Measurement method using latex immunoagglutination method, reagent, and kit

Abstract

To provide a technique for accurately measuring a measurement target substance in a sample with high sensitivity when making measurement using a 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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method, a reagent and a kit for measuring by latex immunoagglutination. This application claims priority based on Japanese Patent Application No. 2020-061066, filed on March 30, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] In the field of clinical testing, latex immunoagglutination is a method for measuring a substance to be measured in a sample. The latex immunoagglutination method is a measurement method in which, for example, latex particles carrying antibodies 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] The immunoagglutination method using a carrier, typified by the latex agglutination method, has become the mainstream of immunoagglutination methods because it has better detection sensitivity and the like than methods that detect agglutination reactions without using a carrier. However, it is sometimes pointed out that the detection sensitivity of the immunoagglutination method is insufficient compared to other measurement methods such as chemiluminescence immunoassay.

[0004] Patent Document 1 discloses an insoluble carrier particle turbidimetric immunoassay reagent and an insoluble carrier particle turbidimetric immunoassay kit that can provide accurate measurement results by suppressing the action of plasma components that are involved in the agglutination reaction of insoluble carrier particles such as latex and affect the measurement value, thereby stabilizing the agglutination reaction and stabilizing the absorbance of the reaction solution, as well as an insoluble carrier particle turbidimetric immunoassay method that uses the reagent and kit. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2002 / 003068 Summary of the Invention [Problem to be solved by the invention]

[0006] However, it has been found that the technique described in Patent Document 1 is insufficient for improving detection sensitivity. An object of the present invention is to provide a method, a reagent and a kit capable of measuring a target substance in a sample with high sensitivity and accuracy when the measurement is performed by latex immunoagglutination. [Means for solving the problem]

[0007] That is, the present invention relates to the following [1] to

[16] . [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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts. [2] The method according to [1], wherein the content of the glycine-based buffer (A) in the step is 0.01 to 10 mass %. [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 the salts (C) in the above step is 0.01 to 20 mass %. [5] The method according to any one of [1] to [4], wherein (A) the glycine-based buffer is bicine or tricine. [6] The method according to any one of [1] to [5], further comprising (D) polyethylene glycol being present in the step. [7] The method according to [6], wherein the content of (D) polyethylene glycol in the above step is 0.01 to 10 mass%.

[0008] [8] (A) Glycine-based buffer; (B) dipropylene glycol or its derivatives; (C) Salts; 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: [9] The reagent according to [8], wherein the content of the glycine-based buffer (A) 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 %.

[10] The reagent according to [8] or [9], 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 %.

[11] The reagent according to any one of [8] to

[10] , wherein the content of the (C) salts 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 20 mass %.

[12] The reagent according to any one of [8] to

[11] , wherein the (A) glycine-based buffer is bicine or tricine.

[13] The reagent according to any one of [8] to

[12] , further comprising (D) polyethylene glycol.

[14] The reagent according to

[13] , wherein the content of the (D) polyethylene glycol 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 %.

[0009]

[15] (A) Glycine-based buffer; (B) dipropylene glycol or a derivative thereof; and (C) a first reagent containing a salt; and a second reagent containing latex particles carrying an antibody that specifically recognizes the substance to be measured, for measuring a substance to be measured in a sample using a latex immunoagglutination method.

[0010]

[16] (A) Glycine-based buffer; (B) dipropylene glycol or its derivatives; (C) Salts; and (D) a first reagent containing polyethylene glycol; a second reagent containing 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: Effect of the Invention

[0011] According to the present invention, when measurement is performed by latex immunoagglutination, a substance to be measured in a specimen can be detected with high sensitivity and accuracy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The method, reagent and kit for measuring by the latex immunoagglutination assay 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 with "~" indicates a range including the numerical values ​​before and after "~" 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 the solution 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.

[0013] "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.

[0014] "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.

[0015] "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.

[0016] 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 capable of binding to an antibody, and may be, for example, in a free form as the substance itself, or in a complex form with another substance (e.g., a protein, etc.).

[0017] "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.

[0018] "(A) Glycine-based buffer" Examples of the glycine-based buffer that is component (A) include buffers such as bicine represented by the following [Chemical formula 1], or tricine represented by the following [Chemical formula 2].

[0019] [ka]

[0020] [ka]

[0021] "(B) Dipropylene glycol or its derivatives" The dipropylene glycol, which is 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. Examples of dipropylene glycol derivatives include dipropylene glycol monoalkyl ether and dipropylene glycol dialkyl ether.

[0022] "(C) Salts" The salt as component (C) is not particularly limited as long as it is a salt that enables the measurement method of the present invention, and examples thereof include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, lithium bromide, sodium bromide, potassium bromide, calcium bromide, magnesium bromide, ammonium bromide, etc., with potassium chloride and sodium chloride being preferred.

[0023] "(D) Polyethylene glycol" The polyethylene glycol, which is the component (D), is not particularly limited as long as it is a salt that enables the measurement method of the present invention, and a commercially available product can be used. The number average molecular weight of the polyethylene glycol may be 500 to 10,000, 1,000 to 5,000, or 1,500 to 3,000. The number average molecular weight of the polyethylene glycol can be calculated from a calibration curve using standard polystyrene by gel permeation chromatography (GPC).

[0024] "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.

[0025] 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.

[0026] "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.

[0027] As the latex particles, polystyrene latex particles are particularly preferably used. The use of latex with a highly hydrophobic surface, such as polystyrene latex particles, allows for smooth adsorption of proteins and peptides. 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 the 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.

[0028] 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.

[0029] "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.

[0030] "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.

[0031] 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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts" 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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts, and the sample, the latex particles carrying an antibody that specifically recognizes the substance to be measured, (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts may be added in any order.

[0032] "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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts (hereinafter referred to as the "contact step").

[0033] 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.

[0034] 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.

[0035] The content of the (A) glycine-based 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, 0.01 to 10 mass %, 0.1 to 5 mass %, 0.5 to 2 mass %, or 0.6 to 1 mass %.

[0036] 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%, 0.005 to 0.1 mass%, or 0.01 to 0.05 mass%.

[0037] The content of the (C) salts 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 20 mass %, 0.1 to 10 mass %, or 0.5 to 5 mass %.

[0038] The content of the latex particles in the solution in which the sample is brought into contact with the antibody-carrying latex particles 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%.

[0039] In the contact step, (D) polyethylene glycol may further be present. In this case, it is sufficient that (D) polyethylene glycol is present together with (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts in a reaction solution in which a substance to be measured in a sample is reacted with latex particles carrying an antibody that specifically recognizes the substance to be measured, and the sample, the latex particles carrying the antibody, (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) salts, and (D) polyethylene glycol may each be added in any order.

[0040] The content of the (D) polyethylene glycol 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, 0.01 to 10 mass%, 0.1 to 8 mass%, 0.5 to 7 mass%, or 3 to 6 mass%.

[0041] The contacting step can further be carried out in the presence of (E) sucrose as described below. (E) Sucrose is a sugar formed by the α-1,2-glycosidic bond between the monosaccharides glucose and fructose, and is a type of disaccharide. The content of the (E) sucrose in the solution in the contacting step may be 0.01 to 10% by mass, 0.1 to 5% by mass, or 0.5 to 3% by mass.

[0042] "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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) a salt. (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).

[0043] In step (1) and / or step (2), the (A) glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts 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.

[0044] 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.

[0045] 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 in an aqueous medium, in the presence of (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts, 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, 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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts may be added and contacted in any order.

[0046] In step (1), the sample may be diluted in advance with a sample dilution solution containing at least one of the components (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) a salt, 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.

[0047] 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.

[0048] "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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, and latex particles carrying an antibody that specifically recognizes the target substance.

[0049] The reagent of the present invention for measuring an analyte in a sample using the latex immunoagglutination method may further contain (D) polyethylene glycol and / or (E) sucrose.

[0050] In a reagent for measuring a substance to be measured in a sample by latex immunoagglutination, examples of the aqueous medium, (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, (D) polyethylene glycol, (E) sucrose, 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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, (D) polyethylene glycol, (E) sucrose, 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.

[0051] 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.

[0052] The content of the glycine-based buffer (A) 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, for example, 0.01 to 10 mass%, 0.1 to 5 mass%, 0.5 to 2 mass%, or 0.6 to 1 mass%. The content of the glycine-based buffer (A) in the reagent is not particularly limited, but may be, for example, 0.01 to 10 mass%, 0.1 to 5 mass%, or 0.5 to 2 mass%.

[0053] 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%, 0.005 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%, 0.005 to 0.1 mass%, or 0.01 to 0.05 mass%.

[0054] The content of the (C) salt 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 20 mass%, 0.1 to 10 mass%, or 0.5 to 5 mass%. The content of the (C) salt in the reagent is not particularly limited, but may be 0.01 to 20 mass%, 0.1 to 10 mass%, or 0.5 to 5 mass%.

[0055] 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.

[0056] The content of the (D) polyethylene glycol 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, for example, 0.01 to 10 mass%, 0.1 to 8 mass%, 0.5 to 7 mass%, or 3 to 6 mass%. The content of the (D) polyethylene glycol in the reagent is not particularly limited, but may be, for example, 0.01 to 10 mass%, 0.1 to 8 mass%, 0.5 to 7 mass%, or 3 to 6 mass%.

[0057] The content of the (E) 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, for example, 0.01 to 10% by mass, 0.1 to 5% by mass, or 0.5 to 3% by mass. The content of the (E) sucrose in the reagent is not particularly limited, but may be, for example, 0.01 to 10% by mass, 0.1 to 5% by mass, or 0.5 to 3% by mass.

[0058] "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.

[0059] Specific embodiments of the kit of the present invention are exemplified below. Kit 1 A first reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) a salt; and A second reagent containing 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 first reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, and (D) polyethylene glycol; and A second reagent containing 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 second reagent containing (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) salts, and 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:

[0060] Kit 4 A first reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) a salt; and A second reagent comprising 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 first reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, and (D) polyethylene glycol; and A second reagent comprising 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 (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, and latex particles carrying a first antibody that specifically recognizes a substance to be measured; and A second 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 7 a first reagent containing latex particles carrying a first antibody that specifically recognizes the substance to be measured; and A second reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, 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 comprising an aqueous medium; and A second reagent comprising: (A) a glycine-based buffer; (B) dipropylene glycol or a derivative thereof; (C) a salt; 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:

[0061] Kit 9 A first reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) a salt; a second reagent containing 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:

[0062] Kit 10 A first reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, and (D) polyethylene glycol; a second reagent containing 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:

[0063] Kit 11 a first reagent comprising an aqueous medium; A second reagent comprising (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, and latex particles carrying a first antibody that specifically recognizes a 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:

[0064] Kit 12 a first reagent comprising an aqueous medium; a second reagent containing latex particles carrying a first antibody that specifically recognizes the substance to be measured; and (A) a glycine-based buffer, (B) dipropylene glycol or a derivative thereof, (C) a salt, 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:

[0065] The contents of (A) glycine-based buffer, (B) dipropylene glycol or its derivatives, (C) salts, (D) polyethylene glycol, 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.

[0066] The content of the (A) glycine-based buffer in the first, second or third reagent is not particularly limited, and may be, for example, 0.01 to 10 mass %, 0.1 to 5 mass %, 0.5 to 2 mass %, or 0.02 to 1.2 mass %.

[0067] 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%, 0.005 to 0.1 mass%, or 0.01 to 0.05 mass%.

[0068] The content of the (C) salt in the first, second or third reagent is not particularly limited, but may be 0.01 to 20% by mass, 0.1 to 10% by mass, or 0.5 to 5% by mass.

[0069] The content of the (D) polyethylene glycol in the first reagent, the second reagent or the third reagent is not particularly limited, and may be, for example, 0.01 to 10 mass%, 0.1 to 8 mass%, 0.5 to 7 mass%, or 4 to 6.5 mass%.

[0070] 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%. EXAMPLES

[0071] 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.

[0072] 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), bicine (Dojindo Laboratories), and dipropylene glycol (Kanto Chemical).

[0073] [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.

[0074] [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.

[0075] [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.

[0076] [Reference Example 4] Preparation of ANP sample ANP samples of 1 ng / mL and 10 ng / mL were 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.

[0077] [Example 1] An ANP measurement reagent 1 having the following composition was prepared. (First Reagent) Phosphate buffer (pH 7.4) 0.01 mol / L Sodium chloride 0.9% by mass BSA 1% by mass Bicine 1% by mass Dipropylene glycol 0.01% by mass (Second Reagent) Phosphate buffer (pH 7.4) 0.01 mol / L 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

[0078] [Example 2] An ANP measurement reagent 2 having the following composition was prepared. (First Reagent) Phosphate buffer (pH 7.4) 0.01 mol / L Sodium chloride 0.9% by mass BSA 1% by mass Bicine 10% by mass Dipropylene glycol 0.01% by mass (Second Reagent) Phosphate buffer (pH 7.4) 0.01 mol / L 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

[0079] [Comparative Example] An ANP measurement reagent 3 having the following composition was prepared. (First Reagent) Phosphate buffer (pH 7.4) 0.01 mol / L Sodium chloride 0.9% by mass BSA 1% by mass (Second Reagent) Phosphate buffer (pH 7.4) 0.01 mol / L 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

[0080] [Example 3] Using an automatic analyzer 3500 (Hitachi High-Technologies Corporation), three concentrations of ANP samples were measured using the measurement reagent 1 (Example 1), the measurement reagent 2 (Example 2), and the measurement reagent 3 (Comparative Example). At a reaction temperature of 37°C, the ANP sample (13 μL) and the first reagent (137 μL) were added to a cell and uniformly stirred, and then held for 5 minutes. Then, while holding at 37°C, 50 μL of the second reagent was added to the reaction solution and uniformly stirred, and the difference [ΔAbs (×10000)] 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 subtracting the ΔAbs (×10000) of the 0 ng / mL sample from the ΔAbs (×10000) of the 1 ng / mL ANP sample and the 10 ng / mL sample are shown in Table 1. At this time, the higher the value in Table 1, the higher the sensitivity of the measurement. From Table 1, it can be seen that the measuring reagent 1 (Example 1) and the measuring reagent 2 (Example 2) of the present invention can measure ANP in a sample with high sensitivity, as compared with the measuring reagent 3 (Comparative Example).

[0081] [Table 1] [Industrial Applicability]

[0082] INDUSTRIAL APPLICABILITY According to the present invention, when a measurement is performed by a latex immunoagglutination method, a substance to be measured in a specimen can be detected with high sensitivity and accuracy, and therefore the present invention is useful for the early detection of various diseases.

Claims

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 glycine-based buffer, (B) dipropylene glycol or a derivative thereof, and (C) salts.

2. The method according to claim 1, wherein the content of the glycine-based buffer (A) in the step is 0.01 to 10% by mass.

3. The method according to claim 1 or 2, wherein the content of the (B) dipropylene glycol or its derivative in the step is 0.0001 to 5 mass%.

4. The method according to any one of claims 1 to 3, wherein the content of the salt (C) in the step is 0.01 to 20 mass%.

5. The method according to any one of claims 1 to 4, wherein the glycine-based buffer (A) is bicine or tricine.

6. The method according to any one of claims 1 to 5, further comprising (D) polyethylene glycol present in said step.

7. The method according to claim 6, wherein the content of the polyethylene glycol (D) in the step is 0.01 to 10% by mass.

8. (A) a glycine-based buffer; (B) dipropylene glycol or a derivative thereof; (C) salts; 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:

9. 9. The reagent according to claim 8, wherein the content of the glycine-based buffer (A) 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 %.

10. The reagent according to claim 8 or 9, 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 %.

11. The reagent according to any one of claims 8 to 10, wherein the content of the salts (C) 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 20 mass %.

12. The reagent according to any one of claims 8 to 11, wherein the glycine-based buffer (A) is bicine or tricine.

13. The reagent according to any one of claims 8 to 12, further comprising (D) polyethylene glycol.

14. The reagent according to claim 13, wherein the content of the polyethylene glycol (D) 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 %.

15. (A) a glycine-based buffer; (B) dipropylene glycol or a derivative thereof; and (C) a first reagent containing a salt; and a second reagent containing latex particles carrying an antibody that specifically recognizes the substance to be measured.

16. (A) a glycine-based buffer; (B) dipropylene glycol or a derivative thereof; (C) salts; and (D) a first reagent containing polyethylene glycol; a second reagent containing 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:

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