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: A latex agglutination immunoassay for N-terminal pro-brain natriuretic peptide (NT-proBNP) in a sample, which comprises reacting NT-proBNP in the sample with latex particles to which antibodies or fragments thereof that recognize NT-proBNP are bound, wherein the reaction is carried out in an aqueous solvent having a pH of pH6. 0 or less.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 conducted by the present inventors to solve the above-mentioned problems, they discovered that a latex agglutination immunoassay method in which NT-proBNP in a sample is reacted in an aqueous medium with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP, wherein the reaction is carried out in an aqueous medium at a pH of 6.0 or less, enables highly sensitive and accurate measurement of NT-proBNP, and 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 in an aqueous medium with latex particles to which an antibody or its antibody fragment that recognizes NT-proBNP is bound, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less. [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 bound to an antibody or antibody fragment thereof that recognizes NT-proBNP, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less. [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 antibody fragment thereof that recognizes NT-proBNP, and latex particles bound to a second antibody or antibody fragment thereof that recognizes NT-proBNP, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less. [4] The method according to any one of [1] to [3], wherein the aqueous medium having a pH of 6.0 or less is an aqueous medium having a pH of 5.0 to 6.0. [5] The method according to any one of [1] to [4], wherein the aqueous medium contains a sensitizer. [6] The method according to [5], wherein the sensitizer is a hydrophilic polymer. [7] The method according to [5] or [6], wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v). [8] The method according to any one of [1] to [7], wherein the sample contains a rheumatoid factor. [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 NT-proBNP in the sample is reacted in an aqueous medium with latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[10] 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, 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 an antibody or its antibody fragment that recognizes NT-proBNP, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[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, 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 the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[12] The method according to any one of [9] to

[11] , wherein the aqueous medium having a pH of 6.0 or less is an aqueous medium having a pH of 5.0 to 6.0.

[13] The method according to any one of [9] to

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

[14] The method according to

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

[15] The method according to

[13] or

[14] , wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v).

[16] The method according to any one of [9] to

[15] , wherein the contaminant is a rheumatoid factor.

[17] A latex agglutination immunoassay reagent for NT-proBNP in a sample containing latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP, wherein the pH of the reagent in solution state is 6.0 or less.

[18] 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, and latex particles bound to a second antibody or its antibody fragment that recognizes NT-proBNP, wherein the pH of the reagent in solution is 6.0 or less.

[19] The reagent according to

[17] or

[18] , wherein the pH of the reagent in a solution state is 5.0 to 6.0.

[20] The reagent according to any one of

[17] to

[19] , wherein the reagent contains a sensitizer.

[21] The reagent according to

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

[22] The reagent according to

[20] or

[21] , wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v).

[23] 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 solution state of the reagent obtained by mixing the first reagent and the second reagent has a pH of 6.0 or less.

[24] The kit according to

[23] , wherein the pH of the solution of the mixed first and second reagents is 5.0 to 6.0.

[25] The kit according to

[23] or

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

[26] The kit according to any one of

[23] to

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

[27] The kit according to

[25] or

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

[28] The kit according to any one of

[25] to

[27] , wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v). [Effects of the Invention]

[0012] 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

[0013] 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.

[0014] In this embodiment, "% (w / v)" means the percentage of mass (g) based on volume (100 mL).

[0015] 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.

[0016] [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. Examples of Good's buffers include 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPS), and O), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), N-[ Tris(hydroxymethyl)methyl)-2-hydroxy-3-aminopropanesulfonic acid (TAPSO), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid (HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid [(H)EPPS], N-[tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid N,N-bis(2-hydroxyethyl)glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and the like. The concentration of the buffer solution is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L.

[0017] The aqueous medium may contain salts, metal ions, sugars, proteins, surfactants, and the like. Examples of salts include sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. Examples of metal ions include sodium ions, magnesium ions, manganese ions, and zinc ions. Examples of sugars include mannitol and sorbitol. Examples of proteins include bovine serum albumin (hereinafter referred to as BSA). Examples of surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. 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 this range is preferred because it reduces the possibility of nonspecific reactions and may also reduce the possibility of reactions with various immunoglobulins contained in the sample, resulting in variability in measured values. Furthermore, the aqueous medium may contain a sensitizer, as described below.

[0018] [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 preferably includes whole blood, plasma, serum, ascites, etc. The sample may also contain a rheumatoid factor, which will be described later. The sample in this embodiment may be an aqueous medium containing NT-proBNP, for example, NT-proBNP diluted with phosphate buffered saline (50 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 6.0, hereinafter referred to as PBS).

[0019] [Latex agglutination immunoassay method] In this embodiment, the latex agglutination immunoassay method involves reacting a sample containing NT-proBNP with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in an aqueous medium to form a complex containing NT-proBNP and the latex particles bound to the antibody or antibody fragment thereof that recognizes NT-proBNP, thereby selectively agglutinating the latex particles, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less. The agglutination can be detected by measuring absorbance, scattered light, etc.

[0020] [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.

[0021] 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.

[0022] 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.

[0023] [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.

[0024] [Table 1]

[0025] NT-proBNP can be prepared according to known methods, for example, by expressing it in Escherichia coli or the like using genetic recombination techniques.

[0026] [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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 HM146 anti-NT-proBNP antibody (manufactured by EastCoast Biosciences) and 4NT1cc-Mab-15F11cc anti-NT-proBNP antibody (manufactured by Hytest Biosciences). In this embodiment, the monoclonal antibody recognizing NT-proBNP is also referred to as an "anti-NT-proBNP antibody."

[0031] [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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] [pH In this embodiment, the reaction between NT-proBNP in the sample and latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound is carried out in an aqueous medium having a pH of 6.0 or less. In this embodiment, the reaction between NT-proBNP in the sample and latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound is carried out at a pH of 6.0 or less, but may also be at a pH of 5.5 or less, or at a pH of 5.0 or less. Furthermore, in this embodiment, the reaction between NT-proBNP in a sample and latex particles to which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound may be carried out at a pH of 5.0 to 6.0, 5.2 to 6.0, 5.4 to 6.0, 5.5 to 6.0, 5.6 to 6.0, or 5.8 to 6.0, or may be at a pH of 5.5 to 5.6, 5.5 to 5.7, 5.5 to 5.8, or 5.5 to 5.9, but is preferably at a pH of 5.0 to 6.0. The pH of the aqueous medium can be adjusted, for example, using the above-mentioned buffer, hydrochloric acid, sodium hydroxide, or the like.

[0038] [Sensitizer] In this embodiment, the aqueous medium may contain a sensitizer. The sensitizer can improve sensitivity by promoting aggregation of the target component, NT-proBNP, with latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP. Examples of sensitizers include hydrophilic polymers and salts. 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 molecular weight 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., molecular weight 450,000 to 650,000 (manufactured by Sigma-Aldrich). The sensitizer improves the agglutination reaction (sensitivity) of NT-proBNP, the component to be measured, while also enhancing the agglutination reaction (non-specific reaction) of substances in the sample other than the component to be measured.

[0039] [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.

[0040] Contaminants can 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. In particular, the latex agglutination immunoassay method for NT-proBNP in a sample of this embodiment can reduce non-specific reactions caused by rheumatoid factor in a sample containing rheumatoid factor as an impurity.

[0041] [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 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, and the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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).

[0048] The reaction may be carried out in an aqueous medium containing a sensitizer. In this case, the concentration of the sensitizer in the reaction is not particularly limited as long as it is a concentration that allows the measurement of NT-proBNP according to the present embodiment, and may be, for example, 0.01% (w / v) or more, 0.05% (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, 0.9% (w / v) or more, 1% (w / v) or more, 1.1% (w / v) or more, 1.2% (w / v) or more, 1.3% (w / v) or more, 1.4% (w / v) or more, or 1.5% (w / v) or more. Furthermore, the concentration of the sensitizer 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, 4.5% (w / v) or less, 4% (w / v) or less, 3.5% (w / v) or less, 3 (w / v) or less, 2.9% (w / v) or less, 2.8% (w / v) or less, 2.7% (w / v) or less, 2.6% (w / v) or less, or 2.5% (w / v) or less.

[0049] The above values ​​can be freely combined. For example, the concentration of the sensitizer in the above reaction may be 0.01% (w / v) to 10% (w / v), 0.1% (w / v) to 5% (w / v), or 0.4% (w / v) to 3% (w / v). In the above reaction, one type of sensitizer may be present, or two or more types of sensitizers may be present. When two or more types of sensitizers are present, the above concentration refers to the total concentration of the two or more sensitizers.

[0050] 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; A method for latex agglutination immunoassay of NT-proBNP in a sample, comprising:

[0051] 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.

[0052] <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) may be the same as those mentioned above.

[0053] Step (1) may be carried out in the presence of a sensitizer. The sensitizer is not particularly limited, and examples thereof include the sensitizers described above. The concentration of the sensitizer in step (1) is not particularly limited as long as it is a concentration that enables the measurement of NT-proBNP according to the present embodiment, and may be, for example, 0.01% (w / v) or more, 0.05% (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, 0.9% (w / v) or more, 1% (w / v) or more, 1.1% (w / v) or more, 1.2% (w / v) or more, 1.3% (w / v) or more, 1.4% (w / v) or more, or 1.5% (w / v) or more. Furthermore, the concentration of the sensitizer in step (1) 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, 4.5% (w / v) or less, 4% (w / v) or less, 3.5% (w / v) or less, 3 (w / v) or less, 2.9% (w / v) or less, 2.8% (w / v) or less, 2.7% (w / v) or less, 2.6% (w / v) or less, or 2.5% (w / v) or less.

[0054] The above values ​​can be freely combined. For example, the concentration of the sensitizer in step (1) may be 0.01% (w / v) to 10% (w / v), 0.1% (w / v) to 5% (w / v), or 0.4% (w / v) to 3% (w / v). In the above step (1), one type of sensitizer may be present, or two or more types of sensitizers may be present. When two or more types of sensitizers are present, the above concentration means the total concentration of the two or more sensitizers.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] <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. The aqueous medium used in step (2) may be the aqueous medium described above.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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).

[0074] 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.

[0075] The above step (2), step (2A), step (2A-1) or step (2A-2) may be carried out in the presence of a sensitizer. The concentration of the sensitizer in 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 measurement of NT-proBNP according to the present embodiment, and may be, for example, 0.01% (w / v) or more, 0.05% (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, 0.9% (w / v) or more, 1% (w / v) or more, 1.1% (w / v) or more, 1.2% (w / v) or more, 1.3% (w / v) or more, 1.4% (w / v) or more, or 1.5% (w / v) or more. Furthermore, the concentration of the sensitizer in 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, 4.5% (w / v) or less, 4% (w / v) or less, 3.5% (w / v) or less, 3 (w / v) or less, 2.9% (w / v) or less, 2.8% (w / v) or less, 2.7% (w / v) or less, 2.6% (w / v) or less, or 2.5% (w / v) or less.

[0076] The above values ​​can be freely combined. For example, the concentration of the sensitizer in step (2), step (2A), step (2A-1), or step (2A-2) may be 0.01% (w / v) to 10% (w / v), 0.1% (w / v) to 5% (w / v), or 0.4% (w / v) to 3% (w / v). In the above step (2), step (2A), step (2A-1), or step (2A-2), one type of sensitizer may be present, or two or more types of sensitizers may be present. When two or more types of sensitizers are present, the above concentration means the total concentration of the two or more sensitizers.

[0077] The above step (2), step (2A), step (2A-1) or step (2A-2) is 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).

[0078] When the reaction in the above step (2), step (2A), step (2A-1) or step (2A-2) is carried out in an aqueous medium, the pH of the aqueous medium is 6.0 or less. When the reaction in the above step (2), step (2A), step (2A-1) or step (2A-2) is carried out in an aqueous medium, the pH of the aqueous medium may be 5.5 or less, or 5.0 or less. Furthermore, when the reaction in the above step (2), step (2A), step (2A-1) or step (2A-2) is carried out in an aqueous medium, the pH of the aqueous medium is pH 6.0 or lower, and may be pH 5.0 to 6.0, pH 5.2 to 6.0, pH 5.4 to 6.0, pH 5.5 to 6.0, pH 5.6 to 6.0, or pH 5.8 to 6.0, or may be pH 5.5 to 5.6, pH 5.5 to 5.7, pH 5.5 to 5.8, or pH 5.5 to 5.9, but is preferably pH 5.0 to 6.0.

[0079] <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.

[0080] 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.

[0081] 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).

[0082] 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).

[0083] 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.

[0084] 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.

[0085] [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.

[0086] (Aspect 1) 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 NT-proBNP in the sample is reacted in an aqueous medium with latex particles to which an antibody or its antibody fragment that recognizes NT-proBNP is bound, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[0087] (Aspect 2) 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, comprising the steps of: (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 an antibody or antibody fragment thereof that recognizes NT-proBNP, wherein the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[0088] (Aspect 3) 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 the steps of: (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 the reaction is carried out in an aqueous medium having a pH of 6.0 or less.

[0089] In the above-mentioned suppression method, the latex particles to which an antibody that recognizes NT-proBNP or an antibody fragment thereof is bound, the latex particles to which a first antibody that recognizes NT-proBNP or an antibody fragment thereof is bound, and the latex particles to which a second antibody that recognizes NT-proBNP or an antibody fragment thereof is bound can be, for example, the above-mentioned latex particles to which an antibody that recognizes NT-proBNP or an antibody fragment thereof is bound, the latex particles to which a first antibody that recognizes NT-proBNP or an antibody fragment thereof is bound, and the latex particles to which a second antibody that recognizes NT-proBNP or an antibody fragment thereof is bound. In the above-mentioned suppression method, the pH during the reaction is the pH during the reaction in the above-mentioned latex agglutination immunoassay method for NT-proBNP in a sample.Furthermore, in the above-mentioned suppression method, the concentration of each additive, reaction temperature, reaction time, etc. in each step may be, for example, the concentration of each additive, reaction temperature, reaction time, etc. in each step in the above-mentioned latex agglutination immunoassay method for NT-proBNP in a sample.

[0090] [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 measurement reagent is a reagent for measuring NT-proBNP in a sample, which comprises latex particles and an antibody or antibody fragment thereof that recognizes NT-proBNP, and the pH of the reagent in solution state is 6.0 or less. 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.

[0091] Another aspect (aspect 2) of the measurement reagent is a reagent for measuring NT-proBNP in a sample containing latex particles bound to an antibody or its antibody fragment that recognizes NT-proBNP, wherein the pH of the reagent in solution state is 6.0 or less. Another aspect (aspect 3) of the measurement reagent is a reagent for measuring NT-proBNP in a sample, which comprises 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, and the pH of the reagent in solution state is 6.0 or less.

[0092] In the reagents of Aspects 1 to 3, the latex particles, the latex particles bound with an antibody that recognizes NT-proBNP or an antibody fragment thereof, the latex particles bound with a first antibody that recognizes NT-proBNP or an antibody fragment thereof, and the latex particles bound with a second antibody that recognizes NT-proBNP or an antibody fragment thereof can be, for example, the above-mentioned latex particles, the latex particles bound with an antibody that recognizes NT-proBNP or an antibody fragment thereof, the latex particles bound with a first antibody that recognizes NT-proBNP or an antibody fragment thereof, and the latex particles bound with a second antibody that recognizes NT-proBNP or an antibody fragment thereof.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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).

[0097] 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.

[0098] 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.

[0099] The reagent for measuring NT-proBNP of this embodiment may contain a sensitizer. The concentration of the sensitizer 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, 0.01% (w / v) or more, 0.05% (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, 0.9% (w / v) or more, 1% (w / v) or more, 1.1% (w / v) or more, 1.2% (w / v) or more, 1.3% (w / v) or more, 1.4% (w / v) or more, or 1.5% (w / v) or more. Furthermore, the concentration of the sensitizer 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, 4.5% (w / v) or less, 4% (w / v) or less, 3.5% (w / v) or less, 3 (w / v) or less, 2.9% (w / v) or less, 2.8% (w / v) or less, 2.7% (w / v) or less, 2.6% (w / v) or less, or 2.5% (w / v) or less.

[0100] The above values ​​can be freely combined. For example, the concentration of the sensitizer in the reagent for measuring NT-proBNP of this embodiment may be 0.01% (w / v) to 10% (w / v), 0.1% (w / v) to 5% (w / v), or 0.4% (w / v) to 3% (w / v). In the measurement reagent, one type of sensitizer may be present, or two or more types of sensitizers may be present. When two or more types of sensitizers are present, the concentration refers to the total concentration of the two or more sensitizers.

[0101] 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. Examples of the aqueous medium used for dissolution include the aqueous media described above. The aqueous medium may contain the aforementioned salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. In the present embodiment, in the measuring reagent for NT-proBNP in a sample, the pH of the reagent in a solution state is 6.0 or less means that in the case of a liquid reagent, the pH of the liquid reagent is 6.0 or less, and in the case of a freeze-dried measuring reagent, the pH of the liquid reagent is 6.0 or less when the liquid reagent is dissolved in an aqueous medium before measurement to form a liquid reagent, and when the liquid reagent is used for measurement, the pH of the liquid reagent is 6.0 or less. In addition, the pH of the measuring reagent is, for example, 5.5 or less, or 5.0 or less, and the like also have the same meaning. In the reagent for measuring NT-proBNP in a sample of this embodiment, the pH of the reagent in solution is 6.0 or less. In the reagent for measuring NT-proBNP in a sample of this embodiment, the pH of the reagent in solution is 5.5 or less, and may be 5.0 or less. Furthermore, in the reagent for measuring NT-proBNP in a sample of this embodiment, the pH of the reagent in solution may be pH 5.0 to 6.0, pH 5.2 to 6.0, pH 5.4 to 6.0, pH 5.5 to 6.0, pH 5.6 to 6.0, or pH 5.8 to 6.0, or may be pH 5.5 to 5.6, pH 5.5 to 5.7, pH 5.5 to 5.8, or pH 5.5 to 5.9, with pH 5.6 to 6.0 being preferred. 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. 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.

[0102] [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. Hereinafter, preferred embodiments of the kit for measuring NT-proBNP in a sample according to the present embodiment will be exemplified.In the following embodiments, the latex particles to which an antibody recognizing NT-proBNP or its antibody fragment is bound, the latex particles to which a first antibody recognizing NT-proBNP or its antibody fragment is bound, and the latex particles to which a second antibody recognizing NT-proBNP or its antibody fragment is bound can be, for example, the above-mentioned latex particles to which an antibody recognizing NT-proBNP or its antibody fragment is bound, the latex particles to which a first antibody recognizing NT-proBNP or its antibody fragment is bound, and the latex particles to which a second antibody recognizing NT-proBNP or its antibody fragment is bound.

[0103] Measurement kit (1) Reagent 1A comprising an aqueous medium; and Reagent 2A containing latex particles to which an antibody or an antibody fragment thereof that recognizes NT-proBNP is bound. A measurement kit comprising: The kit comprises a solution of the first A reagent and the second A reagent, the solution having a pH of 6.0 or less.

[0104] Measurement kit (2) Reagent 1B comprising an aqueous medium; and a second reagent B comprising latex particles to which a first antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; and a third reagent B comprising latex particles to which a second antibody or an antibody fragment thereof that recognizes NT-proBNP is bound; A measurement kit comprising: A kit in which the pH of the solution of the mixed reagents, ie, the first B reagent, the second B reagent, and the third B reagent, is 6.0 or less.

[0105] Measurement kit (3) Reagent 1C containing an aqueous medium; a second C 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; A measurement kit comprising: A kit in which the pH of the solution of the mixed first and second C reagents is 6.0 or less.

[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 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.

[0107] 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 aforementioned molecules.

[0108] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes NT-proBNP in the second A 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, 0.003% (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 antibodies or antibody fragments thereof that recognize NT-proBNP bound thereto in the second A 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.

[0109] 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 Reagent 2A 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 having an antibody or antibody fragment thereof that recognizes NT-proBNP bound thereto in Reagent 2B, Reagent 3B or Reagent 2C 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.

[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 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 C reagent, 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 first A reagent, the second A reagent, the first B reagent, the second B reagent, the third B reagent, the first C reagent, or the second C reagent may contain a sensitizer. The concentration of the sensitizer in the first A reagent, the second A reagent, the first B reagent, the second B reagent, the third B reagent, the first C reagent, or the second C reagent 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, 0.01% (w / v) or more, 0.05% (w / v) or more, 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, or 0.4% (w / v) or more. The concentration may be 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, 0.9% (w / v) or more, 1% (w / v) or more, 1.1% (w / v) or more, 1.2% (w / v) or more, 1.3% (w / v) or more, 1.4% (w / v) or more, or 1.5% (w / v) or more. The concentration of the sensitizer 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, 4.5% (w / v) or less, 4% (w / v) or less, 3.5% (w / v) or less, 3 (w / v) or less, 2.9% (w / v) or less, 2.8% (w / v) or less, 2.7% (w / v) or less, 2.6% (w / v) or less, or 2.5% (w / v) or less.

[0113] The above values ​​can be freely combined. For example, the concentration of the sensitizer in the reagent may be 0.01% (w / v) to 10% (w / v), 0.1% (w / v) to 5% (w / v), or 0.4% (w / v) to 3% (w / v). In the above reagent, one type of sensitizer may be present, or two or more types of sensitizers may be present. When two or more types of sensitizers are present, the above concentration refers to the total concentration of the two or more sensitizers.

[0114] 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 described above. The aqueous medium may contain the aforementioned salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc.

[0115] When the constituent reagents of the assay kit are in liquid form, at least one antibody selected from the group consisting of 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.

[0116] In the present embodiment, in the measurement kit for NT-proBNP in a sample, the pH of the solution of the first and second reagents means the pH of the solution of the reagents that are mixed together at a mixing ratio determined for each kit before measurement.For example, when the first and second reagents are liquid measurement reagents, it means that the pH of the mixture of the first and second reagents is 6.0 or less; when the first and second reagents are freeze-dried measurement reagents, it means that the first and second reagents are dissolved in aqueous medium before measurement to be liquid, and then mixed together, and the pH of the mixture that is used for measurement is 6.0 or less.The mixing ratio determined for each kit can be, for example, the mixing ratio included in the kit or described in the instruction manual that accompanies the kit. When the constituent reagents of a kit are a first reagent and a second reagent, for example, a kit in which the pH of the solution of the first and second reagents mixed together is 6.0 or less means that the pH of the mixture obtained by mixing the first and second reagents in a mixing ratio determined for each kit is 6.0 or less. The same applies to, for example, a pH of 5.5 or less, a pH of 5.0 or less, etc. In the kit for measuring NT-proBNP in a sample of this embodiment, the pH of the solution of the reagent obtained by mixing the constituent reagents of the kit is 6.0 or less. In the kit for measuring NT-proBNP in a sample of this embodiment, the pH of the solution of the reagent obtained by mixing the constituent reagents of the kit is 5.5 or less, and may be 5.0 or less. Furthermore, in the kit for measuring NT-proBNP in a sample of this embodiment, the pH of the solution of the reagent obtained by mixing the constituent reagents of the kit may be pH 4.0 to 6.0, pH 5.0 to 6.0, pH 5.2 to 6.0, pH 5.4 to 6.0, pH 5.5 to 6.0, pH 5.6 to 6.0, or pH 5.8 to 6.0, or may be pH 5.5 to 5.6, pH 5.5 to 5.7, pH 5.5 to 5.8, or pH 5.5 to 5.9, but is preferably pH 5.0 to 6.0.

[0117] 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 aqueous media described above. 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.

[0118] Examples of NT-proBNP of known concentrations include NT-proBNP solutions prepared by diluting commercially available NT-proBNP with PBS at pH 6.0 or lower. Multiple concentrations of NT-proBNP of known concentrations may also be prepared. Examples of commercially available NT-proBNP include 8NT2 Human recombinant NT-proBNP (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]

[0119] 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.

[0120] Carboxyl-modified polystyrene latex (average particle size 400 nm) (Fujikura Chemical Industries, Ltd.), N,N-bis(2-hydroxyethyl)glycine (Bicine) (Dojindo Laboratories, Ltd.), Sulfo-NHS (Thermo Fisher Scientific), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific), HM146 anti-NT-proBNP antibody (primary antibody) (East Coast), 4NT1cc-Mab-15F11cc anti-NT-proBNP antibody (secondary antibody) (Hytest), Interference Check RF Plus (Sysmex), disodium hydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.), sodium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.), bovine serum albumin (BSA) (Bovogen), 8NT2 human recombinant The following substances were used: NT-proBNP (recombinant NT-proBNP) (Hytest), 2-morpholinoethanesulfonic acid monohydrate (MES) (Dojindo Laboratories), sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), Lipidure-BL103 (NOF Corporation), sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), polyethylene glycol molecular weight 20,000 (PEG20,000) (Fujifilm Wako Pure Chemical Industries, Ltd.), and dextran (derived from Leuconostoc spp., molecular weight 450,000-650,000) (Sigma-Aldrich).

[0121] [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.

[0122] [Experimental Example 2] Preparation of specimens for evaluation An evaluation sample having the following composition was prepared. 50mmol / L phosphate buffer (pH6.0) (Prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate) Recombinant NT-proBNP 1000pg / mL Rheumatoid factor 50 IU / mL For rheumatoid factor, the rheumatoid factor reagent included in Interference Check RF Plus was used.

[0123] [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.

[0124] [Example 1] Preparation of NT-proBNP measurement kit Measurement kit A containing a first reagent and a second reagent having the following compositions: pH5.5(MES) was prepared. (First Reagent) MES 100mmol / L BSA 5g / L Sodium chloride 200mmol / L Lipidure-BL103 1.15% (w / v) The pH was adjusted to 5.5 with an appropriate amount of sodium hydroxide. (Second reagent) 10mmol / L MES aqueous solution (pH5.5) 82%(v / v) First antibody-bound latex particle solution 9% (v / v) Secondary antibody-conjugated latex particle solution 9% (v / v)

[0125] [Example 2] Preparation of NT-proBNP measurement kit Measurement kit A of Example 1 pH5.5(MES) Measurement Kit A except that the pH of the first and second reagents was changed from 5.5 to 6.0. pH5.5(MES) Measurement kit A was used in the same way as pH6.0(MES) was prepared.

[0126] [Comparative Example 1] Preparation of NT-proBNP measurement kit Measurement kit A of Example 1 pH5.5(MES)Measurement Kit A except that MES and pH 5.5 in the first and second reagents are changed to MES and pH 7.0, HEPES and pH 7.0, and HEPES and pH 8.0, respectively. pH5.5(MES) Measurement kit A was used in the same way as pH7.0(MES) , Measurement Kit A pH7.0(HEPES) , and measurement kit A pH8.0(HEPES) were prepared, respectively.

[0127] [Example 3] Preparation of NT-proBNP measurement kit Measurement kit A of Example 1 pH5.5(MES)、 and Example 2 Measurement Kit A pH6.0(MES) Measurement Kit A except that 1.15% (w / v) Lipidure-BL103 in the first reagent was changed to 2.75% (w / v) PEG20,000. pH5.5(MES)、 and measurement kit A pH6.0(MES) Using the same method as above, measurement kit B pH5.5(MES)、 and measurement kit B pH6.0(MES) were prepared, respectively.

[0128] [Comparative Example 2] Preparation of NT-proBNP measurement kit Measurement kit A for Comparative Example 1 pH7.0(MES) , Measurement Kit A pH7.0(HEPES) , and measurement kit A pH8.0(HEPES) Measurement Kit A except that 1.15% (w / v) Lipidure-BL103 in the first reagent was changed to 2.75% (w / v) PEG20,000. pH7.0(MES) , Measurement Kit A pH7.0(HEPES) , and measurement kit A pH8.0(HEPES) Using the same method as above, measurement kit B pH7.0(MES) , Measurement Kit B pH7.0(HEPES) , and measurement kit B pH8.0(HEPES) were prepared, respectively.

[0129] [Example 4] Preparation of NT-proBNP measurement kit Measurement kit A of Example 1 pH5.5(MES)、 and Example 2 Measurement Kit A pH6.0(MES)Measurement Kit A except that 1.15% (w / v) Lipidure-BL103 in the first reagent was changed to 2.25% (w / v) dextran. pH5.5(MES)、 and measurement kit A pH6.0(MES) Using the same method as above, measurement kit C pH5.5(MES)、 and measurement kit C pH6.0(MES) were prepared, respectively.

[0130] [Comparative Example 3] Preparation of NT-proBNP measurement kit Measurement kit A for Comparative Example 1 pH7.0(MES) , Measurement Kit A pH7.0(HEPES) , and measurement kit A pH8.0(HEPES) Measurement Kit A except that 1.15% (w / v) Lipidure-BL103 in the first reagent was changed to 2.25% (w / v) dextran. pH7.0(MES) , Measurement Kit A pH7.0(HEPES) , and measurement kit A pH8.0(HEPES) Using the same method as above, measurement kit C pH7.0(MES) , Measurement Kit C pH7.0(HEPES) , and measurement kit C pH8.0(HEPES) were prepared, respectively.

[0131] [Example 5] Calculation of non-specific reaction rate in NT-proBNP measurement Automated analyzer 3500 (Hitachi High-Tech), measurement kit A pH5.5(MES) The evaluation samples were measured using the following procedure, and the non-specific reaction rate was calculated. (1) Preparation of a calibration curve for NT-proBNP measurement Measurement kit A of Example 1 was used as the measurement kit. pH5.5(MES) The absorbance of the NT-proBNP standards was measured using the following procedure using NT-proBNP standards 1 to 5 (five standards with NT-proBNP contents of 0 pg / mL, 300 pg / mL, 900 pg / mL, 1500 pg / mL, and 2000 pg / mL, respectively) from Experimental Example 3 as samples. Standard 1 (10 μL) and the measurement kit A from Example 1 were added to a reaction cell. pH5.5(MES)The first reagent (90 μL) was added, and the mixture was allowed to react at 37° C. for 5 minutes. The absorbance (E1) of the reaction solution was measured at a dominant wavelength of 600 nm with no secondary wavelength. pH5.5(MES) The second reagent (30 μL) was added and the mixture was further reacted at 37°C for 5 minutes. The absorbance (E2) of the reaction solution was measured at a dominant wavelength of 600 nm with no 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.

[0132] (2) Determination of NT-proBNP concentration measurement values ​​in evaluation samples Measurement kit A of Example 1 pH5.5(MES) Using the evaluation specimen prepared in Experimental Example 2 as a sample, the absorbance difference relative to the NT-proBNP concentration was calculated for the evaluation specimen in the same manner as in (1). The measured NT-proBNP concentration (pg / mL) for the evaluation specimen was determined from the calculated absorbance difference and the calibration curve in (1).

[0133] (3) Evaluation of non-specific reaction rate From the theoretical value of the NT-proBNP concentration in the evaluation sample (1000 pg / mL as described in Experimental Example 2) and the measured value of the NT-proBNP concentration in the evaluation sample determined in (2) above, the following formula (I) was used to calculate the NT-proBNP concentration in Measurement Kit A of Example 1. pH5.5(MES) The non-specific reaction rate was calculated, and the results are shown in Table 2.

[0134]

number

[0135] [Example 6] Calculation of non-specific reaction rate in NT-proBNP measurement Measurement kit A of Example 1 pH5.5(MES) Instead of measurement kit A pH6.0(MES)、 Measurement Kit B pH5.5(MES)、 Measurement Kit B pH6.0(MES)、 Measurement Kit C pH5.5(MES)、 and measurement kit C pH6.0(MES) The non-specific reaction rate for each measurement kit was calculated in the same manner as in Example 5, except that the following was used. The results are shown in Tables 2, 3 and 4.

[0136] [Comparative Example 4] Calculation of non-specific reaction rate in NT-proBNP measurement Measurement kit A of Example 1 pH5.5(MES) Instead of measurement kit A pH7.0(MES) , Measurement Kit A pH7.0(HEPES) , Measurement Kit A pH8.0(HEPES) , Measurement Kit B pH7.0(MES) , Measurement Kit B pH7.0(HEPES) , Measurement Kit B pH8.0(HEPES) , Measurement Kit C pH7.0(MES) , Measurement Kit C pH7.0(HEPES) , and measurement kit C pH8.0(HEPES) The non-specific reaction rate for each measurement kit was calculated in the same manner as in Example 5, except that the following was used. The results are shown in Tables 2, 3 and 4.

[0137] [Table 2]

[0138] [Table 3]

[0139] [Table 4]

[0140] It is desirable that the measured NT-proBNP concentration be the same as the theoretical value of the NT-proBNP concentration. However, in reality, due to the occurrence of non-specific reactions, i.e., agglutination reactions of latex particles not derived from NT-proBNP in the sample, the measured NT-proBNP concentration is higher than the theoretical value of the NT-proBNP concentration. Furthermore, the closer the measured NT-proBNP concentration is to the theoretical value of the NT-proBNP concentration, the lower the non-specific reaction rate. The lower the non-specific reaction rate, the more accurately NT-proBNP can be measured. Furthermore, since the first and second reagents in each measurement kit have the same pH, the pH when the latex agglutination immune reaction is performed is approximately the same as the pH of the first and second reagents. As is clear from Tables 2, 3 and 4, regardless of whether Lipidure-BL103, PEG20,000 or dextran is used as a sensitizer, the lower the pH of the first and second reagents in each measurement kit, i.e., the lower the pH when the latex agglutination immune reaction is performed, the lower the non-specific reaction rate, and the more accurately NT-proBNP can be measured.In addition, it was revealed that, particularly when the pH is lower than 6.0, the non-specific reaction rate is lower and the more accurately NT-proBNP can be measured.Furthermore, it was revealed that the measurement kit of this example can reduce non-specific reactions in the measurement of NT-proBNP in a sample containing rheumatoid factor as a contaminant. [Industrial Applicability]

[0141] 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, comprising reacting the NT-proBNP in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes NT-proBNP in an aqueous medium, The reaction is carried out in an aqueous medium having a pH of 6.0 or less.

2. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) A latex agglutination immunoassay method for NT-proBNP in a sample, comprising the step of reacting NT-proBNP in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes NT-proBNP in an aqueous medium, The reaction is carried out in an aqueous medium having a pH of 6.0 or less.

3. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) A latex agglutination immunoassay method for NT-proBNP in a sample, comprising the steps of: reacting NT-proBNP in the sample in an aqueous medium with latex particles bound to a first antibody that recognizes NT-proBNP or an antibody fragment thereof, and with latex particles bound to a second antibody that recognizes NT-proBNP or an antibody fragment thereof, The reaction is carried out in an aqueous medium having a pH of 6.0 or less.

4. The method according to any one of claims 1 to 3, wherein the aqueous medium having a pH of 6.0 or less is an aqueous medium having a pH of 5.0 to 6.

0.

5. The method according to any one of claims 1 to 3, wherein the aqueous medium is an aqueous medium containing a sensitizer.

6. The method of claim 5 , wherein the sensitizer is a hydrophilic polymer.

7. 6. The method of claim 5, wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v).

8. The method according to any one of claims 1 to 3, wherein the sample contains a rheumatoid factor.

9. 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, which comprises reacting N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) in the sample in an aqueous medium with latex particles to which an antibody that recognizes NT-proBNP or an antibody fragment thereof is bound, comprising: The reaction is carried out in an aqueous medium having a pH of 6.0 or less.

10. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) 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, the method comprising the step of reacting NT-proBNP in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes NT-proBNP in an aqueous medium, The reaction is carried out in an aqueous medium having a pH of 6.0 or less.

11. (1) mixing a sample containing N-terminal pro-brain natriuretic peptide (hereinafter referred to as NT-proBNP) with an aqueous medium; and (2) 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, the method comprising the steps of: reacting NT-proBNP in the sample in an aqueous medium with latex particles bound to a first antibody that recognizes NT-proBNP or an antibody fragment thereof; and latex particles bound to a second antibody that recognizes NT-proBNP or an antibody fragment thereof, The reaction is carried out in an aqueous medium having a pH of 6.0 or less.

12. The method according to any one of claims 9 to 11, wherein the aqueous medium having a pH of 6.0 or less is an aqueous medium having a pH of 5.0 to 6.

0.

13. The method according to any one of claims 9 to 11, wherein the aqueous medium is an aqueous medium containing a sensitizer.

14. The method of claim 13 , wherein the sensitizer is a hydrophilic polymer.

15. 14. The method of claim 13, wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v).

16. The method according to any one of claims 9 to 11, wherein the contaminant is a rheumatoid factor.

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 to an antibody or antibody fragment thereof that recognizes NT-proBNP, A reagent having a pH of 6.0 or less in a solution state.

18. 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 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 reagent having a pH of 6.0 or less in a solution state.

19. The reagent according to claim 17 or 18, wherein the pH of the reagent in a solution state is 5.0 to 6.

0.

20. The reagent according to claim 17 or 18, wherein the reagent contains a sensitizer.

21. The reagent according to claim 20 , wherein the sensitizer is a hydrophilic polymer.

22. The reagent according to claim 20, wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v).

23. 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, A kit in which the pH of a solution of the mixed first and second reagents is 6.0 or less.

24. The kit according to claim 23, wherein the pH of the solution of the mixed first and second reagents is 5.0 to 6.

0.

25. 24. The kit of claim 23, wherein the first reagent further comprises a sensitizer.

26. 24. The kit of claim 23, wherein the second reagent further comprises a sensitizer.

27. The kit according to claim 25 or 26, wherein the sensitizer is a hydrophilic polymer.

28. The kit according to claim 25 or 26, wherein the concentration of the sensitizer is 0.4% (w / v) to 3% (w / v).

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