Measurement method, reagent, kit, and method for suppressing non-specific reaction using latex immunoagglutination method

Abstract

To provide a technique for accurately measuring a measurement target substance in a sample when making measurement using a latex immunoagglutination method, and to provide a method of suppressing non-specific reasons caused by a biological sample while implementing the latex immunoagglutination method.SOLUTION: A method of measuring a measurement target substance in a sample using a latex immunoagglutination method is provided, the method comprising bringing the sample into contact with latex particles carrying an antibody that specifically recognizes the measurement target substance in an aqueous medium under the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring a target substance using a latex immunoagglutination method, a reagent for measuring a target substance in a sample using the latex immunoagglutination method, a kit for measuring a target substance in a sample using the latex immunoagglutination method, and a method for suppressing non-specific reactions in a measurement method using the latex immunoagglutination method. This application claims priority based on Japanese Patent Application No. 2020-061067, filed on March 30, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] As a method for measuring a substance to be measured in a biological sample, the latex immunoagglutination method is frequently used in the field of clinical testing. The latex immunoagglutination method is a measurement method in which, for example, latex particles carrying an antibody against the substance to be measured are used, and the degree of agglutination (turbidity) of the latex particles caused by binding of an antigen, which is the substance to be measured, to the antibody-carrying latex particles is detected by optical means or the like.

[0003] In the latex immunoagglutination method, some components contained in the biological sample may nonspecifically adsorb to latex particles or antibodies, resulting in a positive measurement value even when the substance to be measured is not present in the biological sample, and thus a measurement value different from the true value. As a method for suppressing such non-specific reactions, the addition of human IgM to a reaction solution has been disclosed (Patent Document 1), and coating of particle surfaces with BSA (bovine serum albumin) has also been disclosed (Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 11-287801 [Patent Document 2] JP 2004-151000 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, it has been found that the techniques described in Patent Documents 1 and 2 are insufficient in suppressing non-specific reactions caused by biological samples. An object of the present invention is to provide a method, a reagent and a kit for measuring a target substance in a sample when the measurement is performed by a latex immunoagglutination method, and a method for suppressing non-specific reactions caused by a biological sample in the latex immunoagglutination method. [Means for solving the problem]

[0006] That is, the present invention relates to the following [1] to

[15] . [1] A method for measuring a substance to be measured in a sample using a latex immunoagglutination method, comprising the step of contacting the sample with latex particles carrying an antibody that specifically recognizes the substance to be measured in an aqueous medium in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose. [2] The method according to [1], wherein the latex particles are latex particles polymerized from a monomer component containing at least styrene. [3] The method according to [1] or [2], wherein the content of (B) dipropylene glycol or a derivative thereof in the step is 0.0001 to 5 mass %. [4] The method according to any one of [1] to [3], wherein the content of (C) sucrose in the above step is 0.01 to 10 mass%. [5] The method according to any one of [1] to [4], further comprising the presence of a polyhydric alcohol (D) in the step. [6] The method according to [5], wherein the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerin, polyethylene glycol, and polypropylene glycol.

[0007] [7] (A) Buffer; (B) dipropylene glycol or its derivatives; (C) sucrose; and Latex particles carrying antibodies that specifically recognize the substance to be measured in the sample A reagent for measuring a target substance in a sample using a latex immunoagglutination method, comprising: [8] The reagent according to [7], wherein the latex particles are latex particles polymerized from a monomer component containing at least styrene. [9] The reagent according to [7] or [8], wherein the content of (B) dipropylene glycol or a derivative thereof in a solution in which a sample is brought into contact with latex particles carrying an antibody that specifically recognizes a substance to be measured is 0.0001 to 5 mass %.

[10] The reagent according to any one of [7] to [9], wherein the content of the (C) sucrose in a solution in which a sample is brought into contact with latex particles carrying an antibody that specifically recognizes a substance to be measured is 0.01 to 10 mass %.

[11] The reagent according to any one of [7] to

[10] , further comprising a polyhydric alcohol.

[12] The reagent described in

[11] , wherein the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerin, polyethylene glycol, and polypropylene glycol.

[0008]

[13] A first reagent comprising a buffer and a polyhydric alcohol; and (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a second reagent comprising latex particles carrying an antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising:

[14] The kit described in

[13] , wherein the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerin, polyethylene glycol, and polypropylene glycol.

[0009]

[15] A method for measuring a substance to be measured in a sample using a latex immunoagglutination method, comprising a step of contacting a biological sample containing the substance to be measured with latex particles carrying an antibody that specifically recognizes the substance to be measured, characterized in that the step is carried out in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose, to suppress nonspecific reactions caused by a biological sample. Effect of the Invention

[0010] According to the present invention, when measurement is performed by latex immunoagglutination, a substance to be measured in a sample can be accurately detected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The reagent, kit and method for measuring by the latex immunoagglutination method of the present invention will be described in detail below. Note that the embodiment described below shows one typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow. In this specification, a numerical range indicated using "to" indicates a range including the numerical values ​​before and after "to" as the minimum and maximum values, respectively. Furthermore, when a reagent contains multiple substances corresponding to each component, the amount of each component in the reagent means the total amount of the multiple substances present in the reagent, unless otherwise specified. In this specification, when an antibody and a substance to be measured are expressed as "binding" or "reacting," or when an antibody "recognizes" a substance to be measured, these terms include the meanings usually used in the field of the present invention, and are all used synonymously. Methods for confirming "binding" between an antibody and a substance to be measured include competitive ELISA, sandwich ELISA, and methods utilizing the principle of surface plasmon resonance, which are well known to those skilled in the art.

[0012] "Latex immunoagglutination method" The latex immunoagglutination method is a method in which a sample containing a substance to be measured is contacted in an aqueous medium with latex particles carrying an antibody that specifically recognizes the substance to be measured, the antibody is bound to the substance to be measured, a complex consisting of the substance to be measured, the antibody, and the latex particles is formed, and the latex particles are selectively agglutinated. The agglutination can be detected by measuring absorbance, scattered light, etc.

[0013] "sample" The sample is not particularly limited as long as it may contain the substance to be measured, and examples thereof include any one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice. The sample is preferably whole blood, plasma, serum, or urine. The sample may be an aqueous medium containing the substance to be measured, for example, the substance to be measured diluted with phosphate buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2), etc. Examples of the aqueous medium include the aqueous media described below.

[0014] "Measurement target substance" The substance to be measured is not particularly limited as long as it is a substance that can be measured by the measurement method of the present invention, and examples of the substance include CRP (C-reactive protein); prostate-specific antigen (PSA); ferritin; α2-macroglobulin; β2-microglobulin; myoglobin; fibrin; fibrinogen degradation products; D-dimer; thrombin-antithrombin III complex (TAT); soluble fibrin (SF); soluble interleukin-2 receptor (sIL-2R); natriuretic peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). These include rheumatoid factor (rheumatoid factor); transferrin; haptoglobin; α1-antitrypsin; α1-acidoglycoprotein; hemopexin; antithrombin-III; α-fetoprotein; 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; IgM, etc. The term "substance to be measured in a sample" refers to a substance to be measured that is present in a sample. The substance to be measured is not particularly limited as long as it is in a form that allows it to bind to an antibody, and may be, for example, in a free form as the substance itself, or in a complex form with other substances (e.g., proteins, etc.).

[0015] "Aqueous medium" The aqueous medium is not particularly limited as long as it is an aqueous medium that enables the measurement method of the present invention, and examples thereof include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred. Examples of the buffering agent contained in the buffer solution include, for example, phosphate-based, acetate-based, Tris-based, borate-based, carbonate-based, glycine-based, and Good-based buffer solutions, and Good-based buffer solutions such as HEPES, PIPES, and MOPS are preferably used. The buffer solution may be used alone or in combination of two or more types. In addition, as acids for adjusting the pH of the buffer solution containing the buffer component, organic acids such as acetic acid, as well as ordinary hydrochloric acid, sulfuric acid, and nitric acid, can be used, and as alkalis, sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonium hydroxide can be used. The aqueous medium may contain metal ions, sugars, preservatives, proteins, surfactants, and the like.

[0016] "(A) Buffer" Examples of the buffer of component (A) include phosphate, acetate, Tris, borate, carbonate, glycine, and Good's buffers. The buffer of the reagent in the present invention may be used alone or in combination of two or more. The pH of the buffer is not particularly limited, but may be pH 6 to pH 10, pH 6.5 to pH 8, or pH 6.7 to pH 7.5 so that the insoluble carrier particles do not aggregate. To adjust the pH to the above range, Good's buffers such as HEPES, PIPES, and MOPS are preferably used. In addition, acids that can be used to adjust the pH of a buffer solution containing a buffer component include ordinary hydrochloric acid, sulfuric acid, nitric acid, and organic acids such as acetic acid, and alkalis that can be used include sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, and the like.

[0017] "(B) Dipropylene glycol or its derivatives" The dipropylene glycol of component (B) is of three types: 4-oxa-2,6-heptanediol, 2-(2-hydroxy-propoxy)-propan-1-ol, and 2-(2-hydroxy-1-methyl-ethoxy)-propan-1-ol. These may be used alone or in combination of two or more.

[0018] "(C) Sucrose" (C) Sucrose, a component of sugar, is a sugar formed by the α-1,2-glycosidic bond between the monosaccharides glucose (grape sugar) and fructose (fruit sugar), and is a type of disaccharide.

[0019] "(D) Polyhydric alcohol" The polyhydric alcohol of component (D) preferably contains at least one selected from ethylene glycol, propylene glycol, dipropylene glycol, glycerin, polyethylene glycol, polypropylene glycol, and polyoxyethylene polyoxypropylene block copolymers. The average molecular weight of the polyethylene glycol, polypropylene glycol, and polyoxyethylene polyoxypropylene block copolymer may be 500 to 10,000, 1,000 to 5,000, or 1,500 to 3,000.

[0020] "Antibody that specifically recognizes the substance to be measured" The antibody that specifically recognizes the substance to be measured is not particularly limited as long as it binds to the substance to be measured and enables the measurement method of the present invention. Either a polyclonal or monoclonal antibody can be used, but a monoclonal antibody is preferred.

[0021] In the present invention, the antibody includes not only a full-length antibody but also an antibody fragment. The antibody fragment is a fragment of a desired antibody and has the same reactivity as the original monoclonal antibody. Examples of antibody fragments that can be used in the present invention include Fab, Fab', F(ab')2, Fv, and the like. These fragments can be obtained, for example, by digesting a monoclonal antibody with a protease in a conventional manner, followed by a conventional method for separating and purifying the protein. The antibody can be used by carrying a full-length antibody on latex particles, but it can also be prepared as a Fab' fragment or F(ab')2 fragment and carried on latex particles. Fab' and F(ab')2 are more preferable from the viewpoint of avoiding non-specific reactions to the Fc fragment of the antibody. As the antibody that specifically recognizes the substance to be measured, a commercially available antibody can be used.

[0022] "Latex particles" The latex particles are not particularly limited as long as they are latex particles that enable the measurement method of the present invention, and examples thereof include fine particles of organic polymeric substances, fine particles of inorganic oxides, and fine particles in which the surface of the fine particles serving as the core is surface-treated with an organic substance or the like. Specific examples thereof include synthetic resins (latexes) such as polystyrene, copolymers mainly composed of styrene, polyvinyl chloride, polypropylene, (meth)acrylic resins, polymethyl methacrylate, and silicone rubber, cellulose derivatives such as nitrocellulose, cellulose, and methylcellulose, and inorganic substances such as metals, ceramics, and glass. The latex particles may be used alone or in combination of two or more kinds. Among these, polystyrene-based synthetic polymers and polystyrene-based synthetic polymers copolymerized with acrylic acid-based monomers, monomers having sulfonic acid, and the like as components to impart electric charge are particularly preferred.

[0023] As the latex particles, polystyrene latex particles are particularly preferably used. When latex having a highly hydrophobic surface such as polystyrene latex particles is used, the adsorption of proteins and peptides can be made smooth. In addition, polystyrene latex particles obtained by soap-free polymerization without using 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. In addition, various modified latexes (e.g., carboxylic acid modified latex), magnetic latex (latex containing magnetic particles), etc. can also be used as necessary.

[0024] The shape of the latex particles is not particularly limited, and may be, for example, spherical, elliptical, or irregular. 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, by a laser diffraction particle size distribution analyzer. In this specification, the average particle size (D50) is defined as the particle size at an integrated value of 50% (volume basis) in the particle size distribution.

[0025] "Latex particles carrying antibodies that specifically recognize the substance to be measured" The method of making latex particles carry an antibody that specifically recognizes the substance to be measured may be carried out in accordance with a known method, for example, the antibody and latex particles are suspended in a buffer solution, reacted at 25°C for 1 hour, and then centrifuged, blocked, or other treatments usually performed in this field. Alternatively, a method of carrying the antibody and latex particles by chemical bonding or a method of carrying the antibody by biotin-avidin reaction may also be selected. When binding the antibody to the latex particles, it is preferable to carry out the binding under conditions that allow the antibody to maintain its reactivity and specificity with respect to the substance to be measured.

[0026] "contact" "Contacting a sample with latex particles carrying an antibody that specifically recognizes a substance to be measured" means adding the sample and the latex particles carrying the antibody to one reaction vessel and allowing them to coexist, so that the substance to be measured in the sample and the antibody carried by the latex particles can come into contact with each other. In this case, the sample and the latex particles carrying the antibody may be added in any order for contact.

[0027] The phrase "contacting the sample with latex particles carrying an antibody that specifically recognizes the substance to be measured in an aqueous medium in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose" means that the reaction solution in which the substance to be measured in the sample is reacted with the latex particles carrying an antibody that specifically recognizes the substance to be measured contains (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose, and the sample, the latex particles carrying an antibody that specifically recognizes the substance to be measured, (A) the buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose may be added in any order.

[0028] "Measurement method" The measurement method of the present invention is a method for measuring a target substance in a sample using a latex immunoagglutination method, which includes a step of contacting the sample with latex particles carrying an antibody that specifically recognizes the target substance in an aqueous medium in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose (hereinafter referred to as the "contact step").

[0029] By contacting a sample with latex particles carrying an antibody that specifically recognizes a substance to be measured, the substance to be measured in the sample reacts with the antibody carried by the latex particles, forming a complex between the substance to be measured, the antibody, and the latex particles, and the latex particles agglutinate due to the formation of the complex. The agglutination is then measured by optical means. Examples of methods for optically measuring agglutination include methods that measure absorbance, scattered light intensity, or transmitted light intensity using an optical instrument.

[0030] 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 during which the latex agglutination reaction occurs, or the amount of change per unit time. For example, when measuring absorbance, it can be measured by measuring the rate of change in absorbance per unit time from 30 seconds to 5 minutes after the start of the latex agglutination reaction, or the amount of change in absorbance per unit time. The reaction temperature is preferably 10 to 50°C, more preferably 20 to 40°C. The reaction time can be appropriately determined, and for example, a reaction time of 10 to 15 minutes can be used for measurement in a general-purpose automatic analyzer.

[0031] The content of the (A) buffer in the solution in the contact step is not particularly limited as long as it is a content that enables the measurement method of the present invention, and may be, for example, 0.001 to 10 mass %, 0.01 to 5 mass %, 0.02 to 2 mass %, or 0.5 to 1 mass %.

[0032] The content of the (B) dipropylene glycol or a derivative thereof in the solution in the contacting step is not particularly limited as long as it is a content that enables the measurement method of the present invention, and may be, for example, 0.0001 to 5 mass%, 0.0005 to 0.5 mass%, 0.001 to 0.1 mass%, or 0.01 to 0.05 mass%. The content of the (C) sucrose in the solution in the contact step is not particularly limited as long as it is a content that enables the measurement method of the present invention, and may be, for example, 0.01 to 10 mass%, 0.1 to 5 mass%, 0.5 to 3 mass%, or 0.75 to 1.25 mass%.

[0033] The content of the antibody-carrying latex particles in the solution in the contact step is not particularly limited as long as it is a content that enables the measurement method of the present invention, and may be, for example, 0.001 to 10 mass%, 0.005 to 5 mass%, 0.01 to 2 mass%, or 0.015 to 0.035 mass%.

[0034] In the contact step, the aforementioned (D) polyhydric alcohol may further be present. In this case, it is sufficient that the (D) polyhydric alcohol is present together with (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose in the reaction solution in which the analyte in the sample is reacted with the latex particles carrying an antibody that specifically recognizes the analyte, and the sample, the latex particles carrying the antibody, (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and (D) polyhydric alcohol may each be added in any order.

[0035] The content of the (D) polyhydric alcohol in the solution in the contacting step is not particularly limited as long as it is a content that enables the measurement method of the present invention, and may be, for example, 0.0001 to 10 mass%, 0.0005 to 8 mass%, 0.002 to 7 mass%, or 2 to 5 mass%.

[0036] The contact step can be further carried out in the presence of the following (E) salts: The salts are not limited, but examples thereof include sodium chloride, potassium chloride, and the like.

[0037] "Aspects of the measurement method" One aspect of the measurement method according to this embodiment includes, for example, a method including the following steps. A method for measuring a substance to be measured in a sample using a latex immunoagglutination method, comprising the following steps (1) to (3): step (1) and / or step (2) are carried out in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose. (1) contacting, in an aqueous medium, a substance to be measured in a sample with latex particles carrying a first antibody that specifically recognizes the substance to be measured; (2) adding latex particles carrying a second antibody that specifically recognizes the substance to be measured to the solution of step (1), and reacting the substance to be measured with the latex particles carrying the first antibody and the latex particles carrying the second antibody to generate agglutination; (3) measuring the agglutination produced in step (2).

[0038] In step (1) and / or step (2), the (A) buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose may be added directly to the reaction solution, and each component may be added in any order. Alternatively, each component may be included in the latex particle solution carrying the first antibody, the latex particle solution carrying the second antibody, or a solution for diluting the sample (hereinafter referred to as a "sample dilution solution"), and each component may be included in any solution.

[0039] The first antibody that specifically recognizes the substance to be measured and the second antibody that specifically recognizes the substance to be measured may be antibodies that recognize the same epitope or may be antibodies that recognize different epitopes.

[0040] Step (1) and step (2) may be performed sequentially or simultaneously, but are preferably performed simultaneously. Here, "step (1) and step (2) are performed simultaneously" means that the substance to be measured, the latex particles carrying the first antibody, and the latex particles carrying the second antibody are allowed to coexist in one reaction vessel in an aqueous medium in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose, and are in a state in which they can come into contact with each other. In this case, the substance to be measured in the sample, the latex particles carrying the first antibody, the latex particles carrying the second antibody, (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose may be added and contacted in any order.

[0041] In step (1), the sample may be preliminarily diluted with a sample dilution solution containing at least one of the components (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose, and then allowed to stand for a certain period of time before being brought into contact with latex particles carrying a first antibody that specifically recognizes the substance to be measured.

[0042] By further including the following steps after the step (3), the concentration of the substance to be measured in the sample can be determined. (4) carrying out the steps (1) to (3) using a substance to be measured having a known concentration, and creating a calibration curve showing the relationship between the concentration of the substance to be measured and the measured value; and (5) Determining the concentration of the substance to be measured in the sample from the calibration curve prepared in step (4) and the measured value of the sample.

[0043] "Reagents for measurement" The reagent of the present invention for measuring a target substance in a sample using the latex immunoagglutination method is a reagent used in the measurement method of the present invention, and comprises (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and latex particles carrying an antibody that specifically recognizes the target substance.

[0044] The reagent of the present invention for measuring a target substance in a sample using the latex immunoagglutination method may further contain (D) a polyhydric alcohol and / or (E) a salt.

[0045] In a reagent for measuring a substance to be measured in a sample by latex immunoagglutination, examples of the aqueous medium, (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, (D) a polyhydric alcohol, (E) a salt, latex particles carrying an antibody that specifically recognizes the substance to be measured, latex particles carrying a first antibody that specifically recognizes the substance to be measured, and latex particles carrying a second antibody that specifically recognizes the substance to be measured include the above-mentioned aqueous medium, (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, (D) a polyhydric alcohol, (E) a salt, latex particles carrying an antibody that specifically recognizes the substance to be measured, latex particles carrying a first antibody that specifically recognizes the substance to be measured, and latex particles carrying a second antibody that specifically recognizes the substance to be measured.

[0046] The reagent of the present invention may be in a lyophilized state or in a liquid state. When a lyophilized reagent is used, it is dissolved in an aqueous medium in advance and then subjected to measurement. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins, surfactants, etc. The liquid reagent contains an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, proteins, surfactants, etc.

[0047] The content of the (A) buffer in the solution in which the sample is brought into contact with the latex particles carrying the antibody is not particularly limited as long as it is a content that enables the measurement method of the present invention, and may be, for example, 0.001 to 10 mass%, 0.01 to 5 mass%, 0.02 to 2 mass%, or 0.5 to 1 mass%. The content of the (A) buffer in the reagent is not particularly limited, and may be, for example, 0.001 to 10 mass%, 0.01 to 5 mass%, 0.02 to 2 mass%, or 0.5 to 1 mass%.

[0048] The content of the (B) dipropylene glycol or a derivative thereof in the solution in which the sample is brought into contact with the latex particles carrying the antibody is not particularly limited, but may be 0.0001 to 5 mass%, 0.0005 to 0.5 mass%, 0.001 to 0.1 mass%, or 0.01 to 0.05 mass%. The content of the (B) dipropylene glycol or a derivative thereof in the reagent is not particularly limited, but may be 0.0001 to 5 mass%, 0.0005 to 0.5 mass%, 0.001 to 0.1 mass%, or 0.01 to 0.05 mass%.

[0049] The content of the (C) sucrose in the solution in which the sample is brought into contact with the latex particles carrying the antibody is not particularly limited, but may be 0.01 to 10 mass%, 0.1 to 5 mass%, 0.5 to 3 mass%, or 0.75 to 1.25 mass%. The content of the (C) sucrose in the reagent is not particularly limited, but may be 0.01 to 10 mass%, 0.1 to 7 mass%, 0.5 to 5 mass%, or 0.75 to 1.25 mass%.

[0050] The content of the latex particles in the solution in which the sample is brought into contact with the latex particles carrying the antibody is not particularly limited, and may be, for example, 0.001 to 10 mass%, 0.005 to 5 mass%, 0.01 to 2 mass%, or 0.015 to 0.035 mass%. The content of the latex particles in the reagent is not particularly limited, and may be, for example, 0.001 to 10 mass%, 0.005 to 5 mass%, 0.01 to 2 mass%, or 0.015 to 0.035 mass%. When used at the above concentration content, the latex particle agglutination reaction can be further stabilized.

[0051] The content of the (D) polyhydric alcohol in the solution in which the sample is brought into contact with the latex particles carrying the antibody is not particularly limited, and may be, for example, 0.0001 to 10 mass%, 0.0005 to 8 mass%, 0.002 to 7 mass%, or 2 to 5 mass%. The content of the (D) polyhydric alcohol in the reagent is not particularly limited, and may be, for example, 0.0001 to 10 mass%, 0.0005 to 8 mass%, 0.002 to 7 mass%, or 2 to 5 mass%.

[0052] "A kit for measuring" The reagent of the present invention may be in the form of a kit from the viewpoints of storage, transportation, distribution, etc. The form of the kit is not particularly limited as long as it enables the measurement method of the present invention, and examples thereof include a two-reagent kit, a three-reagent kit, etc.

[0053] Specific embodiments of the kit of the present invention are exemplified below. Kit 1 (A) a first reagent comprising a buffer; and (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a second reagent comprising latex particles carrying an antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 2 (A) a first reagent comprising a buffer and (D) a polyhydric alcohol; and (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a second reagent comprising latex particles carrying an antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 3 a first reagent comprising an aqueous medium; and (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a second reagent comprising latex particles carrying an antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising:

[0054] Kit 4 (A) a first reagent comprising a buffer; and A second reagent comprising: (A) a buffer; (B) dipropylene glycol or a derivative thereof; (C) sucrose; latex particles carrying a first antibody that specifically recognizes the substance to be measured; and latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 5 (A) a first reagent comprising a buffer and (D) a polyhydric alcohol; and A second reagent comprising: (A) a buffer; (B) dipropylene glycol or a derivative thereof; (C) sucrose; latex particles carrying a first antibody that specifically recognizes the substance to be measured; and latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 6 a first reagent comprising an aqueous medium; and A second reagent comprising: (A) a buffer; (B) dipropylene glycol or a derivative thereof; (C) sucrose; latex particles carrying a first antibody that specifically recognizes the substance to be measured; and latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising:

[0055] Kit 7 (A) a first reagent containing a buffer and latex particles carrying a first antibody that specifically recognizes a substance to be measured; and A second reagent containing (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 8 a first reagent containing latex particles carrying a first antibody that specifically recognizes the substance to be measured; and A second reagent containing (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising:

[0056] Kit 9 (A) A first reagent containing a buffer; (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a second reagent comprising latex particles carrying a first antibody that specifically recognizes the substance to be measured; and A third reagent containing latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 10 (A) a first reagent comprising a buffer and (D) a polyhydric alcohol; (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a second reagent comprising latex particles carrying a first antibody that specifically recognizes the substance to be measured; and A third reagent containing latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising:

[0057] Kit 11 (A) A first reagent containing a buffer; a second reagent containing latex particles carrying a first antibody that specifically recognizes the substance to be measured; and (A) a buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, and a third reagent comprising latex particles carrying a second antibody that specifically recognizes the substance to be measured. A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: Kit 12 (A) a first reagent comprising a buffer and (D) a polyhydric alcohol; a second reagent containing latex particles carrying a first antibody that specifically recognizes the substance to be measured; and A kit for measuring a target substance in a sample using a latex immunoagglutination method, comprising: (A) a buffer; (B) dipropylene glycol or a derivative thereof; (C) sucrose; and a third reagent comprising latex particles carrying a second antibody that specifically recognizes the target substance.

[0058] The contents of the aqueous medium, (A) buffer, (B) dipropylene glycol or a derivative thereof, (C) sucrose, (D) polyhydric alcohol, latex particles carrying an antibody that specifically recognizes the substance to be measured, latex particles carrying a first antibody that specifically recognizes the substance to be measured, and latex particles carrying a second antibody that specifically recognizes the substance to be measured in the solution in which the sample is brought into contact with the latex particles carrying the antibody are the same as those described above.

[0059] When the (A) buffer is contained in a different reagent among the first, second, or third reagent in the kit (e.g., Kit 2), each (A) buffer may be different or the same.

[0060] The content of the (A) buffer agent in the first, second or third reagent is not particularly limited, and may be, for example, 0.001 to 10 mass%, 0.01 to 5 mass%, 0.02 to 2 mass%, or 0.1 to 1.5 mass%.

[0061] The content of the (B) dipropylene glycol or a derivative thereof in the first reagent, the second reagent or the third reagent is not particularly limited, and may be 0.0001 to 5 mass%, 0.0005 to 0.5 mass%, 0.001 to 0.1 mass%, or 0.008 to 0.05 mass%.

[0062] The content of the (C) sucrose in the first, second or third reagent is not particularly limited, but may be 0.01 to 10% by mass, 0.1 to 8% by mass, 0.5 to 6% by mass or 3 to 5% by mass.

[0063] The content of the (D) polyhydric alcohol in the first reagent, the second reagent or the third reagent is not particularly limited, and may be, for example, 0.0001 to 10 mass%, 0.0005 to 8 mass%, 0.005 to 7 mass%, or 0.008 to 6 mass%.

[0064] The content of the latex particles carrying the antibody in the first reagent, the second reagent or the third reagent is not particularly limited, and may be, for example, 0.001 to 10 mass%, 0.005 to 5 mass%, 0.01 to 2 mass%, or 0.02 to 0.2 mass%.

[0065] "Method for suppressing non-specific reactions caused by biological samples" The method of the present invention for suppressing nonspecific reactions caused by a biological sample is a method for measuring a substance to be measured in a sample using a latex immunoagglutination method, which comprises a step of contacting a biological sample containing the substance to be measured with latex particles carrying an antibody that specifically recognizes the substance to be measured, and is characterized in that the step is carried out in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose.

[0066] "Non-specific reactions caused by biological samples" refers to the occurrence of a value higher than the true value when a target substance in a biological sample is measured using the latex immunoagglutination method, whereby some component contained in the biological sample non-specifically adsorbs to latex particles or antibodies, causing the latex particles to agglutinate non-specifically. "Non-specific reactions caused by biological samples" also includes a value lower than the true value when some component contained in the biological sample non-specifically adsorbs to latex particles or antibodies, inhibiting the binding between the antibody and the target substance. EXAMPLES

[0067] The present invention will be described in more detail below with reference to examples, comparative examples, and reference examples, but these are not intended to limit the scope of the present invention in any way. In the following examples, comparative examples, and reference examples, reagents from the following manufacturers were used.

[0068] The following drugs were used: KLH (Keyhole limpet hemocyanin; Thermo Fisher Scientific), human ANP (1-28) (Funakoshi), carboxyl group-modified polystyrene latex (particle size 300 nm; Fujikura Chemical), Sulfo-NHS (Thermo Fisher Scientific), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (Thermo Fisher Scientific), disodium hydrogen phosphate (anhydrous) (Kanto Chemical), sodium dihydrogen phosphate (anhydrous) (Kanto Chemical), sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), bovine serum albumin (BSA) (Bovogen), dipropylene glycol (Kanto Chemical), and sucrose (Kanto Chemical).

[0069] [Reference Example 1] Preparation of anti-ANP monoclonal antibody KLH-conjugated human ANP(1-28) was mixed with an adjuvant to form an emulsion, which was then subcutaneously injected into mice (BALB / c). The spleens were removed from mice with elevated serum antibody titers, and fused with mouse myeloma cells (P3-U1) to produce hybridomas according to a standard method. The hybridoma cells were grown in GIT medium (Kohjin Bio) containing 10% BM Condimed H1 Hybridoma Cloning Supplement (10x) (Roche Diagnostics) and HAT, and the desired hybridomas were selected. A single clone producing an antibody that specifically binds to human ANP(1-28) was then obtained using the limiting dilution method. Of the resulting clones, two clones (clone 1 and clone 2) were obtained that produced antibodies capable of sandwich immunoassay of human ANP(1-28). The antibodies produced by each clone were designated the first and second antibodies.

[0070] [Reference Example 2] Preparation of first antibody-bound latex particles The first antibody was bound to the latex particles according to a standard method using carboxyl group-modified polystyrene latex (average particle size 300 nm), EDC, Sulfo-NHS, and the first antibody.

[0071] [Reference Example 3] Preparation of second antibody-bound latex particles The primary antibody was bound to latex particles according to a standard method using carboxyl group-modified polystyrene latex (average particle size 300 nm), EDC, Sulfo-NHS, and the secondary antibody.

[0072] [Reference Example 4] Preparation of ANP sample A 1000 ng / mL ANP sample was prepared using human ANP (1-28) and Determiner CL ANP sample diluent (Hitachi Chemical Diagnostics Systems Co., Ltd.). The Determiner CL ANP sample diluent was used as a 0 ng / mL ANP sample.

[0073] [Reference Example 5] Measurement of ANP concentration in plasma of healthy subjects Ten samples of plasma from healthy individuals were purchased and the ANP concentration was measured using Determiner CL ANP. Among them, healthy individual plasma 1 was selected, which had an ANP concentration below the lower limit of the measurement range (5 pg / mL). Healthy individual plasma 1 was determined to be plasma that did not contain ANP.

[0074] [Example 1] An ANP measurement reagent 1 having the following composition was prepared. (First Reagent) Sodium dihydrogen phosphate (anhydrous) 0.023% by mass Disodium hydrogen phosphate (anhydrous) 0.115% by mass Sodium chloride 0.9% by mass BSA 1% by mass (Second Reagent) Sodium dihydrogen phosphate (anhydrous) 0.023% by mass Disodium hydrogen phosphate (anhydrous) 0.115% by mass Sodium chloride 0.9% by mass BSA 1% by mass Dipropylene glycol 0.01% by mass Sucrose 4% by mass First antibody-bound latex particles: 0.03% by mass Second antibody-bound latex particles: 0.03% by mass

[0075] [Comparative Example] An ANP measurement reagent 2 having the following composition was prepared. (First Reagent) Sodium dihydrogen phosphate (anhydrous) 0.023% by mass Disodium hydrogen phosphate (anhydrous) 0.115% by mass Sodium chloride 0.9% by mass BSA 1% by mass (Second Reagent) Sodium dihydrogen phosphate (anhydrous) 0.023% by mass Disodium hydrogen phosphate (anhydrous) 0.115% by mass Sodium chloride 0.9% by mass BSA 1% by mass First antibody-bound latex particles: 0.03% by mass Second antibody-bound latex particles: 0.03% by mass

[0076] [Example 2] Using an automatic analyzer 3500 (Hitachi High-Technologies Corporation), two concentrations of ANP samples and healthy human plasma 1 were measured using the measurement reagent 1 (Example 1) and the measurement reagent 2 (Comparative Example). At a reaction temperature of 37°C, the sample (13 μL) and the first reagent (137 μL) were added to the cell and stirred uniformly, and then held for 5 minutes. Thereafter, while holding at 37°C, 50 μL of the second reagent was added to the reaction solution and stirred uniformly, and the difference (ΔAbs) between the absorbance at 570 nm of the reaction solution after 30 seconds and the absorbance at 570 nm of the reaction solution after 300 seconds was measured. The values ​​obtained by multiplying ΔAbs by 10,000 are shown in Table 1.

[0077] [Table 1]

[0078] There was no difference in the measurement value of the ANP sample between Measurement Reagent 1 (Example 1) and Measurement Reagent 2 (Comparative Example 2). On the other hand, when healthy person's plasma 1 was measured with Measurement Reagent 2, the measurement value was 1722 [ΔAbs (×10000)], confirming that a non-specific reaction derived from the biological sample occurred. When healthy person's plasma 1 was measured with Measurement Reagent 1, the measurement value was 1290 [ΔAbs (×10000)], confirming that the non-specific reaction derived from the biological sample was suppressed. [Industrial Applicability]

[0079] INDUSTRIAL APPLICABILITY According to the present invention, when a measurement is made by the latex immunoagglutination method, a substance to be measured in a sample can be accurately measured, and therefore the present invention is useful for the early detection of various diseases.

Claims

1. A method for measuring a target substance in a sample using a latex immunoagglutination method, comprising the step of contacting the sample with latex particles carrying an antibody that specifically recognizes the target substance in an aqueous medium in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose, wherein the content of (B) dipropylene glycol or a derivative thereof in the step is 0.0001 to 5 mass %.

2. The method of claim 1 , wherein the latex particles are latex particles polymerized from monomer components including at least styrene.

3. The method according to claim 1 or 2, wherein the content of (C) sucrose in the step is 0.01 to 10% by mass.

4. The method according to any one of claims 1 to 3, further comprising (D) a polyhydric alcohol being present in the step.

5. The method according to claim 4, wherein the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerin, polyethylene glycol, and polypropylene glycol.

6. (A) a buffer; (B) dipropylene glycol or a derivative thereof; (C) sucrose; and Latex particles carrying antibodies that specifically recognize the substance to be measured in the sample A reagent for measuring a target substance in a sample by using a latex immunoagglutination method, comprising: (B) a solution in which the sample is brought into contact with latex particles carrying an antibody that specifically recognizes the target substance, and the content of the dipropylene glycol or a derivative thereof in the solution is 0.0001 to 5 mass %.

7. The reagent according to claim 6 , wherein the latex particles are latex particles polymerized from a monomer component containing at least styrene.

8. The reagent according to claim 6 or 7, wherein the content of the sucrose (C) in a solution in which the sample is brought into contact with the latex particles carrying an antibody that specifically recognizes the substance to be measured is 0.01 to 10 mass %.

9. The reagent according to any one of claims 6 to 8, further comprising a polyhydric alcohol.

10. 10. The reagent according to claim 9, wherein the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerin, polyethylene glycol, and polypropylene glycol.

11. a first reagent comprising a buffer and a polyhydric alcohol; and A kit for measuring a target substance in a sample by using a latex immunoagglutination method, comprising: (A) a buffer; (B) dipropylene glycol or a derivative thereof; (C) sucrose; and a second reagent containing latex particles carrying an antibody that specifically recognizes the target substance, A kit for performing the measurement, in which the content of (B) dipropylene glycol or a derivative thereof in a reaction solution containing the first reagent, the second reagent, and the sample and in which a substance to be measured in the sample is reacted with latex particles carrying an antibody that specifically recognizes the substance to be measured is set to 0.0001 to 5 mass %.

12. The kit according to claim 11, wherein the polyhydric alcohol is at least one selected from the group consisting of ethylene glycol, propylene glycol, glycerin, polyethylene glycol, and polypropylene glycol.

13. A method for measuring a target substance in a sample using a latex immunoagglutination method, comprising the step of contacting a biological sample containing the target substance with latex particles carrying an antibody that specifically recognizes the target substance, wherein the step is carried out in the presence of (A) a buffer, (B) dipropylene glycol or a derivative thereof, and (C) sucrose, and the content of (B) dipropylene glycol or a derivative thereof in the step is 0.0001 to 5 mass %.

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