinations of biotin and avidins (avidin, neutravidin, streptavidin, etc.); Combinations of avidins (avidin, neutravidin, streptavidin, etc.) with biotin; A combination of the Fc region of an antibody that recognizes NT-proBNP and an antibody that binds to the Fc region.

[0043] Examples of linkers include molecules that can covalently bond both functional groups on the surface of latex particles and functional groups possessed by antibodies or antibody fragments thereof that recognize NT-proBNP. For example, molecules that simultaneously have a first reactive group capable of reacting with a functional group possessed by antibodies or antibody fragments thereof that recognize NT-proBNP and a second reactive group capable of reacting with a functional group on the surface of latex particles, where the first reactive group and the second reactive group are different groups, are preferably used. Examples of functional groups possessed by an antibody or antibody fragment thereof that recognizes NT-proBNP and functional groups carried on the surface of latex particles include carboxyl, amino, glycidyl, sulfhydryl, hydroxyl, amide, imino, N-hydroxysuccinyl, and maleimide groups. Examples of reactive groups in linkers include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide ester, pentafluorophenyl (PFP) ester, psoralen, pyridyl disulfide, and vinyl sulfone.

[0044] [Pyridinium derivatives] In this embodiment, the pyridinium derivative is not particularly limited as long as it enables the NT-proBNP measurement method of this embodiment or can be used in the measurement reagent or measurement kit of this embodiment, and examples include a pyridinium salt represented by the following formula (I) (hereinafter referred to as compound (I)).

[0045] [ka]

[0046] (In the formula, R 1 represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X - represents a monovalent anion)

[0047] R 1 In the above, examples of the alkyl in the substituted or unsubstituted alkyl include linear alkyl having 1 to 20 carbon atoms and branched alkyl having 3 to 20 carbon atoms, with linear alkyl having 1 to 10 carbon atoms and branched alkyl having 3 to 10 carbon atoms being preferred, and linear alkyl having 1 to 4 carbon atoms being more preferred. Examples of linear alkyl having 1 to 20 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, and icosyl. Examples of branched alkyls having 3 to 20 carbon atoms include isopropyl, isobutyl, isopentyl, isohexyl, isoheptyl, isooctyl, isononyl, isodecyl, isoundecyl, isododecyl, isotridecyl, isotetradecyl, isopentadecyl, isohexadecyl, isoheptadecyl, isooctadecyl, isononadecyl, isoicosyl, and octyldodecyl. Examples of linear alkyls having 1 to 10 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Examples of branched alkyls having 3 to 10 carbon atoms include isopropyl, isobutyl, isopentyl, isohexyl, isoheptyl, isooctyl, isononyl, and isodecyl. Examples of linear alkyls having 1 to 4 carbon atoms include methyl, ethyl, propyl, and butyl.

[0048] R 1In the above, the alkenyl in the substituted or unsubstituted alkenyl includes, for example, alkenyl having 2 to 20 carbon atoms, and preferably alkenyl having 2 to 10 carbon atoms. Examples of alkenyl having 2 to 20 carbon atoms include vinyl, propyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, oleyl, nonadecenyl, icocenyl, etc. Examples of alkenyl having 2 to 10 carbon atoms include vinyl, propyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.

[0049] R 1 In the above, examples of the substituent in the substituted alkyl and substituted alkenyl include a phenyl group, a hydroxyl group, a sulfo group, a cyano group, a halogen atom, etc. Examples of the halogen atom include a chlorine atom, a bromine atom, an iodine atom, etc.

[0050] X in compound (I) - represents a monovalent anion. Examples of monovalent anions include halogen ions, OH - , PF6 - , BF4 - , CH3CH2OSO3 - , (CF3SO2)2N - Examples of halogen ions include Cl. - , Br - , I - etc.

[0051] Compound (I) is preferably a linear alkylpyridinium having 1 to 4 carbon atoms in the alkyl or a salt thereof, more preferably methylpyridinium or a salt thereof. Examples of linear alkylpyridinium salts include linear alkylpyridinium halides, and examples of halogens include chlorine, bromine, and iodine. Examples of linear alkylpyridinium halides include methylpyridinium halides such as methylpyridinium chloride and methylpyridinium bromide.

[0052] Specific examples (products) of compound (I) include methylpyridinium chloride, 1-dodecylpyridinium chloride, 1-cetylpyridinium chloride, 1-acetonylpyridinium chloride, 1-(3-sulfopropyl)pyridinium hydroxide inner salt, 1-methylpyridinium chloride, 1-ethylpyridinium chloride, 1-propylpyridinium chloride, 1-butylpyridinium chloride, 1-methylpyridinium bromide (all manufactured by Tokyo Chemical Industry Co., Ltd.), 1-hexylpyridinium chloride (manufactured by Kanto Chemical Co., Ltd.), and the like.

[0053] [Non-specific reaction] In latex agglutination immunoassays, non-specific reactions due to impurities in biological samples can occur, making it difficult to obtain accurate measurements. Contaminants that cause nonspecific reactions include lipids, fibrin, lipophilic low-molecular-weight compounds (molecular weight 500 or less), and rheumatoid factor, which is said to be an autoantibody against the Fc region of denatured IgG.

[0054] Contaminants can also be removed from biological samples by known methods. Lipids can be removed, for example, by adsorbing them onto silica. Fibrin can be precipitated by centrifuging the biological sample, and only the supernatant can be collected. Prior to centrifugation, a fibrin production promoter (e.g., calcium ions and / or bovine thrombin) can be added to the biological sample to generate fibrin, followed by centrifugation. Lipid-soluble small molecules can be removed by adsorbing them onto charcoal (activated carbon). Rheumatoid factors can be removed, for example, by adding an enzyme such as pronase, a protease, to the biological sample and allowing it to react. However, if the enzyme remains in the biological sample, it will denature the antibody used in the latex agglutination immunoreaction, reducing its antigen-binding activity. Therefore, the biological sample must be inactivated by boiling or other methods before use in the latex agglutination immunoreaction. As described above, it is possible to remove impurities and reduce non-specific reactions by pretreating a biological sample, but the pretreatment procedure is complicated. Therefore, a latex agglutination immunoassay method that can reduce non-specific reactions without the above-mentioned pretreatment procedure is desired. The latex agglutination immunoassay method for NT-proBNP in a sample of this embodiment can reduce non-specific reactions without the above-mentioned pretreatment procedure.

[0055] [Method for measuring NT-proBNP in samples] The latex agglutination immunoassay method for NT-proBNP in a sample in this embodiment is a method in which, in an aqueous medium, NT-proBNP in the sample is reacted with latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound, in the presence of a pyridinium derivative.

[0056] The time for reacting NT-proBNP in a sample with an antibody or antibody fragment thereof that recognizes NT-proBNP is not particularly limited as long as it allows the NT-proBNP measurement method of this embodiment, and may be, for example, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more. Furthermore, the reaction time may be, for example, 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 9 minutes or less, 7 minutes or less, or 6 minutes or less.

[0057] The above values ​​can be freely combined. For example, the reaction time may be 10 seconds to 1 hour, 1 minute to 10 minutes, or 3 minutes to 6 minutes.

[0058] The temperature at which NT-proBNP in a sample is reacted with an antibody or antibody fragment thereof that recognizes NT-proBNP is not particularly limited as long as it is a temperature that enables the NT-proBNP measurement method of this embodiment, and may be 0°C or higher, 4°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, or 30°C or higher, or may be 50°C or lower, 45°C or lower, or 40°C or lower.

[0059] The above values ​​can be freely combined. For example, the reaction temperature may be 4°C to 50°C, 10°C to 45°C, or 25°C to 40°C. The reaction temperature may also be 37°C.

[0060] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the above reaction is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.003% (w / v) or more, 0.005% (w / v) or more, 0.01% (w / v) or more, 0.011% (w / v) or more, 0.012% (w / v) or more, or 0.013% (w / v) or more. Furthermore, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the above reaction may be, for example, 0.1% (w / v) or less, 0.08% (w / v) or less, 0.06% (w / v) or less, 0.05% (w / v) or less, 0.04% (w / v) or less, 0.03% (w / v) or less, 0.025% (w / v) or less, 0.02% (w / v) or less, or 0.015% (w / v) or less.

[0061] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the above reaction may be 0.001% (w / v) to 0.1% (w / v), 0.003% (w / v) to 0.08% (w / v), 0.005% (w / v) to 0.05% (w / v), or 0.01% (w / v) to 0.02% (w / v).

[0062] The concentration of the pyridinium derivative in the above reaction is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.002% (w / v) or more, 0.003% (w / v) or more, 0.004% (w / v) or more, 0.005% (w / v) or more, 0.006% (w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more, 0.009% (w / v) or more, or 0.001% (w / v) or more. It may be 0.01% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more, 0.04% (w / v) or more, 0.05% (w / v) or more, 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, 0.4% (w / v) or more, 0.5% (w / v) or more, or 0.6% (w / v) or more. Furthermore, the concentration of the pyridinium derivative in the above reaction may be, for example, 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7% (w / v) or less, 6% (w / v) or less, 5% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, 2% (w / v) or less, 1% (w / v) or less, 0.9% (w / v) or less, 0.8% (w / v) or less, or 0.9% (w / v) or less.

[0063] The above values ​​can be freely combined. For example, the concentration of the pyridinium derivative in the above reaction may be 0.001% (w / v) to 10% (w / v), 0.005% (w / v) to 5% (w / v), 0.01% (w / v) to 3% (w / v), or 0.1% (w / v) to 2% (w / v). In the above reaction, one type of pyridinium derivative may be present, or two or more types of pyridinium derivatives may be present. When two or more types of pyridinium derivatives are present, the above concentration means the total concentration of the two or more types of pyridinium derivatives.

[0064] One aspect of the method for measuring NT-proBNP in a sample in this embodiment includes: (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting NT-proBNP in a sample with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium; wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative.

[0065] The measurement method in this embodiment may also include the following step (3) after the above step (2). (3) A step of measuring the agglutination of latex particles caused by binding between NT-proBNP and latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound, thereby obtaining a measurement value.

[0066] <Process (1)> Step (1) is a step of mixing a sample containing NT-proBNP with an aqueous medium. In step (1), the sample containing NT-proBNP may be added to the aqueous medium and mixed, or the aqueous medium may be added to the sample containing NT-proBNP and mixed. The aqueous medium in step (1) is not particularly limited, and examples thereof include the aqueous media described above.

[0067] Step (1) may be carried out in the presence of a pyridinium derivative. The pyridinium derivative is not particularly limited, and examples thereof include the aforementioned pyridinium derivatives. The concentration of the pyridinium derivative in step (1) is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.002% (w / v) or more, 0.003% (w / v) or more, 0.004% (w / v) or more, 0.005% (w / v) or more, 0.006% (w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more, 0.009% (w / v) or more, 0.01% (w / v) or more, 0.02% (w / v) or more, (w / v) or more, 0.03% (w / v) or more, 0.04% (w / v) or more, 0.05% (w / v) or more, 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, 0.4% (w / v) or more, 0.5% (w / v) or more, 0.6% (w / v) or more, 0.7% (w / v) or more, 0.8% (w / v) or more, or 0.9% (w / v) or more. Furthermore, the concentration of the pyridinium derivative in step (1) above may be, for example, 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7% (w / v) or less, 6% (w / v) or less, 5% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, 2% (w / v) or less, or 1% (w / v) or less.

[0068] The above values ​​can be freely combined. For example, the concentration of the pyridinium derivative in step (1) may be 0.001% (w / v) to 10% (w / v), 0.005% (w / v) to 5% (w / v), 0.01% (w / v) to 3% (w / v), or 0.1% (w / v) to 2% (w / v). In the above step (1), one type of pyridinium derivative may be present, or two or more types of pyridinium derivatives may be present. When two or more types of pyridinium derivatives are present, the above concentration means the total concentration of the two or more types of pyridinium derivatives.

[0069] In step (1), the sample containing NT-proBNP may be mixed with an aqueous medium and then maintained at a constant temperature for a certain period of time. The time for which the mixture is held is not particularly limited as long as it allows for the NT-proBNP measurement method of this embodiment, and may be, for example, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more, or 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 9 minutes or less, 7 minutes or less, or 6 minutes or less.

[0070] The above values ​​can be freely combined. For example, the time for which the mixture is held may be from 10 seconds to 1 hour, from 1 minute to 10 minutes, or from 3 minutes to 6 minutes.

[0071] The temperature at which the above-mentioned mixture is maintained is not particularly limited as long as it is a temperature that enables the NT-proBNP measurement method of this embodiment, and may be 0°C or higher, 4°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, or 50°C or lower, 45°C or lower, or 40°C or lower.

[0072] The above values ​​can be freely combined. For example, the temperature at which the mixture is maintained may be 4°C to 50°C, 10°C to 45°C, or 25°C to 40°C. The temperature may also be 37°C.

[0073] <Process (2)> Step (2) is a step of reacting NT-proBNP in a sample with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium. In step (2), the antibody or antibody fragment thereof that recognizes NT-proBNP reacts with NT-proBNP to produce a complex containing NT-proBNP and latex particles bound to the antibody or antibody fragment thereof that recognizes NT-proBNP.

[0074] In step (2), NT-proBNP may be added to latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound, and the reaction may be allowed to proceed. Alternatively, latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound may be added to NT-proBNP, and the reaction may be allowed to proceed.

[0075] In step (2), an antibody or antibody fragment thereof that recognizes two or more types of NT-proBNP may be used instead of an antibody or antibody fragment thereof that recognizes one type of NT-proBNP. When an antibody or antibody fragment thereof that recognizes two types of NT-proBNP is used, step (2) can be carried out as follows. (2A) In an aqueous medium, NT-proBNP in a sample is reacted with latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP, and with latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP. The measurement method in this embodiment may also include the above step (3) after step (2A).

[0076] In step (2A), a first antibody or antibody fragment thereof that recognizes NT-proBNP and a second antibody or antibody fragment thereof that recognizes NT-proBNP react with NT-proBNP to produce a complex containing NT-proBNP, latex particles to which the first antibody or antibody fragment thereof that recognizes NT-proBNP is bound, and latex particles to which the second antibody or antibody fragment thereof that recognizes NT-proBNP is bound.

[0077] In step (2A), the latex particles in the latex particles to which the first antibody or its antibody fragment that recognizes NT-proBNP is bound may be the same as or different from the latex particles in the latex particles to which the second antibody or its antibody fragment that recognizes NT-proBNP is bound.

[0078] In step (2A), the reaction between NT-proBNP and latex particles bound with a first antibody or antibody fragment thereof that recognizes NT-proBNP, and latex particles bound with a second antibody or antibody fragment thereof that recognizes NT-proBNP is not particularly limited as long as it is a reaction that produces a complex containing NT-proBNP, latex particles bound with a first antibody or antibody fragment thereof that recognizes NT-proBNP, and latex particles bound with a second antibody or antibody fragment thereof that recognizes NT-proBNP. For example, Alternatively, latex particles to which a first antibody or antibody fragment thereof that recognizes NT-proBNP is bound may be reacted with the latex particles to form a complex containing NT-proBNP and latex particles to which a first antibody or antibody fragment thereof that recognizes NT-proBNP is bound, and then latex particles to which a second antibody or antibody fragment thereof that recognizes NT-proBNP is bound may be added and allowed to react. Alternatively, latex particles to which a first antibody or antibody fragment thereof that recognizes NT-proBNP is bound and latex particles to which a second antibody or antibody fragment thereof that recognizes NT-proBNP is bound may be mixed and allowed to react with NT-proBNP.

[0079] When NT-proBNP is reacted with latex particles bound to a first antibody or antibody fragment thereof that recognizes NT-proBNP and latex particles bound to a second antibody or antibody fragment thereof that recognizes NT-proBNP, there is no limitation on the order in which NT-proBNP, the latex particles bound to the first antibody or antibody fragment thereof that recognizes NT-proBNP, and the latex particles bound to the second antibody or antibody fragment thereof that recognizes NT-proBNP are added. For example, the order in which NT-proBNP, the latex particles bound to the first antibody or antibody fragment thereof that recognizes NT-proBNP, and the latex particles bound to the second antibody or antibody fragment thereof that recognizes NT-proBNP are added may be any order. Alternatively, latex particles to which a first antibody that recognizes NT-proBNP or a fragment thereof is bound may be added to NT-proBNP, and then latex particles to which a second antibody that recognizes NT-proBNP or a fragment thereof is bound may be added; or latex particles to which a first antibody that recognizes NT-proBNP or a fragment thereof is bound and a second antibody that recognizes NT-proBNP or a fragment thereof may be mixed, and then NT-proBNP may be added.

[0080] Step (2A) may be divided into the following steps (2A-1) and (2A-2). (2A-1) a step of reacting, in an aqueous medium, NT-proBNP in a sample with latex particles bound to a first antibody that recognizes NT-proBNP or an antibody fragment thereof, to produce a complex 1 containing NT-proBNP and latex particles bound to the first antibody that recognizes NT-proBNP or an antibody fragment thereof; and (2A-2) A step of reacting the complex 1 produced in step (1) with latex particles bound to a second antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium to produce a complex 2 containing NT-proBNP, latex particles bound to a first antibody or antibody fragment thereof that recognizes NT-proBNP, and latex particles bound to a second antibody or antibody fragment thereof that recognizes NT-proBNP. The measurement method in this embodiment may also include the above step (3) after step (2A-2).

[0081] In the above step (2), step (2A), step (2A-1), or step (2A-2), an antibody or antibody fragment thereof that recognizes NT-proBNP may be bound to latex particles in an aqueous medium. Methods for binding the antibody or antibody fragment thereof that recognizes NT-proBNP to latex particles include, for example, the binding methods described above.

[0082] The reaction time for step (2), step (2A), step (2A-1) or step (2A-2) is not particularly limited as long as it enables the measurement of NT-proBNP according to the present embodiment, and may be, for example, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more, or 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 9 minutes or less, 7 minutes or less, or 6 minutes or less.

[0083] The above values ​​can be freely combined. For example, the reaction time in step (2), step (2A), step (2A-1), or step (2A-2) may be 10 seconds to 1 hour, 1 minute to 10 minutes, or 3 minutes to 6 minutes.

[0084] The reaction temperature in the above step (2), step (2A), step (2A-1) or step (2A-2) is not particularly limited as long as it is a temperature that enables the measurement of NT-proBNP according to the present embodiment, and may be 0°C or higher, 4°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, or 50°C or lower, 45°C or lower, or 40°C or lower.

[0085] The above values ​​can be freely combined. For example, the temperature at which the mixture is maintained may be 4°C to 50°C, 10°C to 45°C, or 25°C to 40°C. The temperature may also be 37°C.

[0086] The concentration of the latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound in the above step (2), step (2A), step (2A-1) or step (2A-2) is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.003% (w / v) or more, 0.005% (w / v) or more, 0.01% (w / v) or more, 0.011% (w / v) or more, 0.012% (w / v) or more, or 0.013% (w / v) or more. Furthermore, the concentration of latex particles having an antibody or antibody fragment thereof that recognizes NT-proBNP bound thereto in the above step (2), step (2A), step (2A-1) or step (2A-2) may be, for example, 0.1% (w / v) or less, 0.08% (w / v) or less, 0.06% (w / v) or less, 0.05% (w / v) or less, 0.04% (w / v) or less, 0.03% (w / v) or less, 0.025% (w / v) or less, 0.02% (w / v) or less, or 0.015% (w / v) or less.

[0087] The above values ​​can be freely combined. For example, the concentration of latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound in step (2), step (2A), step (2A-1), or step (2A-2) may be 0.001% (w / v) to 0.1% (w / v), 0.003% (w / v) to 0.08% (w / v), 0.005% (w / v) to 0.05% (w / v), or 0.01% (w / v) to 0.02% (w / v).

[0088] In the above step (2A) or step (2A-2), the concentration of latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP means the sum of the concentration of latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP and the concentration of latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP.

[0089] The concentration of the pyridinium derivative in the above step (2), step (2A), step (2A-1) or step (2A-2) is not particularly limited as long as it is a concentration that enables the method for measuring NT-proBNP of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.002% (w / v) or more, 0.003% (w / v) or more, 0.004% (w / v) or more, 0.005% (w / v) or more, 0.006% (w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more, It may be 0.009% (w / v) or more, 0.01% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more, 0.04% (w / v) or more, 0.05% (w / v) or more, 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, 0.4% (w / v) or more, 0.5% (w / v) or more, or 0.6% (w / v) or more. Furthermore, the concentration of the pyridinium derivative in the above step (2), step (2A), step (2A-1) or step (2A-2) may be, for example, 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7% (w / v) or less, 6% (w / v) or less, 5% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, 2% (w / v) or less, 1% (w / v) or less, 0.9% (w / v) or less, 0.8% (w / v) or less, or 0.7% (w / v) or less.

[0090] The above values ​​can be freely combined. For example, the concentration of the pyridinium derivative in step (2), step (2A), step (2A-1), or step (2A-2) may be 0.001% (w / v) to 10% (w / v), 0.005% (w / v) to 5% (w / v), 0.01% (w / v) to 3% (w / v), or 0.1% (w / v) to 2% (w / v). In the above step (2), step (2A), step (2A-1), or step (2A-2), one type of pyridinium derivative may be present, or two or more types of pyridinium derivatives may be present. When two or more types of pyridinium derivatives are present, the above concentration means the total concentration of the two or more types of pyridinium derivatives.

[0091] The above step (2), step (2A), step (2A-1) or step (2A-2) is preferably carried out in an aqueous medium, such as the aqueous medium described above. Furthermore, the above-mentioned salts, sugars, proteins, etc. may be contained in the above step (2), step (2A), step (2A-1) or step (2A-2).

[0092] <Process (3)> Step (3) is a step of optically measuring the aggregation of latex particles accompanying the production of the conjugate of step (2), the conjugate of step (2A), or the conjugate 2 of step (2A-2). Examples of a method for optically measuring aggregation include a method of measuring absorbance, scattered light intensity, or transmitted light intensity using an optical instrument.

[0093] The wavelength for measuring absorbance is usually 340 nm to 1000 nm, preferably 500 nm to 900 nm. The time for measuring the latex agglutination reaction can be measured by measuring the rate of change per unit time over which the latex agglutination reaction is occurring, or by measuring the amount of change over a fixed period of time. For example, when measuring absorbance, the rate of change in absorbance per unit time from 30 seconds to 5 minutes after the start of the latex agglutination reaction can be measured, or by measuring the amount of change in absorbance over a fixed period of time. The reaction temperature is preferably 10 to 50°C, and more preferably 20 to 40°C. The reaction time can be determined appropriately; for example, a general-purpose automatic analyzer can be used to measure a reaction time of 10 to 15 minutes.

[0094] After the above step (3), the concentration of NT-proBNP in the sample can also be determined by carrying out the following steps (4) and (5). (4) performing the above steps (1) to (3) using a sample having a known concentration of NT-proBNP to prepare a calibration curve showing the relationship between the NT-proBNP concentration and the measured value; (5) A step of determining the concentration of NT-proBNP in the sample from the calibration curve prepared in step (4) and the measured value obtained by the measurement in step (3).

[0095] Examples of NT-proBNP of known concentrations include NT-proBNP solutions prepared by diluting commercially available NT-proBNP with PBS. Multiple concentrations of NT-proBNP of known concentrations may also be prepared. Examples of commercially available NT-proBNP include 8NT2 Human recombinant NT-proBNP (manufactured by Hytest).

[0096] The concentration of NT-proBNP in a known concentration of NT-proBNP is not particularly limited as long as it is a concentration that enables measurement of NT-proBNP, and may be, for example, 1 pg / ml or more, 5 pg / ml or more, 10 pg / ml or more, 20 pg / ml or more, 30 pg / ml or more, 40 pg / ml or more, 50 pg / ml or more, 60 pg / ml or more, 70 pg / ml or more, 80 pg / ml or more, 90 pg / ml or more, 100 pg / ml or more, 120 pg / ml or more, 140 pg / ml or more, 160 pg / ml or more, 180 pg / ml or more, 200 pg / ml or more, or 300 pg / ml or more. pg / ml or more, 220 pg / ml or more, 240 pg / ml or more, 260 pg / ml or more, 280 pg / ml or more, 300 pg / ml or more, 320 pg / ml or more, 340 pg / ml or more, 360 pg / ml or more, 380 pg / ml or more, 400 pg / ml or more, 420 pg / ml or more, 440 pg / ml or more, 460 pg / ml or more, 480 pg / ml or more, 500 pg / ml or more, 600 pg / ml or more, 700 pg / ml or more, 800 pg / ml or more, 900 pg / ml or more, or 1000 pg / ml or more. The concentration of NT-proBNP in the known concentration of NT-proBNP is, for example, 150,000 pg / ml or less, 140,000 pg / ml or less, 130,000 pg / ml or less, 120,000 pg / ml or less, 110,000 pg / ml or less, 100,000 pg / ml or less, 90,000 pg / ml or less, 80,000 pg / ml or less, 70,000 pg / ml or less, 60,000 pg / ml or less, 50,000 pg / ml or less, 40,000 pg / ml or less, 30,000 pg / ml or less, 1 or less, 20,000 pg / ml or less, 18,000 pg / ml or less, 16,000 pg / ml or less, 14,000 pg / ml or less, 12,000 pg / ml or less, 11,000 pg / ml or less, 10,000 pg / ml or less, 9,000 pg / ml or less, 8,000 pg / ml or less, 7,000 pg / ml or less, 6,000 pg / ml or less, 5,000 pg / ml or less, 4,000 pg / ml or less, 3,500 pg / ml or less, 3,000 pg / ml or less, or 2,000 pg / ml or less.

[0097] The above values ​​can be freely combined, and may be, for example, 100 pg / ml to 100,000 pg / ml, 100 pg / ml to 50,000 pg / ml, 200 pg / ml to 16,000 pg / ml, 200 pg / ml to 4,000 pg / ml, or 200 pg / ml to 3,500 pg / ml.

[0098] [Method for suppressing non-specific reactions caused by impurities in biological samples] By carrying out the method for measuring NT-proBNP in a sample according to the present embodiment, non-specific reactions caused by impurities in the biological sample can be suppressed. The non-specific reactions are not particularly limited, and examples thereof include the non-specific reactions described above. Examples of the method for suppressing non-specific reactions caused by impurities in a biological sample include the following.

[0099] (Aspect 1) A method for suppressing non-specific reactions caused by impurities in a biological sample in a latex agglutination immunoassay method for NT-proBNP in a sample, in which NT-proBNP in the sample is reacted with latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP in an aqueous medium in the presence of a pyridinium derivative.

[0100] (Aspect 2) (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting NT-proBNP in a sample with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium; a method for suppressing non-specific reactions caused by impurities in a biological sample in a latex agglutination immunoassay method for NT-proBNP in a sample, the method comprising the steps (1) and / or (2) being carried out in the presence of a pyridinium derivative.

[0101] (Aspect 3) (1) mixing a sample containing NT-proBNP with an aqueous medium; and (2) reacting, in an aqueous medium, NT-proBNP in the sample with latex particles bound to a first antibody or an antibody fragment thereof that recognizes NT-proBNP and latex particles bound to a second antibody or an antibody fragment thereof that recognizes NT-proBNP; a method for suppressing non-specific reactions caused by impurities in a biological sample in a latex agglutination immunoassay method for NT-proBNP in a sample, the method comprising the steps (1) and / or (2) being carried out in the presence of a pyridinium derivative.

[0102] In the above-mentioned suppression method, the latex particles having an antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a first antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a second antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, and the pyridinium derivative may be, for example, the above-mentioned latex particles having an antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a first antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a second antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, and the pyridinium derivative. Furthermore, the concentration of each additive, reaction temperature, reaction time, etc. in each step in the above-mentioned inhibition method may be, for example, the concentration of each additive, reaction temperature, reaction time, etc. in each step in the aforementioned latex agglutination immunoassay method for NT-proBNP in a sample.

[0103] [Reagent for measuring NT-proBNP in samples] The reagent for measuring NT-proBNP in a sample of this embodiment is a reagent used in the method for measuring NT-proBNP in a sample of this embodiment. One aspect (Aspect 1) of the measuring reagent is a reagent for measuring NT-proBNP in a sample, which comprises latex particles, an antibody or antibody fragment thereof that recognizes NT-proBNP, and a pyridinium derivative. In embodiment 1, the latex particles and the antibody or antibody fragment thereof that recognizes NT-proBNP can be bound via two affinity substances. Methods for binding the antibody or antibody fragment thereof that recognizes NT-proBNP to the latex particles include, for example, the binding methods described above.

[0104] Another embodiment (embodiment 2) of the measuring reagent is a reagent for measuring NT-proBNP in a sample, which comprises latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound, and a pyridinium derivative. Another aspect (Aspect 3) of the measurement reagent is a reagent for measuring NT-proBNP in a sample, which comprises latex particles bound with a first antibody or its antibody fragment that recognizes NT-proBNP, latex particles bound with a second antibody or its antibody fragment that recognizes NT-proBNP, and a pyridinium derivative.

[0105] In the reagents of Aspects 1 to 3, the latex particles, the latex particles having an antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a first antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a second antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, and the pyridinium derivatives may be, for example, the above-mentioned latex particles, the latex particles having an antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a first antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, the latex particles having a second antibody that recognizes NT-proBNP or an antibody fragment thereof bound thereto, and the pyridinium derivatives.

[0106] The first antibody or antibody fragment thereof that recognizes NT-proBNP, or the second antibody or antibody fragment thereof that recognizes NT-proBNP, may be bound to latex particles directly or indirectly using the aforementioned physical adsorption and / or chemical binding. Examples of indirect binding methods include a method in which the first antibody or the second antibody or antibody fragment thereof that recognizes NT-proBNP is bound to latex particles using the specific binding of the aforementioned pair of affinity substances, or a method in which the first antibody or the second antibody or antibody fragment thereof that recognizes NT-proBNP is bound to latex particles by covalent binding via a linker.

[0107] When a set of affinity substances is used, latex particles to which the first antibody or its antibody fragment that recognizes NT-proBNP is bound, or latex particles to which the second antibody or its antibody fragment that recognizes NT-proBNP is bound, can be produced by binding a first antibody or its antibody fragment that recognizes NT-proBNP bound to one of the affinity substances (A) of the set, or a second antibody or its antibody fragment that recognizes NT-proBNP bound to one of the affinity substances (A), to latex particles to which the other affinity substance (a) of the set is bound. Examples of combinations of Aa include the combinations mentioned above. Examples of the linker include the molecules described above.

[0108] The concentration of latex particles in the reagent of Aspect 1 is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.003% (w / v) or more, 0.005% (w / v) or more, 0.008% (w / v) or more, 0.01% (w / v) or more, 0.015% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more and 0.04% (w / v) or more, or 0.05% (w / v) or more. Furthermore, the concentration of latex particles in the reagent of Aspect 1 may be, for example, 1% (w / v) or less, 0.8% (w / v) or less, 0.5% (w / v) or less, 0.3% (w / v) or less, 0.2% (w / v) or less, 0.1% (w / v) or less, 0.09% (w / v) or less, 0.08% (w / v) or less, 0.07% (w / v) or less, or 0.06% (w / v) or less.

[0109] The above values ​​can be freely combined. For example, the concentration of latex particles in the reagent of embodiment 1 may be 0.001% (w / v) to 1% (w / v), 0.003% (w / v) to 0.5% (w / v), 0.01% (w / v) to 0.1% (w / v), or 0.02% (w / v) to 0.08% (w / v).

[0110] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the reagent of Aspect 2 or Aspect 3 is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.003% (w / v) or more, 0.005% (w / v) or more, 0.008% (w / v) or more, 0.01% (w / v) or more, 0.015% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more and 0.04% (w / v) or more, or 0.05% (w / v) or more. Furthermore, the concentration of latex particles having bound thereto an antibody or antibody fragment thereof that recognizes NT-proBNP in the reagent of Aspect 2 or Aspect 3 may be, for example, 1% (w / v) or less, 0.8% (w / v) or less, 0.5% (w / v) or less, 0.3% (w / v) or less, 0.2% (w / v) or less, 0.1% (w / v) or less, 0.09% (w / v) or less, 0.08% (w / v) or less, 0.07% (w / v) or less, or 0.06% (w / v) or less.

[0111] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the reagent of Aspect 2 or Aspect 3 may be 0.001% (w / v) to 1% (w / v), 0.003% (w / v) to 0.5% (w / v), 0.01% (w / v) to 0.1% (w / v), or 0.02% (w / v) to 0.08% (w / v). In the reagent of Aspect 3, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP means the sum of the concentration of latex particles bound to a first antibody or antibody fragment thereof that recognizes NT-proBNP and the concentration of latex particles bound to a second antibody or antibody fragment thereof that recognizes NT-proBNP.

[0112] The concentration of the pyridinium derivative in the reagent for measuring NT-proBNP of this embodiment is not particularly limited as long as it is a concentration that enables the method for measuring NT-proBNP of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.002% (w / v) or more, 0.003% (w / v) or more, 0.004% (w / v) or more, 0.005% (w / v) or more, 0.006% (w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more, 0.009% (w / v) or more, or The concentration may be 0.01% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more, 0.04% (w / v) or more, 0.05% (w / v) or more, 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, 0.4% (w / v) or more, 0.5% (w / v) or more, or 0.6% (w / v) or more. Furthermore, the concentration of the pyridinium derivative in the NT-proBNP measurement reagent of this embodiment may be, for example, 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7% (w / v) or less, 6% (w / v) or less, 5% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, 2% (w / v) or less, 1% (w / v) or less, 0.9% (w / v) or less, 0.8% (w / v) or less, or 0.7% (w / v) or less.

[0113] The above values ​​can be freely combined. For example, the concentration of the pyridinium derivative in the reagent for measuring NT-proBNP of this embodiment may be 0.001% (w / v) to 10% (w / v), 0.005% (w / v) to 5% (w / v), 0.01% (w / v) to 3% (w / v), or 0.1% (w / v) to 2% (w / v). In the measurement reagent, one type of pyridinium derivative may be present, or two or more types of pyridinium derivatives may be present. When two or more types of pyridinium derivatives are present, the concentration refers to the total concentration of the two or more types of pyridinium derivatives.

[0114] The measurement reagent of this embodiment may be in a freeze-dried state or in a liquid state. When a freeze-dried measurement reagent is used, it is dissolved in an aqueous medium before measurement to form a liquid and then used for measurement. The aqueous medium used for dissolution is not particularly limited, and examples thereof include the aqueous media described above. The aqueous medium may contain the salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. described above. In the liquid test reagent, at least one selected from the group consisting of latex particles, a first antibody or antibody fragment thereof that recognizes NT-proBNP, and a second antibody or antibody fragment thereof that recognizes NT-proBNP is mixed in an aqueous medium. The aqueous medium is not particularly limited, and examples thereof include the aqueous media described above. The aqueous medium may contain the salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. described above.

[0115] [NT-proBNP measurement kit in samples] The reagent for measuring NT-proBNP in a sample of this embodiment can also be in the form of a kit from the viewpoints of storage, transportation, distribution, etc. Below, preferred embodiments of the kit for measuring NT-proBNP in a sample of this embodiment are exemplified.In the following embodiments, the latex particles to which the antibody that recognizes NT-proBNP or its antibody fragment is bound, the latex particles to which the first antibody that recognizes NT-proBNP or its antibody fragment is bound, the latex particles to which the second antibody that recognizes NT-proBNP or its antibody fragment is bound, and the pyridinium derivative can be, for example, the latex particles to which the antibody that recognizes NT-proBNP or its antibody fragment is bound, the latex particles to which the first antibody that recognizes NT-proBNP or its antibody fragment is bound, the latex particles to which the second antibody that recognizes NT-proBNP or its antibody fragment is bound, and the pyridinium derivative.

[0116] Measurement kit (1) Reagent 1A, comprising a pyridinium derivative; and Reagent 2A containing latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound. Including, a measurement kit.

[0117] Measurement kit (2) Reagent 1B comprising an aqueous medium; and Reagent 2B comprising a pyridinium derivative and latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound. Including, a measurement kit.

[0118] Measurement kit (3) Class 1C reagents containing pyridinium derivatives; a second C reagent comprising latex particles to which a first antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; and a third C reagent containing latex particles to which a second antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; Including, a measurement kit.

[0119] Measurement kit (4) a first D reagent comprising an aqueous medium; a second D reagent comprising a pyridinium derivative and latex particles to which a first antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; and a third-dimensional reagent comprising latex particles to which a second antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; Including, a measurement kit.

[0120] Measurement kit (5) Reagent 1E, comprising an aqueous medium; a second E reagent comprising latex particles to which a first antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; and A third E reagent comprising a pyridinium derivative and latex particles to which a second antibody or its antibody fragment that recognizes NT-proBNP is bound. Including, a measurement kit.

[0121] Measurement kit(6) 1F reagents containing pyridinium derivatives; and a second F reagent comprising latex particles to which a first antibody or an antibody fragment thereof that recognizes NT-proBNP is bound, and latex particles to which a second antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; Including, a measurement kit.

[0122] Measurement kit(7) 1G reagent containing an aqueous medium; A second G reagent comprising a pyridinium derivative, latex particles to which a first antibody or an antibody fragment thereof that recognizes NT-proBNP is bound, and latex particles to which a second antibody or an antibody fragment thereof that recognizes NT-proBNP is bound. Including, a measurement kit.

[0123] The first antibody or antibody fragment thereof that recognizes NT-proBNP, or the second antibody or antibody fragment thereof that recognizes NT-proBNP, may be bound to latex particles directly or indirectly using the aforementioned physical adsorption and / or chemical binding. Examples of indirect binding methods include a method in which the first antibody or antibody fragment thereof that recognizes NT-proBNP, or the second antibody or antibody fragment thereof that recognizes NT-proBNP, is bound to latex particles using the specific binding of the aforementioned pair of affinity substances, or a method in which the first antibody or antibody fragment thereof that recognizes NT-proBNP, or the second antibody or antibody fragment thereof is bound to latex particles by covalent binding via a linker.

[0124] When a set of affinity substances is used, latex particles to which the first antibody or antibody fragment thereof that recognizes NT-proBNP is bound, or latex particles to which the second antibody or antibody fragment thereof that recognizes NT-proBNP is bound, can be produced by binding a first antibody or antibody fragment thereof that recognizes NT-proBNP bound to one of the affinity substances (A) of the set, or a second antibody or antibody fragment thereof that recognizes NT-proBNP bound to one of the affinity substances (A), to latex particles to which the other affinity substance (a) of the set is bound. Examples of combinations of Aa include the combinations mentioned above. Examples of the linker include the molecules described above.

[0125] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in Reagent 2A or Reagent 2B is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.003% (w / v) or more, 0.005% (w / v) or more, 0.008% (w / v) or more, 0.01% (w / v) or more, 0.015% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more and 0.04% (w / v) or more, or 0.05% (w / v) or more. Furthermore, the concentration of latex particles having bound thereto an antibody or antibody fragment thereof that recognizes NT-proBNP in the second A reagent or the second B reagent may be, for example, 1% (w / v) or less, 0.8% (w / v) or less, 0.5% (w / v) or less, 0.3% (w / v) or less, 0.2% (w / v) or less, 0.1% (w / v) or less, 0.09% (w / v) or less, 0.08% (w / v) or less, 0.07% (w / v) or less, or 0.06% (w / v) or less.

[0126] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the second A reagent or the second B reagent may be 0.001% (w / v) to 1% (w / v), 0.003% (w / v) to 0.5% (w / v), 0.01% (w / v) to 0.1% (w / v), or 0.02% (w / v) to 0.08% (w / v).

[0127] The concentration of latex particles having an antibody or antibody fragment thereof that recognizes NT-proBNP bound thereto in the 2nd C reagent, the 3rd C reagent, the 2nd D reagent, the 3rd D reagent, the 2nd E reagent, the 3rd E reagent, the 2nd F reagent, or the 2nd G reagent is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.0005% (w / v) or more, 0.001% (w / v) or more, 0.002% (w / v) or more, 0.003% (w / v) or more, 0.005% (w / v) or more, 0.008% (w / v) or more, 0.01% (w / v) or more, 0.015% (w / v) or more, 0.02% (w / v) or more, or 0.025% (w / v) or more. Furthermore, the concentration of latex particles having bound thereto an antibody or antibody fragment thereof that recognizes NT-proBNP in the reagent may be, for example, 1% (w / v) or less, 0.8% (w / v) or less, 0.5% (w / v) or less, 0.3% (w / v) or less, 0.2% (w / v) or less, 0.1% (w / v) or less, 0.09% (w / v) or less, 0.08% (w / v) or less, 0.07% (w / v) or less, 0.06% (w / v) or less, 0.05% (w / v) or less, 0.04% (w / v) or less, or 0.03% (w / v) or less.

[0128] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the reagent may be 0.0005% (w / v) to 0.5% (w / v), 0.002% (w / v) to 0.3% (w / v), 0.005% (w / v) to 0.05% (w / v), or 0.01% (w / v) to 0.04% (w / v). In the second F reagent or the second G reagent, the concentration of latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP means the sum of the concentration of latex particles bound to a first antibody or its antibody fragment that recognizes NT-proBNP and the concentration of latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP.

[0129] The concentration of the pyridinium derivative in the 1A reagent, the 2B reagent, the 1C reagent, the 2D reagent, the 3E reagent, the 1F reagent, or the 2G reagent is not particularly limited as long as it is a concentration that enables the NT-proBNP measurement method of this embodiment, and may be, for example, 0.001% (w / v) or more, 0.002% (w / v) or more, 0.003% (w / v) or more, 0.004% (w / v) or more, 0.005% (w / v) or more, 0.006% (w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more. ) or more, 0.009% (w / v) or more, 0.01% (w / v) or more, 0.02% (w / v) or more, 0.03% (w / v) or more, 0.04% (w / v) or more, 0.05% (w / v) or more, 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, 0.4% (w / v) or more, 0.5% (w / v) or more, or 0.6% (w / v) or more. Furthermore, the concentration of the pyridinium derivative in the reagent may be, for example, 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7% (w / v) or less, 6% (w / v) or less, 5% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, 2% (w / v) or less, 1% (w / v) or less, 0.9% (w / v) or less, 0.8% (w / v) or less, or 0.7% (w / v) or less.

[0130] The above values ​​can be freely combined. For example, the concentration of the pyridinium derivative in the reagent may be 0.001% (w / v) to 10% (w / v), 0.005% (w / v) to 5% (w / v), 0.01% (w / v) to 3% (w / v), or 0.1% (w / v) to 2% (w / v). In the above reagent, one type of pyridinium derivative may be present, or two or more types of pyridinium derivatives may be present. When two or more types of pyridinium derivatives are present, the above concentration means the total concentration of the two or more types of pyridinium derivatives.

[0131] The constituent reagents of the measurement kit of this embodiment may be in a lyophilized state or in a liquid state. When the reagents constituting the assay kit are in a lyophilized state, they are dissolved in an aqueous medium prior to assay to form a liquid. Examples of the aqueous medium include those mentioned above. The aqueous medium may contain the aforementioned salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc.

[0132] When the constituent reagents of the assay kit are in liquid form, at least one selected from the group consisting of a pyridinium derivative, a first antibody or antibody fragment thereof that recognizes NT-proBNP, and a second antibody or antibody fragment thereof that recognizes NT-proBNP is dissolved or mixed in an aqueous medium. Examples of the aqueous medium include the aqueous media described above. The aqueous medium may contain the salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. described above.

[0133] The above-mentioned measurement kits (1) to (7) may contain a sample dilution reagent containing an aqueous medium as a reagent for diluting the sample. Examples of the aqueous medium include the above-mentioned aqueous media. The aqueous medium may contain the above-mentioned salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. Furthermore, the above-mentioned measurement kits (1) to (7) may also contain the above-mentioned washing solution, a standard substance reagent containing NT-proBNP of a known concentration as a standard substance, and an instruction manual describing the measurement method of this embodiment.

[0134] Examples of NT-proBNP of known concentrations include NT-proBNP solutions prepared by diluting commercially available NT-proBNP with PBS. Multiple concentrations of NT-proBNP of known concentrations may also be prepared. Examples of commercially available NT-proBNP include 8NT2 Human recombinant NT-proBNP (manufactured by Hytest). In the case of NT-proBNP of known concentration as a standard substance, the concentration of NT-proBNP may be, for example, the above-mentioned concentrations. [Example]

[0135] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. In the following examples, reagents from the following manufacturers were used.

[0136] Carboxyl-modified polystyrene latex (average particle size 400 nm) (Fujikura Chemical Co., Ltd.), N,N-bis(2-hydroxyethyl)glycine (Bicine) (Dojindo Laboratories), Sulfo-NHS (Thermo Fisher Scientific), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific), BRJNBNPS102 anti-NT-proBNP antibody (primary antibody) (Fapon Biotech), BECBNPS108 anti-NT-proBNP antibody (secondary antibody) (Fapon Biotech), NT-proBNP measurement kit "Eclusis (registered trademark) Reagent NT-proBNP II" (Roche Diagnostics), Otsuka saline injection (physiological saline) (Otsuka Pharmaceutical Factory), 8NT2 Human recombinant The following antibodies were used: NT-proBNP (recombinant NT-proBNP) (Hytest), disodium hydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.), sodium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.), bovine serum albumin (BSA) (Bovogen), 2-morpholinoethanesulfonic acid monohydrate (MES) (Dojindo Laboratories, Inc.), sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), Lipidure-BL103 (NOF Corporation), 1-methylpyridinium chloride (Tokyo Chemical Industry Co., Ltd.), sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), and IIC-IGM-G4 IIC Goat anti-Human IgM, μ chain specific, Antibody (anti-IgM antibody) (Nittobo America, Inc.).

[0137] [Experimental Example 1] Preparation of antibody-bound latex particle solution Sulfo-NHS and EDC were added to 50 mmol / L aqueous bicine solution (pH 9.0) containing 0.5% (w / v) carboxyl-modified polystyrene latex according to the manufacturer's recommended method, and the carboxyl groups on the surface of the latex were reacted with the sulfo-NHS and EDC. The primary antibody was then added to the reacted solution at a concentration of 0.5 mg / mL and stirred at room temperature for 1.5 hours to bind the primary antibody to the latex. The antibody-sensitized solution was then centrifuged at 15,000 g for 20 minutes, and the supernatant was removed to recover the precipitate (first run). The recovered precipitate (first run) was then added to 50 mmol / L aqueous bicine solution (pH 9.0) and ultrasonically dispersed, followed by centrifugation at 15,000 g for 20 minutes. The supernatant was removed and the precipitate (second run) was recovered. Next, 50 mmol / L aqueous bicine solution (pH 9.0) was added to the precipitate (second time) to make the latex concentration 0.5% (w / v), and the mixture was ultrasonically dispersed to obtain a first antibody-bound latex particle solution. The process from adding the aqueous bicine solution to collecting the centrifugal precipitate was repeated twice to thoroughly remove unbound first antibody that had not bound to the latex. Next, a solution of second antibody-bound latex particles was prepared in the same manner except that the second antibody was used instead of the first antibody.

[0138] [Experimental Example 2] Preparation of specimens for evaluation Whole blood collected from three healthy individuals was centrifuged at 800 g for 20 minutes to recover the supernatant, and three human serum samples were prepared. The three human serum samples were measured using the NT-proBNP measurement kit "Eclusis (registered trademark) Reagent NT-proBNP II." The NT-proBNP concentrations of the three human serum samples were 20.5 pg / mL, 46.0 pg / mL, and 20.6 pg / mL, respectively. The three human serum samples were then diluted 10-fold with saline, and 500 pg of recombinant NT-proBNP was added per mL of diluted serum to create evaluation samples 1, 2, and 3, respectively. Therefore, the theoretical NT-proBNP concentrations of evaluation samples 1, 2, and 3 were 502 pg / mL, 505 pg / mL, and 502 pg / mL, respectively.

[0139] [Experimental Example 3] Preparation of NT-proBNP Standards 1 to 5 An NT-proBNP standard having the following composition was prepared. 50mmol / L phosphate buffer (pH6.0) (Prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate) BSA 75g / L Recombinant NT-proBNP (contents listed below) A total of five NT-proBNP standards with recombinant NT-proBNP contents of 0 pg / mL (no additives), 300 pg / mL, 900 pg / mL, 1500 pg / mL, and 2000 pg / mL were prepared and designated NT-proBNP standards 1 to 5.

[0140] [Example 1] Preparation of NT-proBNP measurement kit A measurement kit A containing a first reagent and a second reagent having the following compositions was prepared. (First Reagent) MES 100mmol / L BSA 5g / L Sodium chloride 200mmol / L Lipidure-BL103 0.7% (w / v) 1-Methylpyridinium chloride 1.0% (w / v) The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Second reagent) 10mmol / L Bicine aqueous solution (pH8.0) 82%(v / v) First antibody-bound latex particle solution 9% (v / v) Secondary antibody-conjugated latex particle solution 9% (v / v)

[0141] [Comparative Example 1] Preparation of NT-proBNP measurement kit A measurement kit a containing a first reagent and a second reagent having the following compositions was prepared. (First Reagent) MES 100mmol / L BSA 5g / L Sodium chloride 200mmol / L Lipidure-BL103 0.7% (w / v) The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Second reagent) 10mmol / L Bicine aqueous solution (pH8.0) 82%(v / v) First antibody-bound latex particle solution 9% (v / v) Secondary antibody-conjugated latex particle solution 9% (v / v) Measurement kit a of Comparative Example 1 is the same as measurement kit A of Example 1, except that the first reagent does not contain 1-methylpyridinium chloride.

[0142] [Example 2] Calculation of non-specific reaction rate in NT-proBNP measurement Using an automatic analyzer 3500 (manufactured by Hitachi High-Technologies Corporation) and measurement kit A, evaluation samples 1, 2, and 3 were measured according to the following procedure, and the non-specific reaction rate was calculated. (1) Preparation of a calibration curve for NT-proBNP measurement The measurement kit used was the measurement kit A of Example 1, and the samples used were NT-proBNP standards 1 to 5 of Experimental Example 3 (five standards with NT-proBNP contents of 0 pg / mL, 300 pg / mL, 900 pg / mL, 1500 pg / mL, and 2000 pg / mL, respectively). The absorbance of the NT-proBNP standards was measured according to the following procedure. Standard 1 (10 μL) and the first reagent (90 μL) of the measurement kit A of Example 1 were added to a reaction cell, and the reaction was allowed to proceed at 37°C for 5 minutes. The absorbance (E1) of the reaction solution was measured at a main wavelength of 600 nm without a secondary wavelength. Next, the second reagent (30 μL) of the kit A of Example 1 was added to the reaction solution, and the reaction was allowed to proceed for a further 5 minutes at 37°C. The absorbance (E2) of the reaction solution was measured at a main wavelength of 600 nm without a secondary wavelength. E1 was subtracted from E2 to obtain the absorbance difference ΔE 標準品1 Next, the absorbance difference ΔE was calculated in the same manner except that Standards 2 to 5 were used instead of Standard 1. 標準品2 , absorbance difference ΔE 標準品3 , absorbance difference ΔE 標準品4 , and absorbance difference ΔE 標準品5 The calculated absorbance difference ΔE 標準品1 , absorbance difference ΔE 標準品2 , absorbance difference ΔE 標準品3 , absorbance difference ΔE 標準品4 , and absorbance difference ΔE 標準品5 Using this, a calibration curve showing the relationship between NT-proBNP concentration (pg / mL) and absorbance was created according to the spline calibration curve setting method preset in the automatic analyzer 3500.

[0143] (2) Determination of NT-proBNP concentration measurements in evaluation samples 1, 2, and 3 Using the measurement kit A of Example 1 and the evaluation specimens 1, 2, and 3 prepared in Experimental Example 2 as samples, the absorbance difference relative to the NT-proBNP concentration was calculated for the evaluation specimens 1, 2, and 3 in the same manner as in (1). The measured NT-proBNP concentration values ​​(pg / mL) for the evaluation specimens 1, 2, and 3 were determined from the calculated absorbance difference and the calibration curve of (1).

[0144] (3) Evaluation of non-specific reaction rate The non-specific reaction rate for measurement kit A of Example 1 was calculated using the following formula (I) from the theoretical values ​​of NT-proBNP concentrations in evaluation specimens 1, 2, and 3 (502 pg / mL, 505 pg / mL, and 502 pg / mL, respectively, as described in Experimental Example 2) and the measured NT-proBNP concentrations in evaluation specimens 1, 2, and 3 determined in (2) above. The results are shown in Table 2.

[0145]

number

[0146] [Comparative Example 2] Calculation of non-specific reaction rate in NT-proBNP measurement The non-specific reaction rate for measurement kit a was calculated in the same manner as in Example 2, except that measurement kit a of Comparative Example 1 was used instead of measurement kit A of Example 1. The results are shown in Table 2.

[0147] [Table 2]

[0148] Although it is desirable for the measured NT-proBNP concentration to be the same as the theoretical value of NT-proBNP concentration, in reality, the measured NT-proBNP concentration will be higher than the theoretical value due to the occurrence of non-specific reactions, i.e., agglutination reactions of latex particles not derived from NT-proBNP in the sample. Furthermore, the closer the measured NT-proBNP concentration is to the theoretical value of NT-proBNP concentration, the lower the non-specific reaction rate, and the lower the non-specific reaction rate, the more accurately NT-proBNP can be measured. As is clear from Table 2, the non-specific reaction rate (Example 2) when using the measurement kit A of Example 1 was lower than the non-specific reaction rate (Comparative Example 2) when using the measurement kit a of Comparative Example 1. Therefore, it was revealed that when using a measurement kit containing a pyridinium derivative, the non-specific reaction rate is lower than when using a measurement kit not containing a pyridinium derivative, and NT-proBNP can be measured more accurately.

[0149] [Example 3] Preparation of NT-proBNP measurement kit A measurement kit B containing a first reagent and a second reagent having the following compositions was prepared. (First Reagent) MES 100mmol / L BSA 5g / L Sodium chloride 200mmol / L Lipidure-BL103 0.7% (w / v) 1-Methylpyridinium chloride 1.0% (w / v) Anti-IgM antibody 0.0375%(w / v) The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Second reagent) 10mmol / L Bicine aqueous solution (pH8.0) 82%(v / v) First antibody-bound latex particle solution 9% (v / v) Secondary antibody-conjugated latex particle solution 9% (v / v) Measurement kit B of Example 3 is the same as measurement kit A of Example 1, except that anti-IgM antibody is added to the first reagent.

[0150] [Comparative Example 3] Preparation of NT-proBNP measurement kit A measurement kit b containing a first reagent and a second reagent having the following composition was prepared. (First Reagent) MES 100mmol / L BSA 5g / L Sodium chloride 200mmol / L Lipidure-BL103 0.7% (w / v) Anti-IgM antibody 0.0375%(w / v) The pH was adjusted to 6.0 with an appropriate amount of sodium hydroxide. (Second reagent) 10mmol / L Bicine aqueous solution (pH8.0) 82%(v / v) First antibody-bound latex particle solution 9% (v / v) Secondary antibody-conjugated latex particle solution 9% (v / v) Measurement kit b of Comparative Example 3 is the same as measurement kit B of Example 3, except that the first reagent does not contain 1-methylpyridinium chloride. Furthermore, measurement kit b of Comparative Example 3 is the same as measurement kit a of Comparative Example 1, except that anti-IgM antibody is added to the first reagent.

[0151] [Example 4] Calculation of non-specific reaction rate in NT-proBNP measurement The non-specific reaction rate for measurement kit B was calculated in the same manner as in Example 2, except that measurement kit B of Example 3 was used instead of measurement kit A of Example 1. The results are shown in Table 3.

[0152] [Comparative Example 4] Calculation of non-specific reaction rate in NT-proBNP measurement The non-specific reaction rate for measurement kit b of Comparative Example 3 was calculated using the same procedure as in Example 2, except that measurement kit b of Comparative Example 3 was used instead of measurement kit A of Example 1. The results are shown in Table 3. For comparison, the non-specific reaction rate for Comparative Example 2, which did not contain 1-methylpyridinium chloride and anti-IgM antibody, and the non-specific reaction rate for Example 2, which contained 1-methylpyridinium chloride but did not contain anti-IgM antibody, are also shown.

[0153] [Table 3]

[0154] As is clear from Table 3, the non-specific reaction rate when using the measurement kit B of Example 3 (Example 4) was lower than the non-specific reaction rate when using the measurement kit a of Comparative Example 1 (Comparative Example 2) and the measurement kit b of Comparative Example 3 (Comparative Example 4). Therefore, it was revealed that the non-specific reaction rate was lower when using a measurement kit containing both a pyridinium derivative and an anti-IgM antibody than when using a measurement kit that did not contain both a pyridinium derivative and an anti-IgM antibody. Furthermore, it was revealed that the non-specific reaction rate was lower when using a measurement kit containing both a pyridinium derivative and an anti-IgM antibody than when using a measurement kit that did not contain a pyridinium derivative but contained an anti-IgM antibody, and NT-proBNP could be measured more accurately. Furthermore, as shown in Table 3, the measurement kit containing anti-IgM antibody but not pyridinium derivative (measurement kit b) sometimes had a high non-specific reaction rate for some samples, such as evaluation sample 2, but the measurement kits containing pyridinium derivative (measurement kit A and measurement kit B) were found to have a low non-specific reaction rate for all evaluation samples, regardless of the presence or absence of anti-IgM antibody. Furthermore, when using a measurement kit containing both a pyridinium derivative and anti-IgM antibody, the non-specific reaction rate was reduced by 35% for evaluation sample 1, but when using a measurement kit containing only a pyridinium derivative (measurement kit A), the non-specific reaction rate was reduced by 7%, and when using a measurement kit containing only anti-IgM antibody (measurement kit b), the non-specific reaction rate was reduced by only 13%. In other words, it was revealed that a measurement kit containing both a pyridinium derivative and an anti-IgM antibody has a synergistically lower non-specific reaction rate than a measurement kit containing either a pyridinium derivative or an anti-IgM antibody alone, and can measure NT-proBNP more accurately. [Industrial Applicability]

[0155] The present invention provides a method, reagent and kit for measuring NT-proBNP in a sample, which are effective for diagnosing and monitoring heart failure and enable highly sensitive and accurate measurement.

Claims

1. A latex agglutination immunoassay method for N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) in a sample, in which N-terminal pro-brain natriuretic peptide (NT-proBNP) in the sample is reacted with latex particles bound to an antibody or an antibody fragment thereof that recognizes NT-proBNP in an aqueous medium in the presence of a pyridinium derivative.

2. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) reacting NT-proBNP in a sample with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium; wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative.

3. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) reacting NT-proBNP in a sample with latex particles bound to a first antibody or an antibody fragment thereof that recognizes NT-proBNP, and with latex particles bound to a second antibody or an antibody fragment thereof that recognizes NT-proBNP, in an aqueous medium; wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative.

4. The method according to any one of claims 1 to 3, wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

5. The method according to any one of claims 1 to 3, wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

6. The method according to any one of claims 1 to 3, wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I): 【Chemistry 1】 (In the formula, R １ represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X － represents a monovalent anion)

7. The method according to any one of claims 1 to 3, wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group having 1 to 4 carbon atoms or a salt thereof.

8. The method according to any one of claims 1 to 3, wherein the pyridinium derivative is methylpyridinium or a salt thereof.

9. A method for suppressing non-specific reactions caused by impurities in a biological sample in a latex agglutination immunoassay of NT-proBNP in a sample, in which N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) in a sample is reacted with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium in the presence of a pyridinium derivative.

10. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) reacting NT-proBNP in a sample with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium; wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative.

11. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) reacting NT-proBNP in a sample with latex particles bound to a first antibody or an antibody fragment thereof that recognizes NT-proBNP, and with latex particles bound to a second antibody or an antibody fragment thereof that recognizes NT-proBNP, in an aqueous medium; wherein steps (1) and / or (2) are carried out in the presence of a pyridinium derivative.

12. The method according to any one of claims 9 to 11, wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

13. The method according to any one of claims 9 to 11, wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

14. The method according to any one of claims 9 to 11, wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I): 【Chemistry 2】 (In the formula, R １ represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X － represents a monovalent anion)

15. The method according to any one of claims 9 to 11, wherein the pyridinium derivative is a linear alkylpyridinium having 1 to 4 alkyl carbon atoms or a salt thereof.

16. The method according to any one of claims 9 to 11, wherein the pyridinium derivative is methylpyridinium or a salt thereof.

17. A latex agglutination immunoassay reagent for N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) in a sample, comprising latex particles bound with an antibody or antibody fragment thereof that recognizes NT-proBNP, and a pyridinium derivative.

18. A latex agglutination immunoassay reagent for measuring N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) in a sample, comprising latex particles bound with a first antibody or an antibody fragment thereof that recognizes NT-proBNP, latex particles bound with a second antibody or an antibody fragment thereof that recognizes NT-proBNP, and a pyridinium derivative.

19. The reagent according to claim 17 or 18, wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

20. The reagent according to claim 17 or 18, wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

21. The reagent according to claim 17 or 18, wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I): 【Transformation 3】 (In the formula, R １ represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X － represents a monovalent anion)

22. The reagent according to claim 17 or 18, wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group having 1 to 4 carbon atoms or a salt thereof.

23. 19. The reagent according to claim 17 or 18, wherein the pyridinium derivative is methylpyridinium or a salt thereof.

24. A latex agglutination immunoassay kit for measuring N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) in a sample, comprising: a first reagent containing an aqueous medium; and a second reagent containing latex particles bound to a first antibody or an antibody fragment thereof that recognizes NT-proBNP, and latex particles bound to a second antibody or an antibody fragment thereof that recognizes NT-proBNP, wherein the first reagent and / or the second reagent contain a pyridinium derivative.

25. The kit according to claim 24, wherein the concentration of the pyridinium derivative is 0.001% (w / v) to 10% (w / v).

26. The kit according to claim 24, wherein the concentration of the pyridinium derivative is 0.01% (w / v) to 3% (w / v).

27. The kit according to any one of claims 24 to 26, wherein the pyridinium derivative is a pyridinium salt represented by the following formula (I): 【Chemistry 4】 (In the formula, R １ represents a substituted or unsubstituted alkyl or a substituted or unsubstituted alkenyl; X － represents a monovalent anion)

28. The kit according to any one of claims 24 to 26, wherein the pyridinium derivative is a linear alkylpyridinium having an alkyl group of 1 to 4 carbon atoms or a salt thereof.

29. The kit according to any one of claims 24 to 26, wherein the pyridinium derivative is methylpyridinium or a salt thereof.

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