Latex agglutination immunoassay method, measurement reagent, and measurement kit for component to be measured in sample

Abstract

To provide a method, a reagent and a kit for accurately measuring a component to be measured in a sample.SOLUTION: A method for measuring an analyte in a sample by a latex agglutination immunoassay, comprising the step of reacting the analyte in the sample with latex particles to which antibodies or fragments thereof that recognize the analyte are bound, wherein the reaction is performed under the presence of a lithium salt, and the antibodies or fragments thereof are IgG1 or fragments thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a latex agglutination immunoassay method for measuring a target component in a sample, a measurement reagent, and a measurement kit. The present invention also relates to a method for suppressing non-specific reactions caused by impurities in a biological sample in a latex agglutination immunoassay. [Background technology]

[0002] Latex agglutination immunoreactions are commonly used in the field of clinical testing as a method for measuring a target component in a sample. Measurement methods utilizing latex agglutination immunoreactions include, for example, using latex particles bound to antibodies that specifically recognize the target component, and detecting the degree of agglutination (turbidity) of the latex particles, which occurs when an antigen, the target component, binds to the antibody-bound latex particles, by optical means or the like. Furthermore, reagents for use in these measurement methods are commercially available from many manufacturers.

[0003] Examples of latex agglutination immunoassay methods for measuring components in a sample include latex agglutination immunoassay methods for brain natriuretic peptide (hereinafter also referred to as BNP), also known as B-type natriuretic peptide, N-terminal pro-brain natriuretic peptide (hereinafter also referred to as NT-proBNP), etc. BNP is derived from the BNP gene, and 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 physiologically inactive NT-proBNP consisting of the 76th amino acid sequence from the N-terminus of proBNP, and physiologically active BNP consisting of the 77th to 108th amino acid sequence of proBNP.

[0004] 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.

[0005] A reported latex agglutination immunoassay method for NT-proBNP is one in which latex particles bound to an antibody that binds to NT-proBNP in a sample are used to induce an antigen-antibody reaction with the NT-proBNP in the sample, and the change in turbidity due to agglutination of the latex particles is measured as absorbance (Patent Document 1).

[0006] Because latex agglutination immunoassays do not require B / F separation, they are also used in general-purpose automated analyzers for biochemical testing. However, latex agglutination immunoassays suffer from 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 2). [Prior art documents] [Patent documents]

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

[0009] An object of the present invention is to provide an accurate method for measuring a target component in a sample, a measuring reagent, and a measuring kit. [Means for solving the problem]

[0010] As a result of intensive investigations to solve the above-mentioned problems, the present inventors have found that an accurate measurement of a target component can be achieved by a latex agglutination immunoassay method in which a target component in a sample is reacted in an aqueous medium with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component, wherein the reaction is carried out in the presence of a lithium salt and the antibody or antibody fragment thereof is IgG1 or a fragment thereof, and have completed the present invention.

[0011] That is, the present invention includes the following aspects. [1] A latex agglutination immunoassay method for a target component in a sample, comprising reacting the target component in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component in an aqueous medium, the reaction is carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof. [2] A latex agglutination immunoassay method for a target component in a sample, comprising: (1) a step of mixing a sample containing the target component with an aqueous medium; and (2) a step of reacting the target component in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component in the aqueous medium, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof. [3] A latex agglutination immunoassay method for a target component in a sample, comprising: (1) mixing a sample containing the target component with an aqueous medium; and (2) reacting, in the aqueous medium, the target component in the sample with latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component, and with latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof. [4] The method according to any one of [1] to [3], wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L. [5] The method according to any one of [1] to [4], wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L. [6] The method according to any one of [1] to [5], wherein the component to be measured is NT-proBNP. [7] The method according to any one of [1] to [6], wherein the aqueous medium contains a sensitizer. [8] The method according to [7], wherein the sensitizer is a hydrophilic polymer. [9] A method for suppressing non-specific reactions caused by impurities in a biological sample in a reaction between a component to be measured in a sample and an antibody or an antibody fragment thereof that recognizes the component to be measured, the method comprising: reacting the component to be measured in an aqueous medium with latex particles bound to the antibody or the antibody fragment thereof; the reaction is carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[10] A method for suppressing non-specific reactions caused by impurities in a biological sample in a reaction between a component to be measured and an antibody or an antibody fragment thereof, the method comprising: (1) a step of mixing a sample containing the component to be measured with an aqueous medium; and (2) a step of reacting the component to be measured in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the component to be measured, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[11] A method for suppressing non-specific reactions caused by contaminants in a biological sample in a reaction between a component to be measured and the first antibody or antibody fragment, and the second antibody or antibody fragment, comprising: (1) a step of mixing a sample containing the component to be measured with an aqueous medium; and (2) a step of reacting the component to be measured in the sample in the aqueous medium with latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured, and latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

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

[11] , wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

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

[12] , wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

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

[13] , wherein the component to be measured is NT-proBNP.

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

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

[16] The method according to

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

[17] A latex agglutination immunoassay reagent for a target component in a sample, comprising latex particles bound to an antibody or antibody fragment thereof that recognizes the target component, the reagent comprises a lithium salt; A reagent wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[18] A latex agglutination immunoassay reagent for a target component in a sample, comprising: latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component; and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, the reagent comprises a lithium salt; A reagent in which the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[19] The reagent according to

[17] or

[18] , wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

[20] The reagent according to any one of

[17] to

[19] , wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

[21] The reagent according to any one of

[17] to

[20] , wherein the component to be measured is NT-proBNP.

[22] The reagent according to any one of

[17] to

[21] , wherein the reagent contains a sensitizer.

[23] The reagent according to

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

[24] A latex agglutination immunoassay kit for a target component in a sample, comprising: a first reagent containing an aqueous medium; latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component; and a second reagent containing latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, the first reagent and / or the second reagent contain a lithium salt; A kit wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[25] The kit according to

[24] , wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

[26] The kit according to

[24] or

[25] , wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

[27] The kit according to any one of

[24] to

[26] , wherein the component to be measured is NT-proBNP.

[28] The kit according to any one of

[24] to

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

[29] The kit according to any one of

[24] to

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

[30] The kit according to

[28] or

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

[0012] An accurate method for measuring a target component 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" a component to be measured, or when an antibody is expressed as "recognizing" a component to be measured, these 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 a component to be measured 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. The aqueous medium may contain salts, metal ions, sugars, proteins, surfactants, and the like. Examples of salts include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc. Examples of metal ions include sodium ions, magnesium ions, manganese ions, zinc ions, etc. Examples of sugars include mannitol and sorbitol. Examples of proteins include bovine serum albumin (hereinafter referred to as BSA). Examples of surfactants include anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants. The aqueous medium may also contain a sensitizer. The sensitizer can improve sensitivity by promoting aggregation between the target component and latex particles bound to an antibody or antibody fragment thereof that recognizes the target component. Examples of sensitizers include hydrophilic polymers. 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. Specific examples (products) of polyethylene glycol include 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 not only improves the agglutination reaction (sensitivity) of the component to be measured, but also enhances the agglutination reaction (non-specific reaction) of substances in the sample other than the component to be measured. The concentration of the sensitizer in the aqueous medium is preferably 0.1 to 5% (w / v), more preferably 0.4 to 3% (w / v). The aqueous medium may also contain an anti-IgM antibody. The anti-IgM antibody can suppress, for example, nonspecific agglutination reactions (hereinafter also referred to as nonspecific reactions) caused by components in the sample other than the component to be measured. The anti-IgM antibody may be derived from any animal species, including rodents such as mice and rats, and mammals such as humans, monkeys, sheep, goats, rabbits, pigs, and cows. The anti-IgM antibody may be a polyclonal antibody or a monoclonal antibody. The concentration of the anti-IgM antibody in the aqueous medium is preferably 3 μg / ml to 10 mg / ml. A concentration of the anti-IgM antibody in the aqueous medium within the above range is preferred because it reduces the possibility of nonspecific reactions and also reduces the possibility of reactions with various immunoglobulins contained in the sample, which can lead to variations in measured values.

[0017] [sample] The sample in this embodiment is not particularly limited as long as it is a sample that may contain the component to be measured, and examples include any one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, ascites, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice, and preferred examples include whole blood, plasma, serum, ascites, etc. The sample in this embodiment may be an aqueous medium containing the component to be measured, such as the component to be measured diluted with phosphate-buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2, hereinafter referred to as PBS).

[0018] [Measurement target components] The components to be measured in this embodiment are not particularly limited, and examples thereof include CRP (C-reactive protein), prostate-specific antigen (PSA), ferritin, α2-macroglobulin, β-2 microglobulin, myoglobin, fibrin, fibrinogen degradation products, D-dimer, thrombin-antithrombin III complex (TAT), soluble fibrin (SF), soluble interleukin-2 receptor (sIL-2R), atrial natriuretic peptide (ANP), BNP, NT-proBNP, C-type natriuretic peptide (CNP), CEA (carcinoembrionic antigen), HBs-Ag (hepatitis B envelope antigen), anti-HBs (anti-hepatitis B envelope antibody), HBe-Ag (hepatitis B e antigen), anti-HBe (anti-hepatitis B e antibody), anti-HBc (anti-hepatitis B core antibody), IgG, IgA, and IgM. The term "measurement target component in a sample" refers to the measurement target component present in the sample. The measurement target component is not particularly limited, and may be, for example, the measurement target component in a free form or in a complex form with other substances (e.g., proteins, etc.).

[0019] [NT-proBNP] NT-proBNP, exemplified as a component to be measured in this embodiment, is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1. NT-proBNP is produced by cleaving a BNP precursor (proBNP) consisting of the amino acid sequence represented by SEQ ID NO: 2 into BNP consisting of the amino acid sequence represented by SEQ ID NO: 3 and NT-proBNP. The amino acid sequences of NT-proBNP, proBNP, and BNP are shown in Table 1.

[0020] [Table 1]

[0021] NT-proBNP can be prepared according to known methods, for example, by expressing it in Escherichia coli or the like using recombinant DNA technology.

[0022] [Latex agglutination immunoassay method] In this embodiment, the latex agglutination immunoassay method is a method in which a sample containing a target component is reacted in an aqueous medium with latex particles bound to an antibody or antibody fragment thereof that recognizes the target component to form a complex containing the target component and the latex particles bound to the antibody or antibody fragment thereof that recognizes the target component, thereby selectively agglutinating the latex particles. The agglutination can be detected by measuring absorbance, scattered light, etc.

[0023] [Latex particles] In this embodiment, the latex particles are not particularly limited as long as they enable the measurement method of the present embodiment for the target component of measurement or are usable 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 core particles are surface-treated with an organic substance or the like. Specific examples include synthetic resins such as polystyrene, copolymers containing styrene as a main component, polyvinyl chloride, polypropylene, (meth)acrylic resin, and polymethyl methacrylate. The above-mentioned latex particles may be used alone or in combination of two or more types. Among these, polystyrene-based synthetic polymers and polystyrene-based synthetic polymers copolymerized with acrylic acid-based monomers, sulfonic acid-containing monomers, or the like as components for imparting electric charge are particularly preferred.

[0024] 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.

[0025] 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.

[0026] [Lithium salts] In this embodiment, the lithium salt is not particularly limited as long as it enables the measurement method of the component to be measured of this embodiment or can be used in the measurement reagent or measurement kit of this embodiment, and examples include lithium chloride, lithium bromide, lithium carbonate, lithium citrate, etc. The lithium salt may be ionized into lithium ions and counter ions (anions) of the lithium ions in an aqueous medium, or may not be ionized, or a portion of the lithium salt may be ionized.

[0027] [Antibody or antibody fragment thereof that recognizes the component to be measured] In this embodiment, the antibody that recognizes the analyte component is not particularly limited as long as it enables the measurement method of the analyte component of this embodiment or is usable in the measurement reagent or measurement kit of this embodiment, and examples thereof include antibodies of the IgG1 subclass. Examples of IgG1 in this embodiment include mouse IgG1, rat IgG1, goat IgG1, sheep IgG1, horse IgG1, human IgG1, and humanized IgG1 antibodies. Furthermore, the antibody that recognizes the analyte component of this embodiment may be a chimeric antibody combining two animal species, such as a chimeric antibody having the constant region of mouse IgG1, rat IgG1, goat IgG1, sheep IgG1, horse IgG1, or human IgG1.

[0028] The antibody that recognizes the component to be measured may be a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, or the like, with monoclonal antibodies being preferred from the viewpoints of homogeneity and stability.

[0029] In this embodiment, an antibody fragment refers to a portion of an IgG1 that recognizes the component to be measured, 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.

[0030] In this embodiment, the first antibody or antibody fragment thereof that recognizes the component to be measured and the second antibody or antibody fragment thereof that recognizes the component to be measured may bind to the same or different sites (epitopes) of the component to be measured. Furthermore, the first antibody or antibody fragment thereof that recognizes the component to be measured and the second antibody or antibody fragment thereof that recognizes the component to be measured may be the same antibody or different antibodies. When the first antibody or antibody fragment thereof that recognizes the component to be measured and the second antibody or antibody fragment thereof that recognizes the component to be measured are different antibodies, both antibodies may be IgG1 or a fragment thereof, or either one of the antibodies may be IgG1 or a fragment thereof. Examples of IgG1 or a fragment thereof include the aforementioned IgG1 or a fragment thereof.

[0031] In this embodiment, the antibody or antibody fragment thereof that recognizes the component to be measured can be obtained by a conventional antibody production method using the component to be measured or a microorganism that produces the component to be measured 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 that recognizes the component to be measured. An example of a commercially available monoclonal antibody that recognizes the component to be measured is a commercially available monoclonal antibody that recognizes NT-proBNP. Commercially available monoclonal antibodies that recognize NT-proBNP include, for example, HM146 anti-NT-proBNP antibody (manufactured by EastCoast), HM148 anti-NT-proBNP antibody (manufactured by EastCoast), HM899 anti-NT-proBNP antibody (manufactured by EastCoast), A10756508P anti-NT-proBNP antibody (manufactured by BiosPacific), 4NT1cc-Mab-15F11cc anti-NT-proBNP antibody (manufactured by Hytest), 1308 SPTN-5 anti-NT-proBNP antibody (manufactured by MedixBiochemica), etc. In this embodiment, the monoclonal antibody that recognizes NT-proBNP is also referred to as an "anti-NT-proBNP antibody."

[0032] [Latex particles bound to antibodies or antibody fragments that recognize the components to be measured] In this embodiment, latex particles bound to an antibody or antibody fragment thereof that recognizes the component to be measured refer to latex particles to which an antibody or antibody fragment thereof that recognizes the component to be measured (including a first antibody or antibody fragment thereof that recognizes the component to be measured, and a second antibody or antibody fragment thereof that recognizes the component to be measured, as described below) is bound.

[0033] The method of binding an antibody or antibody fragment thereof that recognizes a target component to latex particles is not particularly limited as long as it enables the measurement method for the target component of this embodiment or is usable for the measurement reagent or measurement kit of this embodiment, and examples include physical adsorption and chemical bonding. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, and hydrophobic bonding. Examples of chemical bonding include covalent bonding and coordinate bonding.

[0034] The antibody or antibody fragment thereof that recognizes the component to be measured may be bound to the latex particles directly or indirectly using the aforementioned physical adsorption and / or chemical binding. Examples of indirect binding methods include a method in which an antibody or antibody fragment thereof that recognizes NT-proBNP is bound to the 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 that recognizes NT-proBNP is bound to the latex particles by a covalent bond via a linker.

[0035] When a set of affinity substances is used, latex particles to which an antibody or its antibody fragment that recognizes the component to be measured is bound can be produced by binding an antibody or its antibody fragment 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.

[0036] 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 the component to be measured and an antibody that binds to the Fc region.

[0037] Examples of linkers include molecules that can covalently bond functional groups on the surface of latex particles to functional groups carried by antibodies or antibody fragments thereof that recognize the components to be measured. For example, a molecule that simultaneously has a first reactive group that can react with a functional group carried by antibodies or antibody fragments thereof that recognize the components to be measured, and a second reactive group that can react with a functional group on the surface of latex particles, where the first reactive group and the second reactive group are different groups, is preferably used. Examples of functional groups possessed by antibodies or antibody fragments thereof that recognize the component to be measured, and functional groups carried on the surface of latex particles, include carboxyl groups, amino groups, glycidyl groups, sulfhydryl groups, hydroxyl groups, amide groups, imino groups, N-hydroxysuccinyl groups, maleimide groups, etc. 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, vinyl sulfone, etc.

[0038] [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.

[0039] Contaminants can also be removed from biological samples by known methods. Lipids can be removed, for example, by adsorbing them onto silica. Fibrin can be precipitated by centrifuging the biological sample, and only the supernatant can be collected. Prior to centrifugation, a fibrin production promoter (e.g., calcium ions and / or bovine thrombin) can be added to the biological sample to generate fibrin, followed by centrifugation. Lipid-soluble small molecules can be removed by adsorbing them onto charcoal (activated carbon). Rheumatoid factors can be removed, for example, by adding an enzyme such as pronase, a protease, to the biological sample and allowing it to react. However, if the enzyme remains in the biological sample, it will denature the antibody used in the latex agglutination immunoreaction, reducing its antigen-binding activity. Therefore, the biological sample must be inactivated by boiling or other methods before use in the latex agglutination immunoreaction. As described above, it is possible to remove impurities and reduce non-specific reactions by pretreating a biological sample, but the pretreatment procedure is complicated. Therefore, a latex agglutination immunoassay method that can reduce non-specific reactions without the above-mentioned pretreatment procedure is desired. The latex agglutination immunoassay method for NT-proBNP in a sample of this embodiment can reduce non-specific reactions without the above-mentioned pretreatment procedure.

[0040] [Method for measuring target components in samples] The latex agglutination immunoassay method for a target component in a sample in this embodiment is a latex agglutination immunoassay method for a target component in a sample, which comprises reacting the target component in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component in an aqueous medium, the reaction is carried out in the presence of a lithium salt; The method wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof. In the latex agglutination immunoassay method of this embodiment for measuring a component to be measured in a sample, the component to be measured may be, for example, NT-proBNP.

[0041] The time for reacting the target component in the sample with the antibody or antibody fragment thereof that recognizes the target component is not particularly limited as long as it allows the measurement of the target component 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. 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.

[0042] 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.

[0043] The temperature at which the component to be measured in the sample is reacted with the antibody or antibody fragment thereof that recognizes the component to be measured is not particularly limited as long as it is a temperature that enables the measurement method of the component to be measured 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.

[0044] 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.

[0045] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes the component to be measured in the above reaction is not particularly limited as long as it is a concentration that enables the measurement method of the component to be measured 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 the component to be measured 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.

[0046] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes the component to be measured 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).

[0047] The concentration of the lithium salt in the above reaction is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured in this embodiment, and may be, for example, 0.01 mmol / L or more, 0.05 mmol / L or more, 0.1 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, 50 mmol / L or more, 60 mmol / L or more, 70 mmol / L or more, 80 mmol / L or more, 90 mmol / L or more, 100 mmol / L or more, or 150 mmol / L or more. Furthermore, the concentration of the lithium salt in the above reaction may be, for example, 1000 mmol / L or less, 900 mmol / L or less, 800 mmol / L or less, 700 mmol / L or less, 600 mmol / L or less, 500 mmol / L or less, 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, or 200 mmol / L or less.

[0048] The above values ​​can be freely combined. For example, the concentration of the lithium salt in the above reaction may be 0.01 mmol / L to 1000 mmol / L, 1 mmol / L to 500 mmol / L, 10 mmol / L to 300 mmol / L, or 50 mmol / L to 200 mmol / L. In the above reaction, one type of lithium salt may be present, or two or more types of lithium salts may be present. When two or more types of lithium salts are present, the above concentration means the total concentration of the two or more types of lithium salts.

[0049] One aspect of the method for measuring a target component in a sample in this embodiment is: A latex agglutination immunoassay method for a target component in a sample, comprising: (1) a step of mixing a sample containing the target component with an aqueous medium; and (2) a step of reacting the target component in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component, in the aqueous medium, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[0050] 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 the component to be measured and latex particles to which an antibody or an antibody fragment thereof that recognizes the component to be measured is bound, thereby obtaining a measurement value.

[0051] <Process (1)> Step (1) is a step of mixing a sample containing a component to be measured with an aqueous medium. In step (1), a sample containing the component to be measured may be added to an aqueous medium and mixed, or an aqueous medium may be added to a sample containing the component to be measured and mixed. The aqueous medium in step (1) is not particularly limited, and examples thereof include the aqueous media described above.

[0052] Step (1) may be carried out in the presence of a lithium salt. The lithium salt is not particularly limited, and examples thereof include the lithium salts described above. The concentration of the lithium salt in step (1) is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured according to the present embodiment, and may be, for example, 0.01 mmol / L or more, 0.05 mmol / L or more, 0.1 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, 50 mmol / L or more, 60 mmol / L or more, 70 mmol / L or more, 80 mmol / L or more, 90 mmol / L or more, 100 mmol / L or more, or 150 mmol / L or more. The concentration of the lithium salt in step (1) may be, for example, 1000 mmol / L or less, 900 mmol / L or less, 800 mmol / L or less, 700 mmol / L or less, 600 mmol / L or less, 500 mmol / L or less, 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, or 200 mmol / L or less.

[0053] The above values ​​can be freely combined. For example, the concentration of the lithium salt in the above reaction may be 0.01 mmol / L to 1000 mmol / L, 1 mmol / L to 500 mmol / L, 10 mmol / L to 300 mmol / L, or 50 mmol / L to 200 mmol / L. In the above step (1), one type of lithium salt may be present, or two or more types of lithium salts may be present. When two or more types of lithium salts are present, the above concentration means the total concentration of the two or more types of lithium salts.

[0054] In step (1), a sample containing a component to be measured 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 the measurement of the component to be measured by the 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.

[0055] 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.

[0056] The temperature at which the mixture is maintained is not particularly limited as long as it allows the measurement of the component to be measured in 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.

[0057] 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.

[0058] <Process (2)> Step (2) is a step in which, in an aqueous medium, a component to be measured in a sample is reacted with latex particles to which an antibody or an antibody fragment thereof that recognizes the component to be measured is bound. In step (2), the antibody or antibody fragment thereof that recognizes the component to be measured reacts with the component to be measured, producing a complex containing the component to be measured and latex particles to which the antibody or antibody fragment thereof that recognizes the component to be measured is bound.

[0059] In step (2), the target component may be added to latex particles to which an antibody or an antibody fragment thereof that recognizes the target component is bound, and the reaction may be allowed to proceed. Alternatively, latex particles to which an antibody or an antibody fragment thereof that recognizes the target component is bound may be added to the target component and the reaction may be allowed to proceed.

[0060] In step (2), two or more types of antibodies or antibody fragments thereof that recognize the component to be measured may be used, instead of an antibody or antibody fragment thereof that recognizes one type of component to be measured. When two types of antibodies or antibody fragments thereof that recognize the component to be measured are used, step (2) can be carried out as follows. (2A) In an aqueous medium, a component to be measured in a sample is reacted with latex particles bound to a first antibody or an antibody fragment thereof that recognizes the component to be measured, and with latex particles bound to a second antibody or an antibody fragment thereof that recognizes the component to be measured. The measurement method in this embodiment may also include the above step (3) after step (2A).

[0061] In step (2A), a first antibody or antibody fragment thereof that recognizes the component to be measured and a second antibody or antibody fragment thereof that recognizes the component to be measured react with the component to be measured, thereby producing a complex containing the component to be measured, latex particles to which the first antibody or antibody fragment thereof that recognizes the component to be measured is bound, and latex particles to which the second antibody or antibody fragment thereof that recognizes the component to be measured is bound.

[0062] In step (2A), the latex particles in the latex particles to which the first antibody or its antibody fragment that recognizes the component to be measured 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 the component to be measured is bound.

[0063] In step (2A), the reaction between the component to be measured, the latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound, and the latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound is not particularly limited as long as it is a reaction that produces a complex containing the component to be measured, the latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound, and the latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound. For example, Alternatively, the component to be measured may be mixed with latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured to generate a complex containing the component to be measured and latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured, and then latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured are added and allowed to react. Alternatively, the component to be measured may be mixed with latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured and latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured to cause a reaction.

[0064] When a component to be measured is mixed with latex particles bound to a first antibody or an antibody fragment thereof that recognizes the component to be measured and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the component to be measured, and the mixture is reacted with the component to be measured, the order in which the component to be measured, the latex particles bound to the first antibody or an antibody fragment thereof that recognizes the component to be measured, and the latex particles bound to the second antibody or an antibody fragment thereof that recognizes the component to be measured are added is not limited. For example, the order in which the component to be measured, the latex particles bound to the first antibody or an antibody fragment thereof that recognizes the component to be measured, and the second antibody or an antibody fragment thereof that recognizes the component to be measured are added is not limited. Latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound may be added; alternatively, latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound to the component to be measured, and then latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is added; or latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound and latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound may be mixed, and then the component to be measured may be added.

[0065] Step (2A) may be divided into the following steps (2A-1) and (2A-2). (2A-1) a step of reacting a target component in a sample in an aqueous medium with latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component, thereby generating a complex 1 containing the target component and latex particles bound to the first antibody or an antibody fragment thereof that recognizes the target component; and (2A-2) A step of reacting, in an aqueous medium, the complex 1 produced in step (1) with latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured, to produce a complex 2 containing the component to be measured, latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured, and latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured. The measurement method in this embodiment may also include the above step (3) after step (2A-2).

[0066] In the above step (2), step (2A), step (2A-1), or step (2A-2), an antibody or an antibody fragment thereof that recognizes the component to be measured may be bound to the latex particles in an aqueous medium. Examples of the method for binding the antibody or the antibody fragment thereof that recognizes the component to be measured to the latex particles include the binding methods described above.

[0067] 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 the component to be measured 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.

[0068] 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.

[0069] The reaction temperature for 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 the component to be measured in 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.

[0070] 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.

[0071] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes the component to be measured in step (2), step (2A), step (2A-1), or step (2A-2) above is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured according to the present 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 the component to be measured 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.

[0072] 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 the component to be measured 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).

[0073] In the above step (2A) or step (2A-2), the concentration of latex particles bound to an antibody or its antibody fragment that recognizes the component to be measured means the sum of the concentration of latex particles bound to a first antibody or its antibody fragment that recognizes the component to be measured and the concentration of latex particles bound to a second antibody or its antibody fragment that recognizes the component to be measured.

[0074] The concentration of the lithium salt 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 the component to be measured according to the present embodiment, and may be, for example, 0.01 mmol / L or more, 0.05 mmol / L or more, 0.1 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, 50 mmol / L or more, 60 mmol / L or more, 70 mmol / L or more, 80 mmol / L or more, 90 mmol / L or more, 100 mmol / L or more, or 150 mmol / L or more. The concentration of the lithium salt in the above step (2), step (2A), step (2A-1) or step (2A-2) may be, for example, 1000 mmol / L or less, 900 mmol / L or less, 800 mmol / L or less, 700 mmol / L or less, 600 mmol / L or less, 500 mmol / L or less, 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, or 200 mmol / L or less.

[0075] The above values ​​can be freely combined. For example, the concentration of the lithium salt in step (2), step (2A), step (2A-1), or step (2A-2) may be 0.01 mmol / L to 1000 mmol / L, 1 mmol / L to 500 mmol / L, 10 mmol / L to 300 mmol / L, or 50 mmol / L to 200 mmol / L. In the above step (2), step (2A), step (2A-1), or step (2A-2), one type of lithium salt may be present, or two or more types of lithium salts may be present. When two or more types of lithium salts are present, the above concentration means the total concentration of the two or more types of lithium salts.

[0076] The above step (2), step (2A), step (2A-1) or step (2A-2) is preferably carried out in an aqueous medium, such as the aqueous medium described above. Furthermore, the above-mentioned salts, sugars, proteins, etc. may be contained in the above step (2), step (2A), step (2A-1) or step (2A-2).

[0077] <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.

[0078] 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.

[0079] After the above step (3), the concentration of the component to be measured 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 the component to be measured, and creating a calibration curve showing the relationship between the concentration of the component to be measured and the measured value; (5) A step of determining the concentration of the component to be measured in the sample from the calibration curve prepared in step (4) and the measured value obtained by the measurement in step (3).

[0080] Examples of the measurement target component of known concentration include a solution obtained by diluting a commercially available measurement target component with PBS. A plurality of concentrations of the measurement target component of known concentration may be prepared. Examples of the commercially available measurement target component include commercially available NT-proBNP. Examples of commercially available NT-proBNP include 8NT2 Human recombinant NT-proBNP (manufactured by Hytest).

[0081] When the component to be measured is NT-proBNP and the component to be measured of known concentration is NT-proBNP of known concentration, the concentration of NT-proBNP in the 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, or 140 pg / ml or more. , 160pg / ml or more, 180pg / ml or more, 200pg / ml or more, 220pg / ml or more, 240pg / ml or more, 260pg / ml or more, 280pg / ml or more, 300pg / ml or more, 320pg / ml or more, 340pg / ml or more, 360pg / ml or more, 380pg / ml or more, 400pg / ml or more, 420pg / ml or more, 440pg / ml or more, 460pg / ml or more, 480pg / ml or more, 500pg / ml or more, 600pg / ml or more, 700pg / ml or more, 800pg / ml or more, 900pg / ml or more, or 1000pg / 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.

[0082] 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.

[0083] [Method for suppressing non-specific reactions caused by impurities in biological samples] By performing the method for measuring a target component in a sample according to the present embodiment, non-specific reactions caused by impurities in a biological sample can be suppressed in a latex agglutination immune reaction. 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.

[0084] (Aspect 1) A method for suppressing non-specific reactions caused by impurities in a biological sample in a reaction between a target component to be measured in a sample and an antibody or antibody fragment thereof that recognizes the target component to be measured, the method comprising: the reaction is carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[0085] (Aspect 2) A method for suppressing non-specific reactions caused by contaminants in a biological sample in a reaction between a component to be measured and an antibody or an antibody fragment thereof, the method comprising: (1) a step of mixing a sample containing the component to be measured with an aqueous medium; and (2) a step of reacting the component to be measured in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the component to be measured, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[0086] (Aspect 3) A method for suppressing non-specific reactions caused by contaminants in a biological sample in reactions between a component to be measured and the first antibody or antibody fragment, and the second antibody or antibody fragment, comprising: (1) a step of mixing a sample containing the component to be measured with an aqueous medium; and (2) a step of reacting the component to be measured in the sample in the aqueous medium with latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured, and latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[0087] In the above-mentioned suppression method, the latex particles bound with an antibody or antibody fragment thereof that recognizes the component to be measured, the latex particles bound with a first antibody or antibody fragment thereof that recognizes the component to be measured, the latex particles bound with a second antibody or antibody fragment thereof that recognizes the component to be measured, and the lithium salt can be, for example, the above-mentioned latex particles bound with an antibody or antibody fragment thereof that recognizes the component to be measured, the latex particles bound with a first antibody or antibody fragment thereof that recognizes the component to be measured, the latex particles bound with a second antibody or antibody fragment thereof that recognizes the component to be measured, and the lithium salt. Furthermore, the concentration of each additive, reaction temperature, reaction time, etc. in each step in the above-mentioned inhibition method may be, for example, the concentration of each additive, reaction temperature, reaction time, etc. in each step in the aforementioned latex agglutination immunoassay method for the component to be measured in a sample.

[0088] In aspects 1 and 2, the reaction between the target component and the antibody or antibody fragment thereof refers to an immune reaction in which the target component in a sample is reacted in an aqueous medium with latex particles to which an antibody or antibody fragment thereof that recognizes the target component is bound, and the antibody or antibody fragment thereof that recognizes the target component binds to the target component. In aspect 3, the reaction between the component to be measured and the first antibody or antibody fragment that recognizes the component to be measured and the second antibody or antibody fragment that recognizes the component to be measured refers to an immune reaction in which the component to be measured in a sample is reacted in an aqueous medium with latex particles to which the first antibody or its antibody fragment is bound and latex particles to which the second antibody or its antibody fragment is bound, and the first antibody or antibody fragment and the second antibody or antibody fragment bind to the component to be measured.

[0089] In the method of this embodiment for suppressing non-specific reactions caused by impurities in a biological sample in the reaction between the component to be measured and an antibody or an antibody fragment thereof, the component to be measured may be, for example, NT-proBNP.

[0090] [Measuring reagents for measuring target components in samples] The measurement reagent for a target component in a sample of this embodiment is a reagent used in the method for measuring a target component in a sample of this embodiment. One aspect (Aspect 1) of the measurement reagent is a reagent for measuring a target component in a sample, which comprises latex particles and an antibody or antibody fragment thereof that recognizes the target component, the reagent comprising a lithium salt, and the antibody or antibody fragment thereof being IgG1 or a fragment thereof. In embodiment 1, the latex particles and the antibody or antibody fragment thereof that recognizes the component to be measured can be bound via two affinity substances. Methods for binding the antibody or antibody fragment thereof that recognizes the component to be measured to the latex particles include, for example, the binding methods described above.

[0091] Another aspect (Aspect 2) of the measurement reagent is a latex agglutination immunoassay reagent for a target component in a sample, comprising latex particles bound to an antibody or antibody fragment thereof that recognizes the target component, the reagent comprises a lithium salt; The antibody or antibody fragment thereof is an IgG1 or a fragment thereof. Another aspect (aspect 3) of the measurement reagent is a latex agglutination immunoassay reagent for a target component in a sample, comprising latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component, and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, the reagent comprises a lithium salt; In this reagent, the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

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

[0093] The first antibody or its antibody fragment that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured, may be bound to the latex particles directly or indirectly using the aforementioned physical adsorption and / or chemical bond. Examples of indirect binding methods include a method in which the first antibody that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured, is bound to the latex particles using the specific binding of the aforementioned pair of affinity substances, or a method in which the first antibody that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured, is bound to the latex particles by a covalent bond via a linker.

[0094] When a set of affinity substances is used, latex particles bound with the first antibody or its antibody fragment that recognizes the component to be measured bound to one of the affinity substances (A) of the set, or the second antibody or its antibody fragment that recognizes the component to be measured bound to one of the affinity substances (A) of the set, can be bound to latex particles bound to the other affinity substance (a) of the set, to produce latex particles bound with the first antibody or its antibody fragment that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured. 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 measurement of the component to be measured according to the present 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 the component to be measured in the reagent of Aspect 2 or Aspect 3 is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured according to the 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 conjugated with an antibody or antibody fragment thereof that recognizes the component to be measured 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 the component to be measured 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 the component to be measured means the sum of the concentration of latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured and the concentration of latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured.

[0099] The concentration of the lithium salt in the measurement reagent for the component to be measured in this embodiment is not particularly limited as long as it is a concentration that enables the measurement method for the component to be measured in this embodiment, and may be, for example, 0.01 mmol / L or more, 0.05 mmol / L or more, 0.1 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, 50 mmol / L or more, 60 mmol / L or more, 70 mmol / L or more, 80 mmol / L or more, 90 mmol / L or more, 100 mmol / L or more, or 150 mmol / L or more. Furthermore, the concentration of lithium salt in the measurement reagent for the component to be measured in this embodiment may be, for example, 1000 mmol / L or less, 900 mmol / L or less, 800 mmol / L or less, 700 mmol / L or less, 600 mmol / L or less, 500 mmol / L or less, 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, or 200 mmol / L or less.

[0100] The above values ​​can be freely combined. For example, the concentration of the lithium salt in the measurement reagent for the component to be measured according to this embodiment may be 0.01 mmol / L to 1000 mmol / L, 1 mmol / L to 500 mmol / L, 10 mmol / L to 300 mmol / L, or 50 mmol / L to 200 mmol / L. In the measurement reagent, one type of lithium salt may be present, or two or more types of lithium salts may be present. When two or more types of lithium salts are present, the concentration refers to the total concentration of the two or more types of lithium salts.

[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. The aqueous medium used for dissolution is not particularly limited, and examples thereof include the aqueous media described above. The aqueous medium may contain the salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. described above. In the liquid test reagent, latex particles, a first antibody or antibody fragment thereof that recognizes the component to be measured, and at least one antibody selected from the group consisting of a second antibody or antibody fragment thereof that recognizes the component to be measured are mixed in an aqueous medium. The aqueous medium is not particularly limited, and examples thereof include the aqueous media described above. The aqueous medium may contain the salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. described above.

[0102] In this embodiment, the component to be measured in the measuring reagent for the component to be measured in the sample may be, for example, NT-proBNP.

[0103] [Measurement kit for components to be measured in samples] The reagent for measuring the component to be measured in the sample of this embodiment can be in the form of a kit from the viewpoint of storage, transportation, distribution, etc. Preferred embodiments of the kit for measuring a target component in a sample according to this embodiment are described below. In the following embodiments, the latex particles bound with an antibody or antibody fragment thereof that recognizes the target component, the latex particles bound with a first antibody or antibody fragment thereof that recognizes the target component, the latex particles bound with a second antibody or antibody fragment thereof that recognizes the target component, and the lithium salt can be, for example, the latex particles bound with an antibody or antibody fragment thereof that recognizes the target component, the latex particles bound with a first antibody or antibody fragment thereof that recognizes the target component, the latex particles bound with a second antibody or antibody fragment thereof that recognizes the target component, and the lithium salt. Furthermore, the component to be measured in the kit for measuring a component to be measured in a sample of this embodiment may be, for example, NT-proBNP.

[0104] Measurement kit (1) Reagent 1A containing a lithium salt; and Reagent 2A contains latex particles to which an antibody or its antibody fragment that recognizes the component to be measured is bound. Including, a measurement kit. Here, the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[0105] Measurement kit (2) Reagent 1B comprising an aqueous medium; and Reagent 2B contains a lithium salt and latex particles to which an antibody or an antibody fragment thereof that recognizes the component to be measured is bound. Including, a measurement kit. Here, the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

[0106] Measurement kit (3) 1C reagents containing lithium salts; a second C reagent containing latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound; and a third C reagent containing latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound; Including, a measurement kit. Here, the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[0107] Measurement kit (4) a first D reagent comprising an aqueous medium; a second D reagent comprising a lithium salt and latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound; and a third reagent containing latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound; Including, a measurement kit. Here, the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[0108] Measurement kit (5) Reagent 1E, comprising an aqueous medium; a second E reagent containing latex particles to which a first antibody or an antibody fragment thereof that recognizes the component to be measured is bound; and a third E reagent containing a lithium salt and latex particles to which a second antibody or an antibody fragment thereof that recognizes the component to be measured is bound; Including, a measurement kit. Here, the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[0109] Measurement kit(6) A 1F reagent containing a lithium salt; and a second F reagent comprising latex particles bound to a first antibody or an antibody fragment thereof that recognizes the component to be measured, and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the component to be measured; Including, a measurement kit. Here, the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[0110] Measurement kit(7) 1G reagent containing an aqueous medium; a second G reagent comprising a lithium salt, latex particles bound with a first antibody or an antibody fragment thereof that recognizes the component to be measured, and latex particles bound with a second antibody or an antibody fragment thereof that recognizes the component to be measured; Including, a measurement kit. Here, the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

[0111] The first antibody or its antibody fragment that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured, may be bound to the latex particles directly or indirectly using the aforementioned physical adsorption and / or chemical bond. Examples of indirect binding methods include a method in which the first antibody or its antibody fragment that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured, is bound to the latex particles using the specific binding of the aforementioned pair of affinity substances, or a method in which the first antibody or its antibody fragment that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured, is bound to the latex particles by a covalent bond via a linker.

[0112] When a set of affinity substances is used, latex particles bound with the first antibody or its antibody fragment that recognizes the component to be measured bound to one of the affinity substances (A) of the set, or the second antibody or its antibody fragment that recognizes the component to be measured bound to one of the affinity substances (A) of the set, can be bound to latex particles bound to the other affinity substance (a) of the set, thereby producing latex particles bound with the first antibody or its antibody fragment that recognizes the component to be measured, or the second antibody or its antibody fragment that recognizes the component to be measured. Examples of combinations of Aa include the combinations mentioned above. Examples of the linker include the molecules described above.

[0113] The concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes the component to be measured in the second A reagent or the second B reagent is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured in the 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 bound to an antibody or an antibody fragment thereof that recognizes the component to be measured in the second A reagent or the second B reagent may be, for example, 1% (w / v) or less, 0.8% (w / v) or less, 0.5% (w / v) or less, 0.3% (w / v) or less, 0.2% (w / v) or less, 0.1% (w / v) or less, 0.09% (w / v) or less, 0.08% (w / v) or less, 0.07% (w / v) or less, or 0.06% (w / v) or less.

[0114] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or an antibody fragment thereof that recognizes the component to be measured in the second A reagent or the second B reagent may be 0.001% (w / v) to 1% (w / v), 0.003% (w / v) to 0.5% (w / v), 0.01% (w / v) to 0.1% (w / v), or 0.02% (w / v) to 0.08% (w / v).

[0115] The concentration of latex particles bound to an antibody or an antibody fragment thereof that recognizes the component to be measured in the 2nd C reagent, the 3rd C reagent, the 2nd D reagent, the 3rd D reagent, the 2nd E reagent, the 3rd E reagent, the 2nd F reagent, or the 2nd G reagent is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured by the 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 bound to an antibody or antibody fragment thereof that recognizes the component to be measured 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.

[0116] The above values ​​can be freely combined. For example, the concentration of latex particles bound to an antibody or antibody fragment thereof that recognizes the component to be measured in the reagent may be 0.0005% (w / v) to 0.5% (w / v), 0.002% (w / v) to 0.3% (w / v), 0.005% (w / v) to 0.05% (w / v), or 0.01% (w / v) to 0.04% (w / v). In the second F reagent or the second G reagent, the concentration of latex particles bound to an antibody or its antibody fragment that recognizes the component to be measured means the sum of the concentration of latex particles bound to a first antibody or its antibody fragment that recognizes the component to be measured and the concentration of latex particles bound to a second antibody or its antibody fragment that recognizes the component to be measured.

[0117] The concentration of the lithium salt in the 1A reagent, the 2B reagent, the 1C reagent, the 2D reagent, the 3E reagent, the 1F reagent, or the 2G reagent is not particularly limited as long as it is a concentration that enables the measurement of the component to be measured in this embodiment, and may be, for example, 0.01 mmol / L or more, 0.05 mmol / L or more, 0.1 mmol / L or more, 0.5 mmol / L or more, 1 mmol / L or more, 5 mmol / L or more, 10 mmol / L or more, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, 50 mmol / L or more, 60 mmol / L or more, 70 mmol / L or more, 80 mmol / L or more, 90 mmol / L or more, 100 mmol / L or more, or 150 mmol / L or more. The concentration of the lithium salt in the reagent may be, for example, 1000 mmol / L or less, 900 mmol / L or less, 800 mmol / L or less, 700 mmol / L or less, 600 mmol / L or less, 500 mmol / L or less, 450 mmol / L or less, 400 mmol / L or less, 350 mmol / L or less, 300 mmol / L or less, 250 mmol / L or less, or 200 mmol / L or less.

[0118] The above values ​​can be freely combined. For example, the concentration of the lithium salt in the reagent may be 0.01 mmol / L to 1000 mmol / L, 1 mmol / L to 500 mmol / L, 10 mmol / L to 300 mmol / L, or 50 mmol / L to 200 mmol / L. In the reagent, one type of lithium salt may be present, or two or more types of lithium salts may be present. When two or more types of lithium salts are present, the above concentration refers to the total concentration of the two or more types of lithium salts.

[0119] The constituent reagents of the measurement kit of this embodiment may be in a lyophilized state or in a liquid state. When the reagents constituting the assay kit are in a lyophilized state, they are dissolved in an aqueous medium prior to assay to form a liquid. Examples of the aqueous medium include those mentioned above. The aqueous medium may contain the aforementioned salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc.

[0120] When the reagent components of the assay kit are liquid, at least one selected from the group consisting of a lithium salt, a first antibody or antibody fragment thereof that recognizes the component to be measured, and a second antibody or antibody fragment thereof that recognizes the component to be measured 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.

[0121] The above measurement kits (1) to (7) may contain a sample dilution reagent containing an aqueous medium as a reagent for diluting the sample. Examples of the aqueous medium include the above-mentioned aqueous media. The aqueous medium may contain the above-mentioned salts, sugars, proteins, surfactants, sensitizers, anti-IgM antibodies, etc. Furthermore, the above measurement kits (1) to (7) may also contain the above-mentioned washing solution, a standard substance reagent containing a known concentration of the component to be measured as a standard substance, and an instruction manual describing the measurement method of this embodiment.

[0122] Examples of the measurement target component of known concentration include P solution, which is obtained by diluting a commercially available measurement target component with PBS. A plurality of concentrations of the measurement target component of known concentration may be prepared. Examples of the commercially available measurement target component include commercially available NT-proBNP. 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]

[0123] 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.

[0124] 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), anti-NT-proBNP antibody (listed in Table 2), 8NT2 human recombinant The following substances were used: NT-proBNP (recombinant NT-proBNP) (manufactured by Hytest), disodium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), sodium dihydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), bovine serum albumin (BSA) (manufactured by Bovogen), Lipidure-BL103 (manufactured by NOF Corporation), lithium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), potassium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and Otsuka saline injection (physiological saline) (manufactured by Otsuka Pharmaceutical Factory, Inc.).

[0125] [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 Sulfo-NHS and EDC. Antibody A listed in Table 2 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 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 to recover the precipitate (second run). Next, a 50 mmol / L aqueous bicine solution (pH 9.0) was added to the precipitate (second time) so that the concentration of the latex became 0.5% (w / v), and the mixture was ultrasonically dispersed to obtain a solution of antibody A-bound latex particles. The process from adding the aqueous bicine solution to collecting the centrifugal precipitate was repeated twice to thoroughly remove unbound antibody A that had not bound to the latex. Next, antibody B-bound latex particle solutions, antibody C-bound latex particle solutions, antibody D-bound latex particle solutions, antibody a-bound latex particle solutions, and antibody b-bound latex particle solutions were prepared in the same manner as above, except that antibodies B, C, D, a, and b shown in Table 2 were used instead of antibody A shown in Table 2.

[0126] [Table 2]

[0127] [Experimental Example 2] Preparation of specimens for evaluation Whole blood collected from four healthy individuals was centrifuged at 800 g for 20 minutes, and the supernatant was collected to prepare four human serum samples.

[0128] [Experimental Example 3] Preparation of NT-proBNP Standards 1 to 4 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 four NT-proBNP standards with recombinant NT-proBNP contents of 0 pg / mL (no additives), 50 pg / mL, 125 pg / mL, and 500 pg / mL were prepared and designated NT-proBNP standards 1 to 4.

[0129] [Example 1] Preparation of NT-proBNP measurement kit Measurement kit A containing a first reagent and a second reagent having the following compositions: リチウム (Using antibody A and lithium chloride) was prepared. (First Reagent) Phosphate buffer 50mmol / L (pH6.0) (Prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate) BSA 10g / L Lipidure-BL103 1.1% (w / v) Lithium chloride 100mmol / L (Second reagent) 10mmol / L Bicine aqueous solution (pH8.0) 82%(v / v) Antibody A-conjugated latex particle solution 18% (v / v)

[0130] [Comparative Example 1] Preparation of NT-proBNP measurement kit Measurement kit A of Example 1 except that the 100 mmol / L lithium chloride in the first reagent was replaced with another chloride of the same concentration, or no chloride was added in place of lithium chloride. リチウム Assay kit A was prepared in the same manner as in ナトリウム (Using antibody A and sodium chloride), Measurement Kit A カリウム (Using antibody A and potassium chloride), and measurement kit A なし(Antibody A was used, and no chloride was added to replace lithium chloride) was prepared.

[0131] [Comparative Example 2] Preparation of NT-proBNP measurement kit The same procedure as in Example 1 was repeated except that the antibody A-bound latex particle solution of the second reagent was replaced with an antibody a-bound latex particle solution of the same concentration. リチウム Assay kit a was prepared in the same manner as in リチウム was prepared. In addition, the same procedure as for the above-mentioned measurement kit a was repeated except that the 100 mmol / L lithium chloride in the first reagent was replaced with another chloride of the same concentration, or no chloride was added in place of lithium chloride. リチウム Assay kit a was prepared in the same manner as in ナトリウム , measurement kit a カリウム , and measurement kit a なし was prepared.

[0132] [Example 2] Measurement of absorbance difference due to non-specific reaction in latex agglutination immunoreaction Automated analyzer 3500 (Hitachi High-Tech), measurement kit A リチウム The absorbance difference resulting from non-specific reactions was measured using the following procedure. Measurement kit: Measurement kit A リチウム Using physiological saline as a sample, the absorbance against physiological saline (blank) was measured according to the following procedure. リチウム The first reagent (92 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 570 nm with no secondary wavelength. リチウム The second reagent (34 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 570 nm with no secondary wavelength. E1 was subtracted from E2 to obtain the absorbance difference ΔE 生理食塩水 Next, the absorbance difference ΔE was calculated in the same manner as above, except that human serum 1 to 4 in Experimental Example 2 were used instead of physiological saline. ヒト血清1 , absorbance difference ΔE ヒト血清2, absorbance difference ΔE ヒト血清3 , and absorbance difference ΔE ヒト血清4 The absorbance difference ΔE was then calculated. ヒト血清1 absorbance difference ΔE 生理食塩水 The absorbance difference due to the non-specific reaction in human serum 1 was calculated by subtracting the absorbance difference ΔE ヒト血清2 , absorbance difference ΔE ヒト血清3 , and absorbance difference ΔE ヒト血清4 For each of these, the absorbance difference ΔE 生理食塩水 The absorbance difference due to non-specific reactions in each of human sera 2, 3, and 4 was calculated by subtracting the absorbance difference. The results are shown in Table 3.

[0133] [Comparative Example 3] Measurement of absorbance due to non-specific reaction in latex agglutination immunoreaction Measurement Kit A リチウム Instead, measurement kit A ナトリウム , Measurement Kit A カリウム , Measurement Kit A なし , measurement kit a リチウム , measurement kit a ナトリウム , measurement kit a カリウム , measurement kit a なし The absorbance difference resulting from the non-specific reaction in each measurement kit for human serum 1, 2, 3, or 4 was calculated in the same manner as in Example 2, except that the following was used. The results are shown in Table 3.

[0134] [Table 3]

[0135] Since each measurement kit contains only one type of latex particle bound to anti-NT-proBNP antibodies, although NT-proBNP binds to the latex particles bound to anti-NT-proBNP antibodies, multiple latex particles bound to anti-NT-proBNP antibodies cannot bind to a single NT-proBNP molecule. Therefore, it was determined that the increase in absorbance in the above test was not due to the latex agglutination reaction caused by NT-proBNP, but rather due to a nonspecific reaction. Therefore, the lower the absorbance difference calculated above (the closer to 0), the more suppressed the nonspecific reaction was determined to be. As is clear from Table 3, for each measurement kit using antibody A, which is IgG1, the absorbance difference due to non-specific reactions was lower when lithium chloride was used compared to when sodium chloride or potassium chloride was used, and when no chloride was used. Therefore, it was revealed that when a measurement kit using antibody A, which is IgG1, and lithium salt was used, non-specific reactions were suppressed more than when a measurement kit using IgG1 and sodium chloride, a measurement kit using IgG1 and potassium chloride, or a measurement kit using IgG1 but without chloride was used. In other words, it was revealed that the measurement kit using IgG1 as the antibody and lithium salt enabled accurate measurement of the target component. Furthermore, in each measurement kit using antibody a, which is IgG2a, the absorbance difference resulting from non-specific reactions did not decrease when lithium chloride was used compared to when sodium chloride or potassium chloride was used.

[0136] [Example 3] Preparation of NT-proBNP measurement kit Assay kit A of Example 1 was prepared, except that the antibody A-conjugated latex particle solution of the second reagent was replaced with the antibody B-conjugated latex particle solution, the antibody C-conjugated latex particle solution, and the antibody D-conjugated latex particle solution, all of which had the same concentration. リチウム Assay Kit B was prepared in the same manner as in リチウム , Measurement Kit C リチウム , and measurement kit Dリチウム was prepared.

[0137] [Comparative Example 4] Preparation of NT-proBNP measurement kit Measurement kit B of Example 3 except that the 100 mmol / L lithium chloride in the first reagent was replaced with potassium chloride of the same concentration. リチウム , Measurement Kit C リチウム , and measurement kit D リチウム Assay Kit B was prepared in the same manner as in カリウム , Measurement Kit C カリウム , and measurement kit D カリウム was prepared.

[0138] [Comparative Example 5] Preparation of NT-proBNP measurement kit The same procedure as in Example 1 was repeated except that the antibody A-bound latex particle solution of the second reagent was replaced with an antibody b-bound latex particle solution of the same concentration. リチウム Assay kit b was prepared in the same manner as in リチウム was prepared. In addition, the same procedure as in the above measurement kit b was repeated except that the 100 mmol / L lithium chloride in the first reagent was replaced with potassium chloride of the same concentration. リチウム Assay kit b was prepared in the same manner as in カリウム was prepared.

[0139] [Example 4] Measurement of absorbance due to non-specific reaction in latex agglutination immunoreaction Measurement Kit A リチウム Instead, measurement kit B リチウム , Measurement Kit C リチウム , or measurement kit D リチウム The absorbance difference resulting from the non-specific reaction in each measurement kit for human serum 1, 2, 3, or 4 was calculated in the same manner as in Example 2, except that the following was used. The results are shown in Table 4.

[0140] [Comparative Example 6] Measurement of absorbance due to non-specific reaction in latex agglutination immunoreaction Measurement Kit A リチウム Instead, measurement kit bリチウム , or measurement kit b カリウム The absorbance difference resulting from the non-specific reaction in each measurement kit for human serum 1, 2, 3, or 4 was calculated in the same manner as in Example 2, except that the following was used. The results are shown in Table 4.

[0141] [Table 4]

[0142] As is clear from Table 4, for each measurement kit using IgG1 antibody B, C, or D, the absorbance difference due to non-specific reactions was lower when lithium chloride was used compared to when potassium chloride was used. Therefore, it was revealed that when a measurement kit using IgG1 antibody B, C, or D and a lithium salt was used, non-specific reactions were suppressed compared to when a measurement kit using IgG1 and potassium chloride was used. In other words, it was revealed that the use of a measurement kit using IgG1 as the antibody and a lithium salt enabled accurate measurement of the target component. Furthermore, in each measurement kit using antibody b, which is IgG2a, the absorbance difference resulting from non-specific reactions did not decrease when lithium chloride was used compared to when potassium chloride was used.

[0143] [Example 5] Preparation of NT-proBNP measurement kit Measurement kit AB containing first and second reagents with the following compositions: リチウム (Using antibody A, antibody B and lithium chloride) was prepared. (First Reagent) Phosphate buffer 50mmol / L (pH6.0) (Prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate) BSA 10g / L Lipidure-BL103 1.1% (w / v) Lithium chloride 100mmol / L (Second reagent) 10mmol / L Bicine aqueous solution (pH8.0) 82%(v / v) Antibody A-conjugated latex particle solution 9% (v / v) Antibody B-bound latex particle solution 9% (v / v)

[0144] [Example 6] Measurement of NT-proBNP by latex agglutination immunoreaction Autoanalyzer 3500 (Hitachi High-Technologies Corporation), measurement kit AB for Example 5 リチウム Using the above, NT-proBNP Standards 1 to 4 of Experimental Example 3 were measured according to the following procedure, and the absorbance differences attributable to NT-proBNP in NT-proBNP Standards 1 to 4 were determined. Measurement kit: Measurement kit AB リチウム Using NT-proBNP Standard 1 as a sample, the absorbance difference relative to Standard 1 (NT-proBNP concentration 0 pg / mL, blank) was measured according to the following procedure. Standard 1 (8.8 μL) and Measurement Kit AB were added to the reaction cell. リチウム The first reagent (92 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 570 nm with no secondary wavelength. リチウム The second reagent (34 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 570 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 NT-proBNP Standard 2 (NT-proBNP concentration 50 pg / mL), Standard 3 (NT-proBNP concentration 125 pg / mL), and Standard 4 (NT-proBNP concentration 500 pg / mL) were used instead of Standard 1. 標準品2 , absorbance difference ΔE 標準品3 , and absorbance difference ΔE 標準品4 The absorbance difference ΔE was then calculated. 標準品2 absorbance difference ΔE 標準品1 The absorbance difference due to NT-proBNP in the reference standard 2 was calculated by subtracting the absorbance difference ΔE 標準品3, and absorbance difference ΔE 標準品4 For each of these, the absorbance difference ΔE 標準品1 The absorbance difference attributable to NT-proBNP in each of Standards 3 and 4 was calculated by subtracting the absorbance difference from the absorbance difference in each of Standards 3 and 4. The results are shown in Table 5.

[0145] [Table 5]

[0146] As is clear from Table 5, when a measurement kit using IgG1 antibodies A and B and a lithium salt was used, the absorbance difference increased depending on the NT-proBNP concentration in the sample, and measurement of NT-proBNP in the sample was performed without any problems. From the above, as is clear from Tables 3 and 4, it was revealed that a measurement kit using IgG1 and a lithium salt can suppress nonspecific reactions, and does not suppress reactions to the target component, NT-proBNP, making it possible to accurately measure the target component. [Industrial Applicability]

[0147] The present invention provides a method for measuring a target component in a sample, a measurement reagent, and a measurement kit that enable accurate measurement.

Claims

1. 1. A latex agglutination immunoassay method for a target component in a sample, comprising reacting the target component in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component in an aqueous medium, the method comprising: the reaction is carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

2. A latex agglutination immunoassay method for a target component in a sample, comprising: (1) a step of mixing a sample containing the target component with an aqueous medium; and (2) a step of reacting the target component in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the target component, in the aqueous medium, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

3. A latex agglutination immunoassay method for a target component in a sample, comprising: (1) a step of mixing a sample containing the target component with an aqueous medium; and (2) a step of reacting the target component in the sample in the aqueous medium with latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component, and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

4. The method according to any one of claims 1 to 3, wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

5. The method according to any one of claims 1 to 3, wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

6. The method according to any one of claims 1 to 3, wherein the component to be measured is N-terminal pro-brain natriuretic peptide (NT-proBNP).

7. A method for suppressing non-specific reactions caused by impurities in a biological sample in a reaction between a component to be measured in a sample and an antibody or an antibody fragment thereof that recognizes the component to be measured, the method comprising: reacting the component to be measured in a sample with latex particles bound to the antibody or the antibody fragment thereof in an aqueous medium; the reaction is carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

8. A method for suppressing non-specific reactions caused by contaminants in a biological sample in a reaction between a component to be measured and an antibody or an antibody fragment thereof, the method comprising: (1) a step of mixing a sample containing the component to be measured with an aqueous medium; and (2) a step of reacting the component to be measured in the sample with latex particles bound to an antibody or an antibody fragment thereof that recognizes the component to be measured, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

9. A method for suppressing non-specific reactions caused by contaminants in a biological sample in reactions between the component to be measured and the first antibody or antibody fragment, and the second antibody or antibody fragment, comprising: (1) a step of mixing a sample containing the component to be measured with an aqueous medium; and (2) a step of reacting the component to be measured in the sample in the aqueous medium with latex particles bound to a first antibody or antibody fragment thereof that recognizes the component to be measured, and latex particles bound to a second antibody or antibody fragment thereof that recognizes the component to be measured, Steps (1) and / or (2) are carried out in the presence of a lithium salt; The method, wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

10. The method according to any one of claims 7 to 9, wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

11. The method according to any one of claims 7 to 9, wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

12. The method according to any one of claims 7 to 9, wherein the component to be measured is N-terminal pro-brain natriuretic peptide (NT-proBNP).

13. A latex agglutination immunoassay reagent for a target component in a sample, comprising latex particles bound to an antibody or antibody fragment thereof that recognizes the target component, the reagent comprises a lithium salt; A reagent wherein the antibody or antibody fragment thereof is IgG1 or a fragment thereof.

14. A latex agglutination immunoassay reagent for a target component in a sample, comprising: latex particles bound to a first antibody or an antibody fragment thereof that recognizes the target component; and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, the reagent comprises a lithium salt; A reagent in which the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

15. The reagent according to claim 13 or 14, wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

16. The reagent according to claim 13 or 14, wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

17. The reagent according to claim 13 or 14, wherein the component to be measured is N-terminal pro-brain natriuretic peptide (NT-proBNP).

18. A latex agglutination immunoassay kit for a target component 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 the target component, and latex particles bound to a second antibody or an antibody fragment thereof that recognizes the target component, the first reagent and / or the second reagent contain a lithium salt; A kit wherein the first antibody or antibody fragment and / or the second antibody or antibody fragment is IgG1 or a fragment thereof.

19. The kit according to claim 18, wherein the concentration of the lithium salt is 1 mmol / L to 500 mmol / L.

20. The kit according to claim 18, wherein the concentration of the lithium salt is 10 mmol / L to 300 mmol / L.

21. The kit according to any one of claims 18 to 20, wherein the component to be measured is N-terminal pro-brain natriuretic peptide (NT-proBNP).

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