Kit for measuring symmetrical dimethylarginine and method for measuring symmetrical dimethylarginine

Abstract

The present invention addresses the problem of providing a kit for measuring symmetric dimethylarginine (SDMA) and a method for measuring SDMA, with which it is possible to measure SDMA with high sensitivity and ease. According to the present invention, provided is a kit for measuring SDMA, the kit including: (1) particles modified by a first antibody that is a monoclonal antibody having specific binding to SDMA, the particles having a label; (2) a solid phase support; (3) a first metal thin film formed on a part of the solid phase support; and (4) SDMA that is immobilized on the first metal thin film and that is conjugated to an immunogenic substance via a linker including a first linker.

Description

Kit for measuring symmetric dimethylarginine and method for measuring symmetric dimethylarginine

[0001] The present invention relates to a kit for measuring symmetric dimethylarginine (hereinafter also referred to as SDMA) based on an antigen-antibody reaction using a solid support, and a method for measuring symmetric dimethylarginine.

[0002] Fluorescence detection is widely used as a highly sensitive and easy measurement method in biological measurements, etc. One widely used fluorescence detection method involves contacting a sample with a substance labeled with a fluorescent dye that specifically binds to the target substance, irradiating the sample with excitation light of a specific wavelength, and detecting the emitted fluorescence to confirm the presence of the target substance.

[0003] Furthermore, in order to improve the sensitivity of the fluorescence detection method, a surface plasmon fluorescence (SPF) method has been proposed, which utilizes the effect of electric field enhancement due to plasmon resonance.The SPF method is provided with a sensor chip in which a metal layer is provided in a predetermined region on a transparent support in order to generate plasmon resonance, and excitation light is incident on the interface between the support and the metal film from the side opposite to the surface on which the metal layer is formed of the support at a predetermined angle equal to or greater than the total reflection angle, and the irradiation of this excitation light generates surface plasmons in the metal layer, and the electric field enhancement effect enhances the fluorescence, thereby improving the signal / noise ratio (S / N ratio).

[0004] Symmetric dimethylarginine (SDMA) is known as one of the important renal markers along with urinary protein and urinary creatinine, and since its value increases early in the course of renal function decline, there is a growing need for it as a measurement item (Non-Patent Document 1).

[0005] Patent Documents 1 and 2 describe the detection of SDMA by competitive immunoassay using anti-SDMA polyclonal antibodies obtained from antisera, and describe the detection of SDMA by LC-MS and ELISA. Patent Document 3 describes an antibody that binds to a symmetrically dimethylated arginine analyte and can be used in an immunoassay for detecting the analyte. Patent Document 4 describes the measurement of a target substance in a biological sample by fluorescence detection using surface plasmon excitation, using a kit for measuring the target substance, the kit including labeled particles having a first binding substance and a first blocking agent that are capable of binding to the target substance in the biological sample, and a substrate having a second binding substance and a second blocking agent that are capable of binding to either the target substance or the first binding substance.

[0006] International Publication No. WO2010 / 017089 Japanese Patent Application Laid-Open No. 2022-70879 Japanese Patent Application Laid-Open No. 2023-56523 International Publication No. WO2018 / 181800

[0007] J. VET. Med 2014;25:1676-1683. Comparison of Serum Concentrations of Symmetric Dimethyoarginine and Creatinine as Kidney Function Biomarkers in Cats with Chronic Kidney Disease

[0008] The ELISA reagent shown in Figure 1 of Patent Document 1 is useful as an excellent technology for detecting SDMA, which shows a high correlation with SDMA measurement values ​​measured by liquid chromatography-mass spectrometry (LCMS). However, ELISA measurements generally require complicated steps and a long measurement time (approximately 5 hours), making them unsuitable for point-of-care applications where measurement results are required on the spot in a short time. Meanwhile, anti-SDMA monoclonal antibodies, which are preferably used in diagnostic agents for detecting SDMA quickly, easily, and with high sensitivity, are not currently available commercially.

[0009] An object of the present invention is to provide a kit for measuring SDMA and a method for measuring SDMA, which are capable of measuring SDMA with high sensitivity and ease.

[0010] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by measuring SDMA by contacting a solution containing labeled particles modified with a first antibody, which is a monoclonal antibody having specific binding affinity to SDMA, and a biological sample containing SDMA with a first metal thin film formed on a solid support, the first metal thin film having SDMA conjugated with an immunogenic substance immobilized via a linker including a first linker. The present invention was completed based on these findings. Specifically, the present invention provides the following inventions.

[0011] <1> A kit for measuring symmetric dimethylarginine (SDMA), comprising: (1) labeled particles modified with a first antibody that is a monoclonal antibody having specific binding affinity to SDMA; (2) a solid support; (3) a first metal thin film formed on a portion of the solid support; and (4) SDMA conjugated to an immunogenic substance via a linker comprising a first linker, immobilized on the first metal thin film. <2> The kit according to <1>, wherein the first antibody is an anti-SDMA monoclonal antibody produced by any of the rabbit single cell picking method, the mouse iliac lymph node method, and the rat iliac lymph node method. <3> The kit according to <1> or <2>, wherein the first antibody is an anti-SDMA monoclonal antibody obtained by immunizing a rabbit, mouse, or rat with SDMA conjugated to an immunogenic substance via a linker comprising a second linker. <4> The kit according to any one of <1> to <3>, wherein the reactivity of the first antibody with at least one of asymmetric dimethylarginine (ADMA), methylarginine, and arginine is 25% or less of the reactivity of SDMA with the first antibody. <5> The kit according to <3> or <4>, wherein the linker containing the second linker is a linker without an aromatic ring. <6> The kit according to any one of <3> to <5>, wherein the SDMA conjugated to the immunogenic substance via the linker containing the second linker is linked via a carboxyl group of the SDMA. <7> The kit according to any one of <3> to <6>, wherein the SDMA conjugated to the immunogenic substance via the linker containing the second linker is further connected between the second linker and SDMA via a third linker without an aromatic ring. <8> The kit according to any one of <3> to <7>, wherein the amount of SDMA introduced into the SDMA conjugated to the immunogenic substance via a linker containing a second linker is 3 to 25 equivalents relative to the immunogenic substance. <9> The kit according to any one of <3> to <8>, wherein the amount of SDMA introduced into the SDMA conjugated to the immunogenic substance via a linker containing the second linker is 5 to 15 equivalents relative to the immunogenic substance.<10> The kit according to any one of <1> to <9>, wherein the SDMA conjugated to the immunogenic substance via a linker containing the first linker is linked via a carboxyl group of the SDMA. <11> The kit according to any one of <1> to <10>, wherein the amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via the linker containing the first linker is 3 to 25 equivalents relative to the immunogenic substance. <12> The kit according to any one of <1> to <11>, wherein the amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via the linker containing the first linker is 5 to 15 equivalents relative to the immunogenic substance. <13> The kit according to any one of <1> to <12>, wherein the first antibody having specific binding affinity to SDMA is a rabbit anti-SDMA monoclonal antibody, and further comprising: (5) a second thin metal film formed on a portion of the solid support; and (6) an anti-rabbit IgG antibody immobilized on the second thin metal film as the second antibody not having specific binding affinity to SDMA. <14> The kit according to any one of <1> to <12>, wherein the first antibody having specific binding affinity to SDMA is a mouse anti-SDMA monoclonal antibody, and further comprising: (5) a second thin metal film formed on a portion of the solid support; and (7) an anti-mouse IgG antibody immobilized on the second thin metal film as the second antibody not having specific binding affinity to SDMA. <15> The kit according to any one of <1> to <12>, wherein the first antibody having specific binding affinity to SDMA is a rat anti-SDMA monoclonal antibody, and further comprises: (5) a second metal thin film formed on a part of the solid phase support; and (8) an anti-rat IgG antibody as the second antibody that does not have specific binding affinity to SDMA, immobilized on the second metal thin film.<16> A method for measuring symmetric dimethylarginine (SDMA), comprising: (a) contacting a solution containing labeled particles modified with a first antibody that is a monoclonal antibody having specific binding affinity to SDMA, and a biological sample containing SDMA, with a first metal thin film formed on a solid support, the first metal thin film having SDMA conjugated with an immunogenic substance immobilized via a linker that includes a first linker; and (b) detecting a signal from the first metal thin film.

[0012] According to the kit and method of the present invention, SDMA can be measured with high sensitivity and ease.

[0013] Figure 1 shows a schematic diagram of the sensor chip. Figure 2 shows an exploded view of the sensor chip. Figure 3 shows the calibration curves of Example 1 and Comparative Example 1. Figure 4 shows the results of calculating the SDMA concentrations of test samples A to D from the calibration curves of Example 1 and Comparative Example 1 and plotting them against the SDMA concentrations measured by ELISA.

[0014] Hereinafter, embodiments of the present invention will be described in detail. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0015] [Kit for measuring SDMA] The kit for measuring symmetric dimethylarginine (SDMA) according to the present invention comprises: (1) particles modified with a first antibody, which is a monoclonal antibody having specific binding affinity to SDMA, and having a label; (2) a solid support; (3) a first metal thin film formed on a portion of the solid support; and (4) SDMA conjugated to an immunogenic substance via a linker containing a first linker, which is immobilized on the first metal thin film.

[0016] <SDMA> Increased blood levels of SDMA are known to be associated with cardiovascular disease and renal disease, and in recent years, it has been used in veterinary medicine as a test for the early detection of chronic kidney disease (CKD). SDMA is derived from post-translational modification (methylation) of almost all intracellular proteins containing arginine residues. It is released into the circulating blood after proteolysis or breakdown, and most of it is excreted by the kidneys. Therefore, as the kidney's filtering capacity declines, its value increases. While conventional creatinine (CREA) only increases after approximately 75% of kidney function is lost, SDMA is said to increase after 40% loss.

[0017] Furthermore, SDMA is known to be completely free in the blood, unlike asymmetric dimethylarginine (ADMA), which exists bound to proteins in the blood.

[0018] <First antibody that is a monoclonal antibody having specific binding ability to SDMA> The first antibody of the present invention is a monoclonal antibody having specific binding ability to SDMA. That is, the first antibody of the present invention is an anti-SDMA monoclonal antibody, preferably one in which the individual antibodies have uniform properties against the analyte SDMA.

[0019] The first antibody is preferably an anti-SDMA monoclonal antibody obtained by immunizing a rabbit, mouse, or rat with SDMA conjugated to an immunogenic substance via a linker including the second linker described below.

[0020] SDMA is a low molecular weight compound and is difficult to use alone as an immunogen. However, by binding multiple SDMAs to an immunogenic substance via a linker and using the resulting compound as an immune-inducing substance, it becomes possible to produce antibodies specific to SDMA. The ratio of the number of SDMAs to the immunogenic substance is preferably 3 to 25 equivalents, more preferably 5 to 15 equivalents. Conjugating multiple SDMAs to an immunogenic substance is thought to produce a good immune-inducing effect, making it possible to produce antibodies specific to SDMA.

[0021] <Method for producing antibodies> The monoclonal antibody having specific binding ability to SDMA used in the present invention is preferably an anti-SDMA monoclonal antibody produced by any of the rabbit single cell picking method, mouse iliac lymph node method, or rat iliac lymph node method described below.

[0022] (Rabbit single cell picking method) Rabbits are known to produce antibodies with higher specificity, sensitivity, affinity, and diversity than other animal species such as mice, rats, goats, and donkeys. For example, they are known to exhibit strong immune responses even against limited epitope regions and to exhibit high stability due to the presence of additional disulfide bonds.

[0023] By using the "As One cell picking system" described in "Development of a Fully Automated Single Cell Isolation and Analysis Device" (Bioengineering Society Journal, Vol. 89, No. 2, pp. 72-78, 2011), it is possible to place each cell in a well using a microchamber with tens of thousands to hundreds of thousands of tiny wells, culture the cells, and perform ELISA measurements, thereby making it possible to pick out cells with a high ability to produce the target antibody one by one. For example, this makes it possible to pick out cells with excellent antibody production ability from approximately 200,000 monocloned cells, and this, combined with the selection of rabbits as the animal species, increases the possibility of establishing clones that produce high-performance antibodies compared to conventional methods.

[0024] (Method for producing antibodies by the mouse iliac lymph node method) First, antibody production by the mouse iliac lymph node method requires only one immunization, similar to the rat iliac lymph node method described below. This has the advantage of shortening the lead time from immunization to antibody production by about half compared with the spleen method, which requires two to three immunizations. Furthermore, it also makes it possible to reduce the amount of immunogen used by one-half to one-third (Japanese Patent No. 4098796).

[0025] (Method for producing antibodies using the rat iliac lymph node method) Antibody production using the rat iliac lymph node method, like the mouse iliac lymph node method described above, has the advantages of being able to shorten the lead time from immunization to antibody production and being able to reduce the amount of immunogen used (Japanese Patent Laid-Open No. 6-209769).

[0026] In the present invention, it is possible to select from the three different methods described above as a method for producing a monoclonal antibody that has specific binding affinity to SDMA, which provides excellent supply stability and reduces the risk of running out of manufacturing raw materials.

[0027] (Antibody Specificity) The lower the reactivity of the antibodies of the present invention with compounds structurally similar to SDMA (SDMA analogs) compared to the reactivity with the SDMA to be measured, the higher the specificity and the more preferable they can be used. Analogs to be considered include asymmetric dimethylarginine (ADMA), methylarginine, and arginine. Selecting highly specific antibodies from the antibodies obtained by the above method is preferred, as it improves the accuracy of the kit of the present invention. The reactivity of the anti-SDMA antibodies used in the present invention with at least one (preferably at least two, more preferably all three) of asymmetric dimethylarginine (ADMA), methylarginine, and arginine is preferably 25% or less, more preferably 15% or less, even more preferably 10% or less, and particularly preferably 5% or less, of the reactivity of the antibody with SDMA.

[0028] Here, reactivity refers to a value expressed as a percentage of the rate at which anti-SDMA antibodies react when a certain amount of SDMA or an SDMA analogue is reacted with an anti-SDMA antibody, and can be determined by the following method as described in the Examples below.

[0029] Specifically, the fluorescence intensities B(0) and B(100) when the SDMA concentrations are (1) 0 μg / 100 mL and (2) 100 μg / 100 mL are measured using the following measurement procedure, and the value calculated using the following formula is taken as the reactivity R. The concentrations of SDMA and SDMA-like compounds are evaluated in the range of concentrations exceeding 100 μg / 100 mL. Calculation formula for reactivity R: (Formula 1) Reactivity R (%) = [1 - {(B(100) / B(0)}] x 100

[0030] As experimental conditions, the concentration of the mouse anti-SDMA antibody solution in (3) of the [Measurement Procedure] (1) to (9) described in the Examples is adjusted so that the reactivity R calculated by the above (Equation 1) for an SDMA concentration of 100 μg / 100 mL relative to a concentration of 0 μg / 100 mL is between 60% and 97%. Using the reactivity R of SDMA and SDMA analogs calculated above, the ratio of the reactivity R to SDMA (SDMA) to the reactivity R to SDMA analogs (SDMA analogs) is calculated as a reactivity ratio, which serves as an index of antibody specificity.

[0031] Calculation formula for reactivity ratio: (Formula 2) Reactivity ratio (%) = [R (SDMA analogue) / R (SDMA)] × 100

[0032] <Second Linker> As described above, preferably, the first antibody is an antibody obtained by immunizing a rabbit, mouse, or rat with SDMA conjugated to an immunogenic substance via a linker containing a second linker.

[0033] The second linker in the present invention is a linker contained in a conjugate of an immunogenic substance used as an immunogen in producing an antibody and SDMA, and is a substance that connects the immunogenic substance and Analyte.

[0034] The linker containing the second linker is preferably a linker without an aromatic ring. N-(4-Maleimidobutyryloxy) succinimide (GMBS) is preferred as the second linker. Furthermore, as the linker containing the second linker, a maleimide-modified compound as the second linker to an immunogenic substance such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), as described in paragraph 0087 of Patent Document 1, can also be used. SDMA conjugated to an immunogenic substance via a linker containing the second linker is preferably linked via the carboxyl group of SDMA.

[0035] <Third Linker> SDMA conjugated to an immunogenic substance via a linker containing a second linker is preferably conjugated via a third linker that does not have an aromatic ring between the second linker and SDMA.

[0036] The third linker is a linker that functions as an auxiliary to the second linker, and does not function as the second linker by itself. As the third linker, aminoethanethiol (AET) is preferred.

[0037] <Immunogenic Substance> An immunogenic substance is generally a substance that has the property of acting as an antigen, and in the present invention refers to a substance that binds to SDMA via a linker. Preferred immunogenic substances include BSA (bovine serum albumin), HRP (horseradish peroxidase), KLH (keyhole limpet hemocyanin), etc.

[0038] The amount of SDMA introduced into the SDMA conjugated to the immunogenic substance via a linker containing a second linker is preferably 3 to 25 equivalents, and more preferably 5 to 15 equivalents, relative to the immunogenic substance.

[0039] <Particles Modified with a First Antibody and Having a Label> In the present invention, particles are used that are modified with a first antibody, which is a monoclonal antibody that has specific binding ability to SDMA, and that have a label.

[0040] The material and shape of the particles are not particularly limited, and examples thereof include organic polymer particles such as polystyrene beads and inorganic particles such as glass beads. Specific examples of particle materials include homopolymers obtained by polymerizing monomers such as styrene, methacrylic acid, glycidyl (meth)acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, and butyl methacrylate, as well as copolymers obtained by polymerizing two or more monomers. More preferably, the particles include a polymer selected from the group consisting of polystyrene, polystyrene copolymer, polyacrylate, polyacrylate copolymer, poly(meth)acrylate, and poly(meth)acrylate copolymer. Latex particles, in which the above homopolymers or copolymers are uniformly suspended, may also be used. Other examples of particles include other organic polymer powders, inorganic substance powders, microorganisms, blood cells, cell membrane fragments, liposomes, and microcapsules. Latex particles are preferred.

[0041] When using latex particles, specific examples of latex materials include polystyrene, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, and polyvinyl acetate acrylate. As latex, copolymers containing at least styrene as a monomer are preferred, and copolymers of styrene and acrylic acid or methacrylic acid are particularly preferred. The method for preparing latex is not particularly limited, and it can be prepared by any polymerization method. However, when using the luminescent particles of the present invention labeled with an antibody, the presence of a surfactant makes antibody immobilization difficult. Therefore, emulsifier-free emulsion polymerization, i.e., emulsion polymerization without the use of an emulsifier such as a surfactant, is preferred for preparing latex.

[0042] The label in the labeled particle is preferably a fluorescent substance. Examples of the fluorescent substance include, but are not limited to, BODIPY dyes having 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY mother nucleus) as the mother nucleus, specifically a compound represented by the following (Chemical Formula 1), and compounds having a pyrromethene skeleton capable of emitting light with even higher luminance, specifically a compound represented by the following (Chemical Formula 2).

[0043] The average particle size of labeled particles varies depending on the particle material, SDMA concentration range, measurement equipment, etc., but is preferably in the range of 0.001 to 10 μm (more preferably 0.01 to 1 μm), more preferably 30 to 500 nm, even more preferably 50 to 300 nm, particularly preferably 80 to 200 nm, and most preferably 100 to 150 nm. The average particle size of labeled particles can be measured using a commercially available particle size distribution analyzer, etc. Known methods for measuring particle size distribution include optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electric pulse measurement, chromatography, and ultrasonic attenuation, and devices corresponding to each principle are commercially available. Of these measurement methods, dynamic light scattering is preferred for measuring the average particle size of luminescent particles due to its particle size range and ease of measurement. Commercially available measuring devices using dynamic light scattering include Nanotrac UPA (Nikkiso Co., Ltd.), dynamic light scattering particle size distribution measuring device LB-550 (Horiba, Ltd.), and concentrated particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.). In the present invention, the average particle size is determined as the median diameter (d=50) measured at 25°C under conditions of a viscosity of 0.8872 CP and a refractive index of water of 1.330.

[0044] <Method for Producing Labeled Particles> The method for producing labeled particles is not particularly limited, but they can be produced by mixing a fluorescent substance with particles. For example, labeled particles can be produced by adding a fluorescent substance to particles such as latex particles. More specifically, labeled particles can be produced by adding a solution containing a fluorescent substance to a particle dispersion containing at least one of water and a water-soluble organic solvent (tetrahydrofuran, methanol, etc.) and stirring the mixture.

[0045] In the present invention, a dispersion containing labeled particles may be prepared. The dispersion can be produced by dispersing labeled particles in a dispersion medium. Examples of the dispersion medium include water, an organic solvent, or a mixture of water and an organic solvent. Examples of the organic solvent that can be used include alcohols such as methanol, ethanol, and isopropanol, and ether solvents such as tetrahydrofuran. The solids concentration of the labeled particles in the dispersion is not particularly limited, but is generally 0.1 to 20% by mass, preferably 0.5 to 10% by mass, and more preferably 1 to 5% by mass.

[0046] (Modification of Labeled Particles with First Antibody) Methods for immobilizing a first antibody on labeled particles are described, for example, in JP 2000-206115 A and the protocol accompanying Thermo Fisher's FluoSpheres® polystyrene microspheres F8813. Any known method for preparing immunoagglutination reagents can be used. Furthermore, both physical adsorption and covalent chemical bonding can be used as the principle for immobilizing an antibody on particles. Examples of blocking agents (i.e., first blocking agents) that cover the particle surfaces not coated with the antibody after immobilization of the antibody on the particles include albumin (e.g., BSA), skim milk, casein, soybean-derived components, fish-derived components, polyethylene glycol, and commercially available blocking agents for immune reactions containing the above substances or substances with similar properties to the above substances. These blocking agents can also be pretreated, for example, by partial denaturation using heat, acid, alkali, or the like, as needed. Furthermore, as a blocking agent, an antibody (globulin) that does not bind to SDMA, or a protein that is not used in the test area (protein A, protein G), etc. can also be used.

[0047] A specific method for immobilizing antibodies on particles is exemplified below. An antibody solution adjusted to a concentration of 0.01 to 20 mg / mL is added to a liquid in which particles have been dispersed to a solids concentration of 0.1 to 10% by mass, and mixed. Stirring is continued for 5 minutes to 48 hours at a temperature of 4 to 50°C. The particles are then separated from the solution by centrifugation or other methods, and any antibody contained in the solution that has not bound to the particles is thoroughly removed. The particles are then washed with a buffer solution 0 to 10 times. After mixing the particles and antibody to bind the antibody to the particles, it is desirable to protect the portions of the particle surface to which the antibody is not bound using a blocking agent, such as a component not involved in the antigen-antibody reaction, preferably a protein, more preferably globulin, albumin, Block Ace (registered trademark), skim milk, or casein.

[0048] When immobilizing antigens, antibodies, etc. on particles, a stabilizer can be added as needed. The stabilizer is not particularly limited as long as it stabilizes antigens or antibodies, and may be a synthetic polymer such as sucrose or polysaccharides, or a natural polymer, and commercially available stabilizers such as Immunoassay Stabilizer (Advanced Biotechnologies Inc. (ABI)) can also be used.

[0049] The labeled particles having the first antibody are included in the kit of the present invention, and preferably in a container that is part of the kit, such as a cup. In this case, the biological sample can be injected into the container containing the labeled particles and mixed and stirred to bind the analyte in the biological sample to the first binding substance.

[0050] <SDMA conjugated to an immunogenic substance via a linker containing a first linker, immobilized on a first metal thin film> In the present invention, SDMA bound to an immunogenic substance via a linker as a crosslinker is immobilized on a metal thin film. The SDMA conjugated to the immunogenic substance via a linker containing a first linker is preferably linked via a carboxyl group of the SDMA.

[0051] In order to detect SDMA, an analyte in a sample, with high sensitivity, it is preferable to increase the probability of SDMA being present on the metal thin film, and a conjugate in which multiple SDMAs are bound to an immunogenic substance is preferred. The amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via a linker containing a first linker is preferably 3 to 25 equivalents, more preferably 5 to 15 equivalents, relative to the immunogenic substance.

[0052] The method for immobilizing SDMA conjugated with an immunogenic substance on the first metal thin film can be, for example, a method using physical adsorption or a method using covalent chemical bonding. After immobilizing SDMA conjugated with an immunogenic substance on the first metal thin film, the surface of the solid support that is not coated with SDMA conjugated with an immunogenic substance can be covered with a blocking agent. Examples of blocking agents that can be used include known substances such as BSA, globulin, skim milk, casein, soybean-derived components, fish-derived components, and polyethylene glycol, as well as commercially available blocking agents for immune reactions containing the above substances or substances with the same properties as the above substances. These blocking agents can also be pretreated, for example, by partial denaturation using heat, acid, alkali, or the like, as needed.

[0053] <First Linker> The first linker is a linker contained in a conjugate of an immunogenic substance to be immobilized on a metal surface and SDMA. Preferred first linkers are N-(4-Maleimidobutylyloxy)succinimide (GMBS) and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS).

[0054] <Specific Example of Kit Configuration> Specific examples of more preferred configurations of the kit of the present invention are shown below.

[0055] (A) The first antibody having specific binding affinity to SDMA is a rabbit anti-SDMA monoclonal antibody, and further comprises: (5) a second metal thin film formed on a portion of the solid support; and (6) an anti-rabbit IgG antibody as a second antibody that does not have specific binding affinity to SDMA and is immobilized on the second metal thin film.

[0056] (B) The kit, wherein the first antibody having specific binding affinity to SDMA is a mouse anti-SDMA monoclonal antibody, and further comprising: (5) a second metal thin film formed on a portion of the solid support; and (7) an anti-mouse IgG antibody as a second antibody that does not have specific binding affinity to SDMA and is immobilized on the second metal thin film.

[0057] (C) A kit in which the first antibody having specific binding affinity to SDMA is a rat anti-SDMA monoclonal antibody, and further comprising: (5) a second metal thin film formed on a portion of the solid support; and (8) an anti-rat IgG antibody as a second antibody that does not have specific binding affinity to SDMA and is immobilized on the second metal thin film.

[0058] In the above embodiments (A) to (C), the signal resulting from the particles having the above-mentioned label reacted with either an anti-rabbit IgG antibody, a mouse IgG antibody, or a rat IgG antibody formed on the second metal thin film can serve as a positive control for correcting the signal resulting from the particles having the label reacted with SDMA conjugated to an immunogenic substance via a first linker formed on the first metal thin film.

[0059] <Solid Support> In the present invention, to achieve high-sensitivity measurement, it is preferable to employ a measurement method using surface plasmon fluorescence (SPF) detection, as described below. In this case, it is preferable to use a solid support having a metal film on its surface. The metal constituting the metal film is not particularly limited as long as it can generate surface plasmon resonance. Free electron metals such as gold, silver, copper, aluminum, or platinum are preferred, with gold being particularly preferred. When gold is used, the detection region described below is located on the gold film. The above metals can be used alone or in combination. Furthermore, in consideration of adhesion to the solid support, an intervening layer made of chromium or the like may be provided between the solid support and the metal layer. The thickness of the metal film is optional, but is preferably 1 nm to 500 nm, and particularly preferably 10 nm to 200 nm. If the thickness exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be adequately detected. Furthermore, when an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 0.1 nm to 10 nm.

[0060] The metal film may be formed by a conventional method, such as sputtering, vapor deposition, ion plating, electroplating, or electroless plating, but in order to provide a mixed layer of the solid support material and the metal film and improve the adhesion of the metal film, it is preferable to prepare the metal film by sputtering. In this case, the thickness of the mixed layer of the solid support material and the metal film is not particularly limited as long as sufficient adhesion can be ensured, but it is preferably 10 nm or less.

[0061] The metal film is preferably disposed on a solid support. Here, "disposed on a solid support" refers not only to the case where the metal film is disposed on the solid support in direct contact with the solid support, but also to the case where the metal film is disposed via another layer without direct contact with the solid support. Materials for the solid support that can be used in the present invention include, for example, optical glass such as BK7 (borosilicate glass), a type of common optical glass, or synthetic resins, specifically, materials transparent to laser light such as polymethyl methacrylate, polyethylene terephthalate, polycarbonate, or cycloolefin polymer. Such solid supports are preferably made of materials that do not exhibit anisotropy with respect to polarized light and have excellent processability.

[0062] A preferred embodiment of the solid support for detecting SPF is a solid support in which a gold film is vapor-deposited onto polymethyl methacrylate (PMMA).

[0063] <Other Elements of the Kit> The kit of the present invention is used in a method for measuring SDMA. In the present invention, when measuring SDMA, the kit includes a solid support and a sensor chip including a member holding labeled particles such as fluorescent particles. However, various equipment or devices used for measuring the substance to be measured, such as a surface plasmon excitation device and a fluorescence measurement device, may also be included. Further elements of the kit may include a sample containing a known amount of the substance to be measured, an instruction manual, etc.

[0064] [Method for measuring SDMA] The method for measuring symmetric dimethylarginine (SDMA) according to the present invention comprises the steps of: (a) contacting a solution containing labeled particles modified with a first antibody, which is a monoclonal antibody having specific binding affinity to SDMA, and a biological sample containing SDMA with a first metal thin film formed on a solid support, the first metal thin film having SDMA conjugated with an immunogenic substance immobilized via a linker containing a first linker; and (b) detecting a signal from the first metal thin film.

[0065] The measurement in the present invention is interpreted as the broadest concept as long as it is the measurement of SDMA, and includes the quantification of SDMA, etc. A specific embodiment of the measurement method is a competitive method.

[0066] In the competitive method, a biological sample containing SDMA and anti-SDMA antibody-labeled fluorescent particles are first contacted with a solid support on which SDMA is immobilized. If SDMA is not present in the biological sample, an antigen-antibody reaction occurs on the solid support between the anti-SDMA antibody-labeled fluorescent particles and the SDMA on the solid support. On the other hand, if SDMA is present in the biological sample, an antigen-antibody reaction occurs between the SDMA in the biological sample and the anti-SDMA antibody-labeled fluorescent particles, inhibiting the antigen-antibody reaction between the anti-SDMA antibody-labeled fluorescent particles and the SDMA on the solid support. After the reaction is completed, there are methods that remove the anti-SDMA antibody-labeled fluorescent particles that have not bound to SDMA on the solid support, and methods that do not require removal. However, methods that do not require removal of unreacted particles are preferred from the perspective of measurement time and instrument size. Next, the degree of formation of an immune complex (i.e., a complex between the anti-SDMA antibody-labeled fluorescent particles and the SDMA on the solid support) on the solid support is detected as fluorescence intensity, allowing the concentration of SDMA in the biological sample to be measured.

[0067] The fluorescence measurement format in the competitive method can be either plate reader measurement or flow measurement, and can be performed, for example, by the following method. Multiple samples with different SDMA concentrations and known SDMA amounts are prepared in advance, and these samples are premixed with anti-SDMA antibody-labeled fluorescent particles. This mixture is contacted with the SDMA-immobilized region. Fluorescence signals from the SDMA-immobilized region are measured as multiple fluorescence signals at specific time intervals while the mixture is in contact with the conjugate. From these multiple fluorescence signals, the time change (slope) of the fluorescence intensity at each SDMA concentration is determined. This time change is plotted on the Y-axis and the SDMA concentration on the X-axis, and an appropriate fitting method, such as the least squares method, is used to obtain a relationship equation between the SDMA concentration and the time change in fluorescence intensity. Based on the relationship equation thus obtained, the amount of SDMA contained in the biological sample can be quantified using the results of the time change in fluorescence intensity using the biological sample to be tested.

[0068] The quantification of SDMA is preferably carried out within a short period of time. Specifically, it is preferably carried out within 10 minutes, more preferably within 8 minutes, and even more preferably within 6 minutes. This quantification time preferably includes the time from contacting the sample and anti-SDMA antibody-labeled fluorescent particles with the detection region where SDMA is immobilized to converting the amount of SDMA contained in the biological sample based on the result of the change in the fluorescence intensity over time using the biological sample to be tested, using a relationship between the time change in the fluorescence intensity and the SDMA concentration previously obtained using an appropriate fitting method such as the least squares method.

[0069] (Flow Channel) In a preferred embodiment of the present invention, a mixture of a biological sample that may contain SDMA and labeled particles having a first antibody can be applied to a solid support and developed in a flow channel. The flow channel is not particularly limited as long as it is a passage through which the biological sample and labeled particles having the first antibody flow down to the detection region. A preferred embodiment of the flow channel has a structure that includes a drop port for dropping a biological sample liquid containing labeled particles having the first antibody, a metal membrane as the detection region, and a flow channel beyond the metal membrane, allowing the biological sample to pass over the metal membrane. Preferably, a suction port can be provided on the metal membrane on the opposite side of the drop port.

[0070] (Surface Plasmon Fluorescence Measurement) The method for detecting a label such as fluorescence in the present invention is not particularly limited, but it is preferable to detect the fluorescence intensity using, for example, an instrument capable of detecting fluorescence intensity, specifically, a microplate reader, a biosensor for detecting fluorescence by surface plasmon excitation (SPF), etc. Preferably, fluorescence detection by surface plasmon resonance can obtain label information related to the amount of the substance to be measured.

[0071] The fluorescence measurement may be performed using a plate reader or a flow measurement. The fluorescence detection method using surface plasmon excitation (SPF method) is preferably used because it can provide higher sensitivity than the fluorescence detection method using epi-excitation (epi-fluorescence method), does not require B / F separation, and can therefore reduce the size of the device and shorten the measurement time.

[0072] An example of a surface plasmon fluorescence (SPF) biosensor that can be used is a sensor, such as that described in Japanese Patent Application Laid-Open No. 2008-249361, that includes an optical waveguide formed from a material that transmits excitation light of a predetermined wavelength, a metal film formed on one surface of the optical waveguide, a light source that generates a light beam, an optical system that passes the light beam through the optical waveguide and makes it incident on the interface between the optical waveguide and the metal film at an incident angle that generates surface plasmons, and fluorescence detection means that detects fluorescence generated by excitation by evanescent waves enhanced by the surface plasmons.

[0073] The surface plasmon excitation fluorescence detection (SPF) system using the fluorescent particles of the present invention is an assay method for detecting fluorescence from a fluorescent substance, preferably immobilized on a metal film on a solid support, depending on the amount of SDMA. This method differs from the so-called latex agglutination method, which detects changes in optical transparency as turbidity due to the progress of a reaction in a solution. In the latex agglutination method, antibody-sensitized latex in a latex reagent binds to and agglutinates an antigen in a biological sample through an antibody reaction. These agglutinates grow over time, and the SDMA concentration is quantified from the change in absorbance per unit time obtained by irradiating the agglutinates with near-infrared light. The present invention provides a method for measuring SDMA that is significantly simpler and more sensitive than the latex agglutination method.

[0074] (Normalization) Furthermore, the method of the present invention may include a labeled particle-related labeled information acquisition step of acquiring labeled information related to the amount of labeled particles; and a normalization step of normalizing the labeled information acquired in the measured substance-related labeled information acquisition step of acquiring labeled information related to the amount of SDMA, using the labeled information acquired in the labeled particle-related labeled information acquisition step.

[0075] Here, a mixture containing a biological sample and labeled particles having a first antibody capable of binding to SDMA is contacted with a solid-phase support having a detection area (test area) and a reference area (control area) to generate surface plasmons in the detection area and the reference area, and the intensity of the emitted fluorescence is measured. The step of measuring the intensity of the fluorescence due to the surface plasmons generated in the detection area is a analyte-related label information acquisition step for acquiring label information related to the amount of SDMA, and the step of measuring the intensity of the fluorescence due to the surface plasmons generated in the reference area is a label particle-related label information acquisition step. The rate of increase in the fluorescence intensity acquired in these two steps per unit time is calculated as the rate of change of the fluorescent signal value, and the rate of change of the signal value in the detection area is divided by the rate of change of the signal value in the reference area to form a normalization step.

[0076] The present invention will be explained in more detail below by way of examples. Note that the materials, amounts used, ratios, processing details, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0077] [Synthesis Examples of Raw Materials Used] 1. Preparation of SDMA-Immunogenic Substance Conjugate (1) Preparation of SDMA-AET-GMBS-BSA (Carboxyl Group Crosslinked) Using SDMA (Med Chem Express, Catalog No. HY-101410-50 mg) as a starting material, SDMA-AET-GMBS-BSA (carboxyl group crosslinked) was synthesized according to the procedures described in Scheme 1 and Scheme II of Patent Document 1 (AET: Aminoethanol, GMBS: N-(4-Maleimidobutylyloxy) succinimide, BSA: Bovine Serum Albumin). In Scheme II, maleimide-activated BSA was used as the maleimide-activated protein. Four types of SDMA introduced per BSA molecule were prepared by adjusting the amount of SDMA added, and the amounts were determined to be 6 equivalents, 10 equivalents, 20 equivalents, and 30 equivalents by MALDI-MS measurement.

[0078] (2) Preparation of SDMA-AET-MBS-HRP (carboxyl group crosslinked) SDMA (Med Chem Express, Catalog No. HY-101410-50 mg) was used as the starting material, and SDMA-AET-MBS-HRP (carboxyl group crosslinked) was synthesized according to the procedures described in Scheme 1 and Scheme II of Patent Document 1 (MBS: m-maleimidobenzoyl-N-hydroxysuccinimide ester, HRP: horseradish peroxidase). In Scheme II, maleimide-activated HRP was used as the maleimide-activated protein. The number of SDMA molecules introduced per HRP molecule was determined to be 10 equivalents by MALDI-MS measurement.

[0079] (3) Preparation of SDMA-MBS-BSA (α-amino group crosslinked) SDMA (Med Chem Express, Catalog No. HY-101410-50 mg) was used as a starting material, and SDMA-MBS-BSA (α-amino group crosslinked) was synthesized according to the procedure described in FIG. 2 of Reference 1 and the corresponding text. In FIG. 2 of Reference 1, SDMA was used as the hapten. The number of SDMA introduced per BSA molecule was determined to be approximately 16 equivalents based on the change in molecular weight before and after introduction.

[0080] Cited document 1: Enzyme Immunoassay of Blasticidin S with High Sensitivity: A New and Conventional Method for Preparation of Immunogenic (Hapten-Protein) Conjugates (J.Biochem. 92, 585-590 (1982))

[0081] 2. Preparation of Anti-SDMA Monoclonal Antibodies (1) Preparation of Rabbit Anti-SDMA Monoclonal Antibodies by Rabbit Single Cell Picking Method Three rabbits were immunized with the immunogen described above in 1. (1) SDMA-AET-GMBS-BSA (carboxyl group cross-linked, number of SDMA introduced = 10 equivalents), and peripheral blood mononuclear cells (PBMCs) were collected 10 weeks later. SDMA-positive individuals were then selected by ELISA evaluation of the PBMC culture supernatant. Next, using the AS ONE Cell Picking System (manufactured by AS ONE Co., Ltd.), a single cell assay system based on Reference 2, a microchamber was coated with 1. (2) SDMA-AET-MBS-HRP (carboxyl group cross-linked) to select single cells with high antibody production ability from the obtained positive cells. The antibody gene was then isolated, and recombinant rabbit anti-SDMA monoclonal antibodies were produced.

[0082] Reference 2: Development of a fully automated single cell isolation and analysis device (Bioengineering Society Journal, Vol. 89, No. 2, 72-78, 2011)

[0083] (2) Preparation of mouse anti-SDMA monoclonal antibodies by the iliac lymph node method Three mice were immunized with the immunogen described above in 1. (1) SDMA-AET-GMBS-BSA (carboxyl group cross-linked, number of SDMA introduced = 10 equivalents), and mouse anti-SDMA monoclonal antibodies were prepared as follows.

[0084] 100 μg of immunogen (SDMA-AET-GMBS-BSA (carboxyl group cross-linked, number of SDMA introduced = 10 equivalents)) was injected once per mouse into the base of the tail on both sides. 14 days later, iliac lymph nodes were removed and used to carry out cell fusion with SP2 myeloma cells using polyethylene glycol solution. After cell fusion, the fused cells were plated onto four 96-well culture plates. One week after fusion, the culture supernatant was analyzed by ELISA to determine whether the target antibody was produced by adsorbing SDMA-MBS-BSA (α-amino group cross-linked) as the analysis antigen. Wells with an absorbance of more than 0.3 were considered positive.

[0085] The obtained positive wells were monocloned and screened by ELISA (enzyme-linked immunosorbent assay) to obtain mouse anti-SDMA monoclonal antibodies.

[0086] The obtained mouse anti-SDMA monoclonal antibodies were evaluated for their reactivity to SDMA and SDMA analogues (asymmetric dimethylarginine (ADMA), methylarginine, and arginine).

[0087] Due to clinical significance, the quantitative range of SDMA was set to 0 to 100 μg / 100 mL, and the fluorescence intensities B(0) and B(100) at SDMA concentrations of (1) 0 μg / 100 mL and (2) 100 μg / 100 mL were measured using the following measurement procedure, and the value calculated using the following formula was defined as the reactivity R. The concentrations of SDMA and SDMA-like compounds were evaluated in the range of concentrations exceeding 100 μg / 100 mL.

[0088] Calculation formula for reactivity R: (Formula 1) Reactivity R (%) = [1 - {(B (100) / B (0)}] x 100

[0089] As experimental conditions, the concentration of the mouse anti-SDMA antibody solution in (3) of the measurement procedure below was adjusted so that the reactivity R calculated by the above (Equation 1) for an SDMA concentration of 100 μg / 100 mL relative to a concentration of 0 μg / 100 mL would be between 60% and 97%. Using the reactivity R of SDMA and SDMA analogs calculated above, the ratio of the reactivity R to SDMA (SDMA) to the reactivity R to SDMA analogs (SDMA analogs) was calculated as the reactivity ratio, which serves as an index of antibody specificity.

[0090] Calculation formula for reactivity ratio: (Formula 2) Reactivity ratio (%) = [R (SDMA analogue) / R (SDMA)] × 100

[0091] [Measurement Procedure] (1) Coat the plate with anti-mouse IgG antibody (Fc antibody, 50 μL of a 2.5 μg / mL solution). (2) Wash three times with detergent (PBS / 0.05% Tween 100). (3) Coat a quantity of mouse anti-SDMA antibody solution (50 μL of a 3.3 mg / mL solution) that falls within the reactivity range described above. (4) Coat a solution of one of SDMA, ADMA, N-methylarginine, or arginine adjusted to the following concentrations: (1) 0 μg / 100 mL, (2) 1 μg / 100 mL, (3) 10 μg / 100 mL, (4) 100 μg / 100 mL, or (5) 1000 μg / 100 mL. (5) Coat with HRP-labeled SDMA solution (synthesized in the above "(2) Preparation of SDMA-AET-MBS-HRP (carboxyl group cross-linking)") (50 μL of 1.1 μg / mL solution) and allow to react at room temperature for 30 minutes. (6) Wash three times with detergent (PBS / 0.05% Tween 100). (7) Add substrate (3,3',5,5'-tetramethylbenzidine, Dojindo Laboratories) and incubate in the dark for 30 minutes. (8) Add reaction stopper (2N sulfuric acid) to stop the reaction. (9) Measure the fluorescence intensity B at a wavelength of 450 nm.

[0092] The results are shown in Tables 1 to 4 below. Measurement was performed at an antibody concentration where the reactivity R to SDMA was 87%, and the reactivity ratio (to SDMA) was 1.1% for ADMA, -3.4% for arginine, and 8.7% for methylarginine, all of which were less than 25%, demonstrating the high specificity of this antibody to SDMA.

[0093]

[0094]

[0095]

[0096]

[0097] (3) Preparation of rat anti-SDMA monoclonal antibody by iliac lymph node method Three rats were immunized with the immunogen described in 1. (1) SDMA-AET-GMBS-BSA (carboxyl group cross-linked, number of SDMA introduced = 10 equivalents), and rat anti-SDMA monoclonal antibody was prepared as follows.

[0098] SDMA-AET-GMBS-BSA (carboxyl group crosslinked, number of SDMA introduced = 10 equivalents) was used as an immunogen and injected into the sole of a rat's hind paw together with an immunostimulant. Three weeks later, cells from enlarged lymph nodes were used for cell fusion with mouse myeloma cells. Ten days later, the culture supernatant was analyzed by ELISA using a plate adsorbed with SDMA-MBS-BSA (α-amino group crosslinked) as the analysis antigen to determine whether the target antibody was produced. Wells with an absorbance greater than 0.3 were considered positive, and rat anti-SDMA monoclonal antibodies were extracted from these positive wells.

[0099] (4) Preparation of Rabbit Anti-SDMA Polyclonal Antibody A commercially available rabbit anti-SDMA polyclonal antibody (Merck Millipore, SYM10, Catalog No. 07-412) was purchased.

[0100] [Preparation of SDMA Measurement Reagent (Sensor Chip)] 1. Preparation of Fluorescently Labeled Particles Modified with Anti-SDMA Antibody (1) Preparation of Latex Particles with an Average Particle Diameter of 150 nm 30 g (288 mmol) of styrene (Fujifilm Wako Pure Chemical Industries, Ltd.) and 3 g (42 mmol) of acrylic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) were suspended in 440 mL of ultrapure water, heated to 95°C, and an aqueous solution of 1 g of potassium persulfate (KPS) (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved in 10 mL of ultrapure water was added. The mixture was stirred at 95°C and 250 rpm for 6 hours. Then, the mixture was centrifuged three times at 10,000 rpm for 6 hours to obtain latex particles. Finally, the obtained latex particles were redispersed in ultrapure water. Pure water was added to prepare a diluted solution with a solids concentration of 1% by mass. The average particle size of the latex particles was determined as a median diameter (d=50) measured at 25° C. using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), and was found to be 150 nm.

[0101] (2) Preparation of Fluorescent Latex Particles: 100 mL of an aqueous dispersion of latex particles with an average particle size of 150 nm and a solids concentration of 2% by weight prepared as described above was added to 100 mL of methanol and stirred for 10 minutes at room temperature. Separately, 12 mg of the fluorescent dye 4,4-difluoro-1,3,5,7-tetraphenyl-4-bora-3a,4a,8-triaza-s-indecene (Chemical Formula 3), dissolved in 1 mL of N,N-dimethylformamide (DMF), 9 mL of CHCl3, and 16 mL of ethanol, was slowly added dropwise to the latex solution over 60 minutes. After the addition was complete, the organic solvent was removed under reduced pressure using an evaporator. Then, the mixture was centrifuged and redispersed in phosphate-buffered saline (PBS) three times for purification, yielding fluorescent latex particles.

[0102] (3) Preparation of Fluorescently Labeled Particles Modified with Anti-SDMA Antibody Fluorescent particles modified with anti-SDMA antibody were prepared as follows. 39 μL of a 250 mmol / L MES (2-(N-morpholino)ethanesulfonic acid) buffer solution (pH 6.5) and 82 μL of purified water were added to 150 μL of a 2% by mass (solid content) fluorescent latex particle aqueous solution (average particle diameter 150 nm). 29 μL of 1 mg / mL anti-SDMA monoclonal antibody (antibodies prepared in 2.(1) to 2.(3) and the commercially available antibody in 2.(4)) was added and stirred at room temperature for 15 minutes. Subsequently, 5 μL of a 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Fujifilm Wako Pure Chemical Industries, Ltd.) aqueous solution was added and stirred at room temperature for 2 hours. 8.8 μL of a 2 mol / L aqueous solution of glycine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and stirred for 15 minutes. The mixture was then centrifuged (15,000 rpm, 4°C, 15 minutes) to precipitate the fluorescent latex particles. The supernatant was then removed, and 300 μL of a PBS solution (pH 7.4) was added. The fluorescent latex particles were then redispersed using an ultrasonic cleaner. After another centrifugation (15,000 rpm, 4°C, 15 minutes) to remove the supernatant, 300 μL of a PBS solution (pH 7.4) containing 1% by weight of BSA was added to redisperse the fluorescent latex particles, preparing a 1% by weight dispersion of anti-SDMA antibody-conjugated fluorescent latex particles (1) to (4).

[0103] 2. Preparation of latex particles modified with non-fluorescently labeled anti-CRP antibodies: 300 μL of a 2% by mass (solids concentration) latex particle aqueous solution (average particle diameter 150 nm) was mixed with 96 μL of a 250 mmol / L MES buffer (pH 5.6) solution and 25 mL of ultrapure water, followed by 182 μL of a 5 mg / mL anti-CRP monoclonal antibody (MM50175). The resulting mixture was stirred at room temperature for 15 minutes. Then, 9.6 μL of a 10 mg / mL EDC (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide) aqueous solution was added, and the resulting mixture was stirred at room temperature for 2 hours. 30 μL of a 2 mol / L aqueous solution of glycine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the resulting mixture was stirred for 30 minutes. The mixture was then centrifuged (15,000 rpm, 4°C, 15 minutes) to precipitate the latex particles. The supernatant was removed, and 600 μL of a PBS solution (pH 7.4) was added to the precipitate. The latex particles were then redispersed using an ultrasonic cleaner. After another centrifugation (15,000 rpm, 4°C, 15 minutes) to remove the supernatant, 600 μL of a PBS solution (pH 7.4) containing 1% by weight BSA was added to the precipitate to redisperse the latex particles. In this way, a 1% by weight solution of anti-CRP antibody-bound fluorescent latex particles was prepared.

[0104] 3. Preparation of Dried Particles (Preparation of Sensor Chip Cup) 250 μL of ultrapure water, 454 μL of 20% by weight sucrose aqueous solution, 159 μL of 20% by weight BSA aqueous solution, 166 μL of 1% by weight anti-SDMA antibody-modified fluorescent latex particles (average particle diameter 150 nm) prepared in 1.-(1) to (4) above, and 900 μL of 0.8 mol / L MES buffer (pH = 6.5) were mixed. A cup based on polypropylene (Prime Polymer Co., Ltd., Prime Polypro Random PP Grade) was prepared, and 17 μL of the above mixture was spotted into the cup. The mixture was then dried over 24 hours using a Super Dry dryer (TOYO Living Co., Ltd., Ultra Super Dry 00 Series) until the water content was 12% or less. It was then stored at 25°C and 50% RH (relative humidity) for 15 days to prepare dried particles.

[0105] 4. Preparation of Sensor Chip (1) Spotting onto Sensor Chip Solid Support A gold film for the test area and a gold film for the control area were formed by sputtering on one side of a solid support injection-molded from polymethyl methacrylate (PMMA, Mitsubishi Rayon Co., Ltd., ACRYPET VH-001) as a substrate. Both the test area and the control area were 4 mm wide and 36 nm thick. A solution containing the SDMA-BSA conjugate prepared in 1.-(1) or 1.-(3) (concentration: 10 μg / mL in 150 mmol NaCl) was spotted onto the gold-deposited film in the test area of ​​this solid support and incubated at 25°C for 1 hour to allow physical adsorption and immobilization. A solution (concentration: 10 μg / mL in 150 mmol NaCl) containing an IgG antibody that binds to the antibody of the animal species (rabbit, mouse, rat) of the anti-SDMA antibody to be applied to the fluorescently labeled latex on the sensor chip cup side was spotted on the gold film in the control area of ​​this solid support, and was incubated simultaneously with the test area, followed by physical adsorption and immobilization.

[0106] (2) Washing and Blocking of the Sensor Chip Solid-Phase Support Before assembling the sensor chip solid-phase support prepared in this way into a sensor chip, it was washed three times with 300 μL of a pre-prepared washing solution (PBS solution (pH 7.4) containing 0.05% by mass Tween® 20 (polyoxyethylene (20) sorbitan monolaurate, Fujifilm Wako Pure Chemical Industries, Ltd.)). After washing, 300 μL of a PBS solution (pH 7.4) containing 1% by mass casein (Thermo Scientific) was added to block unadsorbed antibody portions on the gold membrane, and the membrane was left to stand at room temperature for 1 hour. After washing with the above washing solution, 300 μL of Immunoassay Stabilizer (ABI) was added as a stabilizer, and the membrane was left to stand at room temperature for 30 minutes. The solution was then removed and the moisture was completely removed using a dryer.

[0107] (3) Fabrication of Sensor Chip A flow-channel type sensor chip was fabricated by combining the fabricated sensor chip solid support with a cup to achieve the configuration of the second embodiment of JP 2010-190880 A. Schematic diagrams are shown in FIGS. 1 and 2. FIG. 1 is a schematic diagram of sensor chip 1, and FIG. 2 is an exploded view of sensor chip 1. Sensor chip 1 is composed of an upper member (sensor chip cup) 2, an intermediate member 3, and a solid support (sensor chip solid support) 4. A first container 5 and a second container 6 are provided in upper member 2. The first container 5 and the second container 6 are collectively referred to as a container group 7. A flow channel 10 is formed in the solid support, and a detection region 8 and a reference region 9 are formed above flow channel 10.

[0108] [Examples 1 to 7 and Comparative Example 1] 1. Sensor Chip Sensor chips of Examples 1 to 7 and Comparative Example 1 were prepared using the materials shown in Table 5 for the anti-SDMA antibody, SDMA-BSA conjugate, and IgG antibody. Among these, SDMA-AET-GMBS-BSA of Example 6 and SDMA-MBS-BSA of Example 7 generated insoluble matter when returned to room temperature from a frozen storage state, so the sensor chips were prepared by centrifuging and filtering.

[0109]

[0110] 2. Preparation of Test Samples Test samples (specimens) A to D shown in Table 6 were prepared by adding the test substance SDMA (manufactured by Med Chem Express, catalog no. HY-101410-50 mg) at the respective concentrations to charcoal-treated beagle dog serum purchased from Kitayama Labes. The charcoal treatment of the dilutions in Table 6 (zero serum: serum without added SDMA) was carried out by adding 0.6 g of activated charcoal to 30 mL of pooled beagle dog serum, incubating at room temperature for 2.5 hours, and then filtering through a 0.22 μm filter. A concentrated SDMA solution prepared by dissolving 10 mg of SDMA in 10 mL of 7% BSA / PBS was used for the addition of SDMA.

[0111]

[0112] 3. Measurement using a control kit SDMA in the test samples was measured by immunoassay using a commercially available ELISA kit (SDMA ELISA Kit (cat. no. K7780): manufactured by Immundiagnostik AG) according to the instruction manual, and the SDMA concentrations of the test samples (zero serum, A to D) were measured. The measured SDMA concentrations of zero serum and test samples A to D were 3.2 μg / 100 mL, 11.9 μg / 100 mL, 20.8 μg / 100 mL, 44.4 μg / 100 mL, and 78.9 μg / 100 mL (also shown in Table 6). It took approximately 300 minutes to obtain these measurement results.

[0113] 4. SDMA Immunoassay (1) Preparation of a Calibration Curve Using the sensor chips of Example 1 and Comparative Example 1, immunoassays were performed on test samples A to D listed in Table 6, and a calibration curve was prepared. The signal Signal 1 from the first metal film was divided by the signal Signal 2 from the second metal film (both representing the rate of increase in fluorescence intensity at a wavelength of 690 nm), and the resulting value (Signal 1 / Signal 2) was plotted against the common logarithm of the SDMA concentration measured by ELISA for test samples A to D in Table 6 to prepare a calibration curve. The Signal 1, Signal 2, and Signal 1 / Signal 2 values ​​for each test sample in Example 1 and Comparative Example 1 are shown in Table 7. The approximation formula and correlation coefficient for linear approximation are also shown in the figure. Furthermore, the Log (SDMA concentration) calculated from Signal 1 / Signal 2 and the approximation formula, and the SDMA concentration calculated therefrom, are also listed in the table.

[0114]

[0115] The calibration curves of Example 1 and Comparative Example 1 are shown in Figure 3. The calibration curve of Example 1 had a linear approximation correlation coefficient R = 0.9997, indicating a very good correlation. The calibration curve of Comparative Example 1 had a correlation coefficient R = 0.3507, indicating no correlation and therefore could not be used as a calibration curve.

[0116] (2) Conversion of SDMA Concentration The SDMA concentrations of test samples A to D were calculated from the calibration curves of Example 1 and Comparative Example 1 and plotted against the SDMA concentrations measured by ELISA ( FIG. 4 ). For Example 1, the linear function approximation equation for ELISA and SPF had a slope of 1.004, a Y-axis intercept of −0.12, and a correlation coefficient R of 0.9999, indicating excellent agreement with the ELISA results. On the other hand, for Comparative Example 1, the correlation of the calibration curve was poor due to insufficient sensitivity in the measurement range, resulting in poor correlation between the SPF measurement results and ELISA (the slope of the linear function approximation equation was 4.903, the Y-axis intercept of −64.4, and the correlation coefficient R of 0.8160), making it unsuitable for use as a quantitative measurement method.

[0117] Furthermore, while the measurement of commercially available ELISA required 15 steps and approximately 300 minutes, the measurement of Example 1 of the present invention was simple and easy; it involved setting the sensor chip and the measurement sample in a commercially available fluorescent antibody assay device, Fuji DryChem IMMUNO AU10V (manufactured by Fujifilm), pressing the start button, and completing the measurement in 12 minutes.

[0118] REFERENCE SIGNS LIST 1 Sensor chip 2 Upper member 3 Intermediate member 4 Solid support 5 First container 6 Second container 7 Container group 8 Detection area 9 Reference area 10 Flow path

Claims

1. A kit for measuring symmetric dimethylarginine (SDMA), comprising: (1) particles modified with a first antibody, which is a monoclonal antibody having specific binding affinity to SDMA, and having a label; (2) a solid support; (3) a first metal film formed on a portion of the solid support; and (4) SDMA conjugated to an immunogenic substance via a linker including a first linker, fixed on the first metal film.

2. The kit according to claim 1, wherein the first antibody is an anti-SDMA monoclonal antibody produced by any one of the rabbit single cell picking method, the mouse iliac lymph node method, or the rat iliac lymph node method.

3. The kit described in claim 1, wherein the first antibody is an anti-SDMA monoclonal antibody obtained by immunizing a rabbit, a mouse, or a rat with SDMA conjugated to an immunogenic substance via a linker containing a second linker.

4. The kit of claim 1, wherein the reactivity of the first antibody to at least one of asymmetric dimethylarginine (ADMA), methylarginine, and arginine is 25% or less of its reactivity to SDMA.

5. The kit according to claim 3, wherein the linker comprising the second linker is a linker that does not have an aromatic ring.

6. The kit of claim 3, wherein the SDMA conjugated to the immunogenic substance via a linker comprising the second linker is linked via a carboxyl group of the SDMA.

7. The kit of claim 3, wherein the SDMA conjugated to the immunogenic substance via a linker containing the second linker is further connected between the second linker and the SDMA via a third linker that does not have an aromatic ring.

8. The kit according to claim 3, wherein the amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via a linker containing a second linker is 3 to 25 equivalents relative to the immunogenic substance.

9. The kit according to claim 3, wherein the amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via a linker containing the second linker is 5 to 15 equivalents relative to the immunogenic substance.

10. The kit of claim 1, wherein the SDMA conjugated to the immunogenic substance via a linker comprising the first linker is linked via a carboxyl group of the SDMA.

11. The kit according to claim 1, wherein the amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via a linker containing the first linker is 3 to 25 equivalents relative to the immunogenic substance.

12. The kit according to claim 1, wherein the amount of SDMA introduced in the SDMA conjugated to the immunogenic substance via a linker containing the first linker is 5 to 15 equivalents relative to the immunogenic substance.

13. A kit according to any one of claims 1 to 12, wherein the first antibody having specific binding affinity to SDMA is a rabbit anti-SDMA monoclonal antibody, and further comprising: (5) a second thin metal film formed on a portion of the solid support; and (6) an anti-rabbit IgG antibody immobilized on the second thin metal film as a second antibody having no specific binding affinity to SDMA.

14. A kit according to any one of claims 1 to 12, wherein the first antibody having specific binding affinity to SDMA is a mouse anti-SDMA monoclonal antibody, and further comprising: (5) a second metal thin film formed on a portion of the solid support; and (7) an anti-mouse IgG antibody as a second antibody having no specific binding affinity to SDMA, immobilized on the second metal thin film.

15. A kit according to any one of claims 1 to 12, wherein the first antibody having specific binding affinity to SDMA is a rat anti-SDMA monoclonal antibody, and further comprising: (5) a second thin metal film formed on a portion of the solid support; and (8) an anti-rat IgG antibody as a second antibody having no specific binding affinity to SDMA, immobilized on the second thin metal film.

16. A method for measuring symmetric dimethylarginine (SDMA), comprising: (a) contacting a solution containing particles modified with a first antibody, which is a monoclonal antibody having specific binding affinity to SDMA, and having a label, and a biological sample containing SDMA, with a first metal film formed on a solid support, the first metal film having SDMA conjugated with an immunogenic substance fixed via a linker including a first linker; and (b) detecting a signal from the first metal film.

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