Immunochromatographic strips, immunochromatographic kits, detection methods, and developing solutions

Abstract

We offer a novel immunostrip. [Solution] An immunochromatographic test strip for detecting or quantifying leucine-rich α2 glycoprotein (LRG) contained in a sample, comprising: a determination unit equipped with a capture ligand that specifically binds to LRG; and a reaction unit located upstream of the determination unit in the direction in which the sample unfolds, equipped with a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, wherein the antibody is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody, and the capture ligand is Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody.

Description

[Technical Field]

[0001] This invention relates to a novel immunochromatographic strip (test strip), etc. [Background technology]

[0002] Immunoassays, also known as immunological assays, are methods that qualitatively and quantitatively analyze trace components by utilizing specific reactions between antigens and antibodies. Antigen-antibody reactions are widely used in fields such as medicine due to their high sensitivity and selectivity. Various immunoassay methods are known, depending on their measurement principle, and include, for example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), latex agglutination methods (LIA, PA), immunochromatography (ICA), hemagglutination assay (HA), and hemagglutination inhibition assay (HI).

[0003] Immunoassays qualitatively or quantitatively detect antigens or antibodies based on changes (changes in the concentration of antigen, antibody, or complex) that occur when antigens and antibodies react and form a complex.

[0004] The reactivity and specificity of antigens and antibodies when they react and form complexes are important factors that determine the detection capability in immunoassays. Therefore, by selecting and using antigens and antibodies with superior reactivity and specificity, the detection sensitivity of immunoassays can be increased. In other words, if the reactivity and specificity of the antigen and antibody when they react and form a complex are insufficient, the sensitivity of the immunoassay cannot be sufficiently increased.

[0005] On the other hand, when detecting antigens or antibodies, the detection sensitivity is increased by attaching a labeling substance to the antibody, antigen, or complex. Therefore, the labeling ability of the labeling substance is also an important factor that influences the detection capability in immunoassays.

[0006] For example, Patent Document 1 discloses that by using an immunoassay (immunochromatographic strip) that combines a specific labeling substance (a metal-resin composite having a structure in which metal particles are immobilized on resin particles) and a specific antibody in a specific manner, analytes derived from SARS-CoV-2 (the so-called novel coronavirus) contained in a sample can be detected with sufficient sensitivity. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2024-95032 [Patent Document 2] International Publication No. 2016 / 129631 [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide novel immunochromatographic strips (test strips), etc. [Means for solving the problem]

[0009] According to the inventors' research, specific labeling substances such as those disclosed in Patent Document 1 (metal-resin composites having a structure in which metal particles are immobilized on resin particles) did not enable highly sensitive detection of any combination of antibody and antigen.

[0010] Under these circumstances, the inventors have diligently conducted research and have found that, in an immunoassay (immunochromatographic strip), by combining a specific labeling substance (a metal-resin composite having a structure in which metal particles are immobilized on resin particles) and a specific antibody [in a specific manner (constituent elements)], it is possible to achieve sufficient detection sensitivity for leucine-rich α2 glycoprotein, or to suppress false positives (and even false negatives).

[0011] Furthermore, the inventors have discovered that by using a specific type of immunochromatographic strip (an immunochromatographic strip with multiple control lines in the reaction section) in the above-mentioned specific combination, even greater detection sensitivity can be achieved, thus completing the present invention.

[0012] In other words, the present invention relates to the following inventions, etc. [1] An immunochromatographic test strip (e.g., a lateral flow type chromatographic test strip) for detecting or quantifying leucine-rich α2 glycoprotein (LRG) contained in a sample, A determination unit (for example, a determination unit comprising a membrane and a capture ligand that specifically binds to LRG) is provided with a capture ligand that specifically binds to LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a reaction unit (including) equipped with a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite (a composite of metal particles and resin particles) having a structure in which multiple metal particles are immobilized on resin particles, The antibody (the antibody in the labeled antibody) is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. Test strip.

[0013] [2] An immunochromatographic test strip for detecting or quantifying LRG contained in a sample, A determination unit equipped with a capture ligand that specifically binds to the LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, and The antibody is Anti-Human LRG(48A1) Mouse IgG or an antibody having the same antigen recognition site as the said antibody, The capture ligand is Anti-Human LRG(50A1) Mouse IgG or an antibody having the same antigen recognition site as the said antibody, A kit (for detecting or quantifying LRG contained in a sample) that uses (is provided with) a test strip.

[0014] [3] An immunochromatographic test strip (e.g., a lateral flow chromatographic test strip) for detecting or quantifying LRG contained in a sample, A determination section (e.g., a membrane and a determination section formed by immobilizing a capture ligand that specifically binds to LRG on the membrane) provided with (comprising) a capture ligand that specifically binds to LRG, and In the direction in which the sample develops, on the upstream side of the determination section, a reaction section provided with (comprising) a labeled antibody obtained by labeling an antibody that specifically binds to LRG with a resin-metal composite (a composite of metal particles and resin particles) having a structure in which a plurality of metal particles are immobilized on resin particles, The antibody (the antibody in the labeled antibody) is Anti-Human LRG(48A1) Mouse IgG or an antibody having the same antigen recognition site as the said antibody, The capture ligand is Anti-Human LRG(50A1) Mouse IgG or an antibody having the same antigen recognition site as the said antibody, A test strip, and A kit (for detecting or quantifying LRG contained in a sample) that uses (is provided with, comprises) a developing solution (or a sample treatment solution) containing a surfactant (polyoxyethylene-type surfactant) comprising polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 alkylphenyl ether).

[0015] [4] An immunochromatographic test strip (e.g., a lateral flow type chromatographic test strip) for detecting or quantifying LRG contained in a sample, A test strip includes a determination unit (for example, a membrane and a determination unit on which a capture ligand that specifically binds to LRG is immobilized) which is equipped with a capture ligand that specifically binds to LRG, Labeled antibodies that specifically bind to LRGs are labeled using a resin-metal composite (a composite of metal particles and resin particles) having a structure in which multiple metal particles are immobilized on resin particles, Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 A kit that uses (includes, combines, or comprises) a developing solution (or sample processing solution) containing a surfactant (polyoxyethylene-type surfactant) containing an alkylphenyl ether, The antibody (the antibody in the labeled antibody) is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. A kit (for detecting or quantifying LRG contained in a sample).

[0016] [5] A method for detecting or quantifying LRG contained in a sample (immunoassay method), A determination unit (for example, a determination unit comprising a membrane and a capture ligand that specifically binds to LRG) is provided with a capture ligand that specifically binds to LRG, Using an immunochromatographic test strip (or a kit using said test strip), the following steps (I) to (III) are performed: A reaction section (including) containing a labeled antibody, in which an antibody that specifically binds to LRG is labeled using a resin-metal composite (a composite of metal particles and resin particles) having a structure in which multiple metal particles are immobilized on resin particles, is used upstream of the detection section in the direction in which the sample is unfolded. Step (I): A step of bringing the LRG contained in the sample (the sample containing the LRG) into contact with the labeled antibody (contact in the reaction section). Step (II): A step of bringing the complex formed in Step (I), which includes the LRG and the labeled antibody, into contact with the capture ligand (in the detection unit). Step (III): A step to measure the color intensity of the resin-metal composite (color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transitions). Includes, The sample is polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 The developing solution (or sample processing solution) contains a surfactant (polyoxyethylene-type surfactant) containing alkylphenyl ether, The antibody (the antibody in the labeled antibody) is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. Method (immunoassay method).

[0017] [6] A method for detecting or quantifying LRG contained in a sample (immunoassay method), An immunochromatographic test strip (or a kit using said test strip) is used, which includes a determination unit (for example, a membrane and a determination unit on which a capture ligand that specifically binds to LRG is immobilized) equipped with a capture ligand that specifically binds to LRG. The following steps (I) to (III); Step (I): A step of contacting the LRG contained in the sample (a sample containing LRG) with a labeled antibody that specifically binds to LRG, which is labeled with a resin-metal composite (a composite of metal particles and resin particles) having a structure in which multiple metal particles are immobilized on resin particles. Step (II): A step of bringing the complex formed in Step (I), which includes the LRG and the labeled antibody, into contact with the capture ligand (in the detection unit). Step (III): A step to measure the color intensity of the resin-metal composite (color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transitions). Includes, The sample is polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 The developing solution (or sample processing solution) contains a surfactant (polyoxyethylene-type surfactant) containing alkylphenyl ether, The antibody (the antibody in the labeled antibody) is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. Method (immunoassay method).

[0018] [7] A developing solution (or sample processing solution) for detecting or quantifying LRG contained in a sample, Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 The developing solution (or sample processing solution) contains a surfactant (polyoxyethylene-type surfactant) containing alkylphenyl ether, A determination unit equipped with a capture ligand that specifically binds to the LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, and The antibody is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. A developing solution for use with an immunochromatographic test strip, the capture ligand being Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody.

[0019] [8] A developing solution (or sample processing solution) for detecting or quantifying LRG contained in a sample, Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 The developing solution (or sample processing solution) contains a surfactant (polyoxyethylene-type surfactant) containing alkylphenyl ether, A test strip, and a determination unit comprising a capture ligand that specifically binds to LRG, and A developing solution for combining an antibody that specifically binds to LRG with a labeled antibody labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, The antibody is Anti-Human LRG(48A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody. A developing solution in which the capture ligand is Anti-Human LRG(50A1)Mouse IgG or an antibody having the same antigen recognition site as said antibody.

[0020] [9] Polyoxyethylene alkylphenyl ethers are polyoxyethylene linear alkylphenyl ethers (for example, polyoxyethylene linear C 3―24 A kit, method, or developing solution according to any one of [3] to [8], comprising an alkylphenyl ether.

[0021]

[10] A test strip, kit, method, or developing solution according to any one of [1] to [9], wherein the metal particles are silver, nickel, copper, gold, platinum, or an alloy of silver, nickel, copper, gold, platinum, or palladium.

[0022]

[11] In resin-metal composites, A first particle having a portion of the metal particle exposed outside the resin particle, A second particle whose entire body is enclosed within a resin particle, It includes, A test strip, kit, method, or developing solution according to any one of [1] to

[10] , wherein at least some of the first and second particles are distributed three-dimensionally in the surface layer of the resin particles.

[0023]

[12] The test strip, kit, method, or developing solution according to any one of [1] to

[11] , wherein the resin particles are polymer particles having substituents in their structure that are capable of adsorbing metal ions.

[0024]

[13] A test strip, kit, method, or developing solution according to any of [1] to

[12] , wherein the average particle size of the metal particles is in the range of 1 to 100 nm.

[0025]

[14] A test strip, kit, method, or developing solution according to any one of [1] to

[13] , wherein the average particle size of the resin-metal composite is in the range of 30 to 1000 nm.

[0026]

[15] A test strip, kit, method, or developing solution according to any one of [1] to

[14] , wherein the reaction section has a plurality of control lines spaced apart in the direction in which the sample develops.

[0027]

[16] A test strip, kit, method, or developing solution according to

[15] , having two to five control lines.

[0028]

[17] A test strip, kit, or developing solution described in any of [1]-[4], [7], [8] and [9]-

[16] for the detection or diagnosis of Kawasaki disease.

[0029]

[18] A method for diagnosing Kawasaki disease (or a method for diagnosing Kawasaki disease) as described in any of [5], [6] and [9]-

[16] . [Effects of the Invention]

[0030] The present invention can provide novel immunochromatographic strips (test strips), kits, detection (or quantitative) methods, and developing solutions. Such strips (kits, methods, and developing solutions) can detect (or quantify) leucine-rich α2 glycoprotein contained in a sample. Leucine-rich α2 glycoprotein (LRG) is a type of protein found in the blood, and it has been found that LRG levels in the blood increase when Kawasaki disease develops (Patent Document 2, etc.). Kawasaki disease is considered difficult to diagnose quickly because its etiology and pathogenesis are still unknown, there are no specific diagnostic tests, major symptoms vary from person to person, and many cases do not meet the diagnostic criteria. Therefore, according to the strip (kit, method, and developing solution) of the present invention, Kawasaki disease can be tested or diagnosed quickly and easily. In particular, in one aspect of the present invention, sufficient detection sensitivity can be achieved by combining a specific labeling substance with a specific antibody (and in a specific manner). In addition, other embodiments of the present invention can suppress false positives (and even false negatives). [Brief explanation of the drawing]

[0031] [Figure 1] This is a schematic diagram showing the cross-sectional structure of a metal-resin composite used in one embodiment of the present invention. [Figure 2] This is a cross-sectional view showing an immunochromatographic strip used in one embodiment of the present invention. [Modes for carrying out the invention]

[0032] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. All patents, applications, and other publications and information referenced herein are incorporated herein by reference in their entirety.

[0033] <Immunochromatography Strip> The immunochromatographic strip (test strip) of the present invention can be used to detect or quantify leucine-rich α2 glycoprotein (LRG) contained in a sample, and may be a lateral flow type chromatographic strip (test strip) in particular.

[0034] The sample (or specimen) can be anything containing LRG and is not particularly limited, but examples include blood, plasma, serum, urine, semen, cerebrospinal fluid, saliva, oral mucosa, nasal mucus, sweat, tears, ascites, amniotic fluid, feces, blood vessels, organs (e.g., liver), tissues, cells, etc. The sample may contain any of the examples described above, and may be used as is, or may be a solution or extract of the examples described above.

[0035] The sample may be from a human or an animal. The animals are not particularly limited; for example, dogs, cats, etc.

[0036] The method of collecting the sample is not particularly limited, and any known method may be used, for example, a cotton swab may be used.

[0037] The immunochromatographic strip (test strip) of the present invention comprises at least a determination section equipped with a capture ligand that specifically binds to LRG, and may further comprise a reaction section equipped with a labeled antibody (labeled substance).

[0038] The detection unit and the reaction unit each only need to be equipped with (contain) a capture ligand and a labeled antibody, and the form in which the capture ligand and the labeled antibody exist is not particularly limited. For example, the detection unit and the reaction unit may each be a capture ligand and a labeled antibody, and the capture ligand and the labeled antibody may be added (contained) or immobilized (positioned), respectively.

[0039] As for the specific form of the reaction site (labeled antibody), for example, the labeled antibody may be directly contained (attached to, added) to the membrane (forming a reaction site), or the labeled antibody may be contained (attached to, added) to a fixing means provided (immobilized) on the membrane [for example, a pad made of cellulose filter paper, glass fiber, nonwoven fabric, etc. (conjugate pad described later)] (forming a reaction site).

[0040] As for the specific form of the determination unit (capture ligand), for example, the capture ligand may be directly fixed to the membrane (forming the determination unit), or the capture ligand may be fixed to a fixing means provided (fixed) to the membrane (forming the determination unit).

[0041] The method of addition or fixation can be appropriately selected depending on the form of existence, etc., and is not particularly limited. For example, a dispersion containing labeled antibody or capture ligand may be attached to or coated (impregnated) a membrane or fixation means to add or fix the substance (forming a detection area or reaction area).

[0042] The reaction unit is located upstream (on the side of) the determination unit in the direction in which the sample is unfolded (the direction of sample unfolding) [or the determination unit is located downstream (on the side of) the direction in which the sample is unfolded].

[0043] The present invention is characterized by the use of specific capture ligands and labeled antibodies, as will be described in detail later.

[0044] Immunochromatographic strips may typically include a membrane. In such a membrane, the determination section (test section, test line) and the reaction section may be fixed (positioned).

[0045] The membrane is formed from an inert substance (a substance that does not react with analytes, various ligands, etc.) consisting of a fine porous material that exhibits capillary action and causes the sample to unfold simultaneously upon addition.

[0046] Examples of membranes (membrane materials) include fibrous or nonwoven fibrous matrices, films, filter paper, glass fiber filter paper, cloth, and cotton, all composed of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc. Among these, films, filter paper, and glass fiber filter paper composed of cellulose derivatives or nylon are preferred, and more preferably are nitrocellulose films, mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate) films, nylon films, and filter paper.

[0047] The immunochromatographic strip may further include other parts (components, parts) as needed, such as a support section [a support section (support body) for supporting the membrane, for example, a plastic support body], a sample addition section [a sample addition section (sample addition pad, sample pad) located upstream of the determination section and reaction section for adding a sample containing LRG, for example, a sample addition section made of materials such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, or cotton cloth], a liquid absorption section [a liquid absorption section (absorbent section, liquid absorption (absorbent) pad) located downstream of the determination section and reaction section, for example, a pad made of water-absorbent material such as cellulose filter paper, nonwoven fabric, cloth, or cellulose acetate], a conjugate section [for example, a conjugate section (conjugate pad) interposed between the sample addition section and the membrane], and a control section (control line).

[0048] The immunochromatographic strip may have one control section (control line), but preferably it may have multiple control lines, from the viewpoint of enabling more accurate quantification of LRG.

[0049] If the immunochromatographic strip has multiple control lines, the number of control lines may be two or more (for example, two to five), preferably two to four, and more preferably two or three. The multiple control lines may be arranged at equal intervals or at different intervals in the direction of sample development.

[0050] Figure 2 illustrates an example of an immunochromatographic strip [lateral flow type chromatographic strip (test strip)] according to one embodiment of the present invention.

[0051] This test strip is equipped with a membrane 4. The membrane 4 is provided with, in order in the direction of sample deployment, a sample addition section (sample pad) 2, a conjugate section (reaction section, conjugate pad) 3, a test line (determination section) 5, a control line 1 (control section 1) 6, a control line 2 (control section 2) 7, and a liquid absorption section (absorption pad) 8.

[0052] To simplify the operation, the test strip preferably includes a support (packing sheet) 1 that supports the membrane 4. For example, plastic can be used as the support.

[0053] The test strip may have a sample addition section 2 for adding a sample containing LRG. The sample addition section 2 is a part of the test strip for receiving a sample containing LRG. The sample addition section 2 may be formed directly on the membrane 4 upstream of the determination section 5 in the direction in which the sample unfolds, or via a conjugate section 3.

[0054] Here, a specific labeled antibody that specifically binds to LRG is immobilized on the conjugate portion (conjugate pad) 3, and a specific capture ligand that specifically binds to LRG is immobilized on the test line 5, forming the reaction portion and the determination portion, respectively.

[0055] The test strip may have a control section formed downstream of the detection section in the direction in which the sample unfolds, on which a capture ligand that specifically binds to the labeled antibody is immobilized.

[0056] The control unit is not particularly limited as long as it is configured such that the labeled antibody or LRG-labeled antibody conjugate specifically contacts the labeled antibody and the capture ligand. For example, the capture ligand may be directly immobilized on the membrane 4, or the capture ligand may be immobilized on a pad made of cellulose filter paper, glass fiber, nonwoven fabric, etc., which is immobilized on the membrane 4.

[0057] Along with the test line (determination unit) 5, the color intensity is also measured in the control units (control lines) 6 and 7. This allows us to confirm that the sample applied to the test strip has spread, reached the reaction unit and the determination unit, and that the test has been performed correctly.

[0058] As shown in Figure 2, if the test strip has multiple control lines, the labeled antibody and LRG-labeled antibody conjugates moving along the test strip can be captured in almost their entirety in the detection and control sections. More specifically, the detection and control sections can capture the total amount of the moving labeled antibody and LRG-labeled antibody conjugates with a high capture yield of preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more. Furthermore, if the test strip has multiple control lines, any labeled antibody (including LRG-labeled antibody conjugates) that could not be captured by the first control line can be captured by the downstream control line. This allows for accurate determination of the relationship between the color intensity of the LRG-labeled antibody conjugates captured in the detection unit and the color intensity based on the total amount of labeled antibody deployed on the test strip. By correcting based on the calculation formula (1) or (2) described later, LRG can be quantified with high accuracy.

[0059] The amounts of capture ligand immobilized on multiple control lines (e.g., 6 and 7 in Figure 2) may be equal or different. If the amounts of capture ligand immobilized on multiple control lines are different, the amount of capture ligand at the upstream control line (e.g., 6 in Figure 2) may be greater or less than the amount of capture ligand at the downstream control line (e.g., 7 in Figure 2) in the direction of sample development. For example, in Figure 2, by increasing the amount of capture ligand at control line 7 compared to control line 6, it is possible to capture more particles that could not be captured by control line 6. Furthermore, when adjusting the amounts of capture ligand at control lines 6 and 7, it is preferable to adjust the amount of capture ligand so that the color intensity of control lines 6 and 7, as measured by a measuring device such as an immunochromatograph, falls within the range that can be quantitatively measured according to the specifications of the measuring device.

[0060] Furthermore, the initial total amount Q1 of labeled antibody in the reaction region is equal to the amount Q2 that can be captured by the capture ligand in the control region. 0.9 × Q1 ≤ Q2 ≤ 1.0 × Q1 It is preferable that the following relationship exists. Here, the captureable amount Q2 means the sum of the captureable amounts (for example, the sum of the captureable amounts of 6 and 7 in Figure 2) if the test strip has multiple control lines.

[0061] The liquid absorption section 8 may be formed from a pad of an absorbent material such as cellulose filter paper, nonwoven fabric, cloth, or cellulose acetate. The speed at which the sample moves after the front line of the added sample reaches the liquid absorption section 8 will vary depending on the material and size of the liquid absorption section 8. Therefore, by selecting the material and size of the liquid absorption section 8, the optimal speed for LRG detection and quantification can be set.

[0062] [Antibodies or fragments thereof that bind to LRG] In this invention, an antibody or fragment thereof that binds to LRG is used in the labeled antibody and the capture ligand. In particular, in the present invention, it is preferable to select specific antibodies (or fragments thereof) for both the labeled antibody and the capture ligand.

[0063] Specifically, as the labeled antibody, Anti-Human LRG(48A1)Mouse IgG (for example, Anti-Human LRG (48A1) Mouse IgG, Institute of Immunobiological Laboratories, product number 99912-100) is preferred. Alternatively, an antibody having the same antigen recognition site as the aforementioned antibody may be used as the labeled antibody.

[0064] Furthermore, Anti-Human LRG(50A1)Mouse IgG (e.g., Anti-Human LRG (50A1) Mouse IgG, Institute of Immunobiological Sciences, product number 99912-099) is preferred as the capture ligand. Alternatively, a capture ligand having the same antigen recognition site as the antibody may be used.

[0065] Such labeled antibodies and capture ligands specifically bind to LRGs. Commercially available labeled antibodies and capture ligands may be used, or they may be manufactured. When manufacturing, the manufacturing method is not particularly limited, and known methods can be used. Commercially available labeled antibodies and capture ligands can be obtained, for example, from the Institute of Immunobiological Sciences.

[0066] [Labeled antibody] In this invention, a labeled antibody (labeling, labeling substance) is used, which is an antibody that specifically binds to LRG and is labeled with a resin-metal composite (a composite of metal particles and resin particles).

[0067] (resin-metal composite) A resin-metal composite (metal-resin composite) is a composite material containing metal particles and resin particles, and typically has a structure in which metal particles (multiple metal particles) are immobilized on resin particles.

[0068] Figure 1 is a schematic cross-sectional view showing an example of a metal-resin composite 100, and the metal-resin composite will be explained with reference to this figure. The metal-resin composite 100 comprises resin particles 10 and metal particles 20.

[0069] (Structure of metal-resin composites) In the metal-resin composite 100 (or metal-resin composite, hereinafter the same applies to those with the reference numerals shown in Figure 1), metal particles 20 are dispersed or immobilized in resin particles 10. In the example of Figure 1, it is preferable that in the metal-resin composite 100, a portion of the metal particles 20 are distributed three-dimensionally in the surface layer 60 of the resin particles 10, and a portion of the three-dimensionally distributed metal particles 20 are partially exposed outside the resin particles 10, while the remaining portion is enclosed within the resin particles 10.

[0070] Here, "surface layer" refers to the area from the surface of the resin particle 10 to 50% of the particle radius in the depth direction. Furthermore, "three-dimensional distribution" means that the metal particles 20 are dispersed not only in the surface direction of the resin particle 10 but also in the depth direction.

[0071] Here, the metal particles 20 may include one or more selected from metal particles completely enclosed within the resin particles 10 (hereinafter also referred to as "enclosed metal particles 30" or "second particles"), metal particles having a portion embedded within the resin particles 10 and a portion exposed outside the resin particles 10 (hereinafter also referred to as "partially exposed metal particles 40" or "first particles"), and metal particles adsorbed on the surface of the resin particles 10 (hereinafter also referred to as "surface-adsorbed metal particles 50").

[0072] The encapsulated metal particles 30 have their entire surface covered with the resin that makes up the resin particles 10.

[0073] The partially exposed metal particles 40 are, for example, covered with the resin constituting the resin particles 10, with 5% to less than 100% of their surface area covered. From the viewpoint of durability as a labeling material, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. For example, the surface-adsorbed metal particles 50 have more than 0% but less than 5% of their surface area covered by the resin that makes up the resin particles 10.

[0074] For example, when using the metal-resin composite 100 in the present invention, antibodies are immobilized on the surface of the resin particles 10, the surface of the partially exposed metal particles 40, or the surface-adsorbed metal particles 50. In this case, for example, antibodies may be immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, while they may not be immobilized on the encapsulated metal particles 30. However, since all of the metal particles 20, including the encapsulated metal particles 30, exhibit localized surface plasmon absorption, not only the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, but also the encapsulated metal particles 30 contribute to improved visibility as a labeling substance.

[0075] Furthermore, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a larger contact area with the resin particles 10 compared to the surface-adsorbed metal particles 50, and also possess strong physical adsorption forces such as anchoring effects due to their embedded state, making them less likely to detach from the resin particles 10. Therefore, the durability and stability of the metal-resin composite 100 as a labeling material can be improved.

[0076] Furthermore, the amount of metal particles 20 (total of encapsulated metal particles 30, partially exposed metal particles 40, and surface-adsorbed metal particles 50) supported on the metal-resin composite 100 is preferably in the range of 5% to 70% by weight relative to the weight of the metal-resin composite 100. Within this range, the metal-resin composite 100 exhibits particularly excellent visibility, visual determination, and detection sensitivity as a labeling material. More preferably, the amount of metal particles 20 supported is in the range of 15% to 70% by weight.

[0077] Furthermore, it is preferable that 10% to 90% by weight of the metal particles 20 consist of partially exposed metal particles 40 and surface-adsorbed metal particles 50. Within this range, a sufficient amount of antibody can be immobilized on the metal particles 20, resulting in high sensitivity as a labeling substance. It is even more preferable that 20% to 80% by weight of the metal particles 20 consist of partially exposed metal particles 40 and surface-adsorbed metal particles 50.

[0078] Furthermore, it is preferable that 60% to 100% by weight of the metal particles 20 are present in the surface layer 60, and that 5% to 90% by weight of the metal particles 20 present in the surface layer 60 are partially exposed metal particles 40 or surface-adsorbed metal particles 50, as this ensures sufficient immobilization of the antibody on the metal particles 20, thereby increasing the sensitivity as a labeling substance. In other words, it is preferable that 10% to 95% by weight of the metal particles 20 present in the surface layer 60 are encapsulated metal particles 30.

[0079] (Resin particles) The resin particles (the resin constituting the resin particles) 10 are not particularly limited, and any known resin [for example, polystyrene, polyvinyl alcohol, poly(p-styrene sulfonate sodium), melamine resin, urea resin, acrylic resin, cellulose, etc.] may be used. The resin particles 10 are preferably polymer particles having substituents in their structure that are capable of adsorbing metal ions. In particular, nitrogen-containing polymer particles are preferred. Nitrogen atoms in nitrogen-containing polymers are preferred because they have excellent visibility and readily chemically adsorb anionic metal ions, which are precursors to metal particles such as silver, nickel, copper, gold, platinum, and palladium, to which antibodies can be easily immobilized. When metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, some of the generated metal particles 20 become encapsulated metal particles 30 or partially exposed metal particles 40.

[0080] Furthermore, polymers having functional groups such as carboxylic acids, such as acrylic acid polymers, can adsorb cationic metal ions. Therefore, they readily adsorb cationic metal ions, which are precursors to metal particles such as silver, nickel, copper, gold, platinum, and palladium. This makes it possible to form metal particles 20 such as silver, nickel, copper, gold, platinum, and palladium, and to create alloys with any of the above metals. In addition, if the side chain has substituents that adsorb cationic metal ions, it can be widely used in, for example, phenolic resins, epoxy resins, cellulose resins, melamine resins, etc.

[0081] The nitrogen-containing polymers mentioned above are resins having nitrogen atoms in their main chain or side chains, and include, for example, polyamines, polyamides, polypeptides, polyurethanes, polyureas, polyimides, polyimidazoles, polyoxazoles, polypyrroles, and polyanilines. Preferably, they are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. When nitrogen atoms are present in the side chains, a wide range of materials can be used, such as acrylic resins, phenolic resins, epoxy resins, cellulose resins, and melamine resins.

[0082] Furthermore, in the case of resin particles that do not have substituents in their structure that can adsorb metal ions, such as unsubstituted polystyrene particles, metal particles can be immobilized on the particle surface by irradiation of the particle surface with ultrasound or ionizing radiation, or by metal plating of the resin particle surface.

[0083] (metal particles) The structure of the metal particles 20 is not particularly limited, but those that exhibit color development in the state of the metal-resin composite 100 immobilized on the resin particles 10 are preferred because they improve the detection sensitivity (visual determination) as a labeling substance.

[0084] Suitable materials for the metal particles 20 include, for example, silver, nickel, copper, gold, platinum, and palladium. These metals can be used individually or in composites such as alloys.

[0085] Preferably, gold, platinum, or palladium are used, as they offer excellent visibility and facilitate antibody immobilization. These materials develop color through absorption originating from localized surface plasmon resonance. More preferably, gold and platinum are used, as they offer good storage stability.

[0086] Furthermore, from the perspective of obtaining excellent color development when bound to antibodies, gold, platinum, gold alloys, or platinum alloys are the most preferred metal species.

[0087] Here, a gold alloy means an alloy consisting of, for example, gold and other metal species, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more, of gold. A platinum alloy means an alloy consisting of, for example, platinum and other metal species, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more, of platinum.

[0088] Furthermore, the average particle diameter of the metal particles 20 measured by scanning electron microscopy (SEM) observation is preferably in the range of, for example, 1 to 100 nm. If the average particle diameter of the metal particles 20 is too small or too large (for example, less than 1 nm or more than 100 nm), localized surface plasmons are less likely to appear, and sensitivity tends to decrease. The average particle diameter of the metal particles 20 is preferably in the range of 1 nm or more and less than 80 nm (for example, 1 nm or more and less than 70 nm), and more preferably in the range of 1 nm or more and less than 50 nm.

[0089] Furthermore, the average particle size of the metal-resin composite 100 is, for example, within the range of 30 to 1000 nm (e.g., 50 to 1000 nm, 100 to 1000 nm). If the average particle size is too small (e.g., less than 30 nm), for example, when using gold or platinum particles as the metal particles 20, the amount of metal particles 20 that can be supported tends to be small, resulting in a tendency for weaker coloration compared to gold particles of the same size. If the particle size is too large (e.g., exceeding 1000 nm), when used as a labeled antibody, it tends to clog the pores of chromatographic media such as membrane filters and tends to have reduced dispersibility.

[0090] The average particle diameter of the metal-resin composite 100 is preferably in the range of 100 nm or more and less than 700 nm, and more preferably in the range of 100 nm or more and less than 650 nm (for example, 370 nm or more and less than 650 nm, 380 nm or more and less than 650 nm, 390 nm or more and less than 650 nm, 400 nm or more and less than 650 nm, etc.). Here, the particle diameter of the metal-resin composite 100 means the particle diameter of the resin particles 10 plus the length of the protruding portion of the partially exposed metal particles 40 or surface-adsorbed metal particles 50, and can be measured by laser diffraction / scattering, dynamic light scattering, or centrifugal sedimentation.

[0091] The method for producing the metal-resin composite 100 is not particularly limited. For example, a solution containing metal ions can be added to a dispersion of resin particles 10 produced by emulsion polymerization to adsorb the metal ions onto the resin particles 10 (hereinafter referred to as "metal ion-adsorbing resin particles"). Next, the metal ion-adsorbing resin particles can be added to a reducing agent solution to reduce the metal ions and generate metal particles 20, thereby obtaining the metal-resin composite 100. Here, for example, when gold particles are used as the metal particles 20, it is preferable to use an aqueous solution of chloroauric acid (HAuCl4) as the solution containing metal ions. Also, when platinum particles are used as the metal particles 20, it is preferable to use an aqueous solution of chloroplatinic acid (H2PtCl6) as the solution containing metal ions. Note that a metal complex may be used instead of metal ions.

[0092] Furthermore, instead of water, aqueous alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, and t-butanol, or acids such as hydrochloric acid, sulfuric acid, and nitric acid may be used as the solvent for solutions containing metal ions.

[0093] Furthermore, additives such as water-soluble polymer compounds like polyvinyl alcohol, surfactants, alcohols, ethers like tetrahydrofuran, diethyl ether, and diisopropyl ether, polyols like alkylene glycol, polyalkylene glycol, their monoalkyl or dialkyl ethers, and glycerin, and various water-miscible organic solvents like acetone and methyl ethyl ketone may be added to the metal ion-containing solution as needed. Such additives are effective in accelerating the reduction reaction rate of the metal ions and in controlling the size of the resulting metal particles 20.

[0094] Furthermore, there are no particular restrictions on the reducing agent, but it is preferable to use, for example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, hydrazine sulfate, Rochelle salt, etc. Among these, sodium borohydride, dimethylamine borane, and citric acid are more preferred. Surfactants can be added to the reducing agent solution as needed, or the pH of the solution can be adjusted. pH can be adjusted with buffers such as boric acid or phosphoric acid, acids such as hydrochloric acid or sulfuric acid, or alkalis such as sodium hydroxide or potassium hydroxide. Furthermore, by adjusting the reduction rate of metal ions using the temperature of the reducing agent solution, the particle size of the formed metal particles 20 can be controlled.

[0095] Furthermore, when reducing the metal ions in the metal ion adsorbing resin particles to generate metal particles 20, the metal ion adsorbing resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion adsorbing resin particles. However, the former is preferred from the viewpoint of the ease with which encapsulated metal particles 30 and partially exposed metal particles 40 are generated.

[0096] Furthermore, to maintain the dispersibility of the metal-resin composite 100 in water, dispersants such as citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, and DISPERBYK184 (manufactured by Big Chemie Japan Co., Ltd.) may be added. In addition, the pH can be adjusted with buffers such as boric acid and phosphoric acid, acids such as hydrochloric acid and sulfuric acid, and alkalis such as sodium hydroxide and potassium hydroxide to maintain dispersibility.

[0097] (Form of labeled antibody, manufacturing method, etc.) Labeled antibodies are antibodies that specifically bind to LRG and are labeled with a resin-metal composite (a composite of metal particles and resin particles).

[0098] Such labeled antibodies may specifically be those in which the antibody is adsorbed (bound to) (immobilized) onto the surface of a metal-resin composite (e.g., resin particles and / or metal particles).

[0099] As mentioned above, antibodies that bind to LRG or fragments thereof can be used as antibodies, but in particular, Anti-Human LRG(48A1)Mouse IgG may be preferably used as the labeled antibody.

[0100] Labeled antibodies can be manufactured using conventional methods, and some representative methods are described below.

[0101] ·Antibody binding process First, the antibody may be bound (adsorbed, immobilized, or attached) to the metal-resin complex by mixing the antibody with the metal-resin complex (antibody binding step).

[0102] In such an antibody binding process, the antibody and the metal-resin complex may be mixed in the presence of a solvent [water, etc., especially a buffer (binding buffer)].

[0103] The binding of the metal-resin composite and the antibody in this process may be a so-called physical binding, and the antibody may be bound to the surface of the metal particles and / or the resin particles in the metal-resin composite by physical interaction. Here, binding by physical interaction can be, for example, electrostatic bonding, hydrogen bonding, hydrophilic-hydrophobic interactions, and van der Waals forces.

[0104] When using a binding buffer, the type of binding buffer is not particularly limited, but inorganic buffers such as boric acid, carbonates, and phosphates, and organic buffers such as Tris and Good's Buffer can be used. The pH of the buffer is preferably around pH 5 to 10 in order to bind the antibody.

[0105] In the antibody conjugation step, a labeled antibody dispersion can be obtained by mixing the antibody and the metal-resin complex in the presence of a solvent (especially a conjugation buffer) and stirring thoroughly.

[0106] In this process, for example, the antibody and the metal-resin complex can be bound in a solvent (especially a binding buffer). Therefore, the metal-resin complex may be dispersed in the solvent (especially a binding buffer) beforehand, and the antibody may be added thereto, or the antibody may be dispersed in the solvent (especially a binding buffer) beforehand, and the metal-resin complex may be added thereto. Dispersion can be carried out by known methods, but it is preferable to use dispersion means such as sonication, stirring with a rotator, or a vortex mixer. The labeled antibody dispersion obtained in this way can be separated as a solid portion, such as the labeled antibody, by solid-liquid separation means such as centrifugation.

[0107] The method for producing the labeled antibody of this embodiment may further include the following steps, if necessary.

[0108] • Blocking process The blocking step involves dispersing the labeled antibody in a blocking buffer. In the blocking step, the labeled antibody obtained in the antibody binding step is dispersed in a blocking buffer to suppress nonspecific adsorption to the labeled antibody.

[0109] As the blocking buffer, it is preferable to use a solution of a protein that does not bind to the substance to be detected. Examples of proteins that can be used as the blocking buffer include bovine serum albumin (BSA), ovalbumin, casein, and gelatin. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. Known methods can be used to disperse the labeled antibody, but it is preferable to use dispersion means such as sonication or stirring with a rotator. In this way, a labeled antibody dispersion in which the labeled antibody is uniformly dispersed is obtained. From this labeled antibody dispersion, only the labeled antibody can be separated as a solid portion by solid-liquid separation means such as centrifugation. Furthermore, optional processes such as washing and preservation can be carried out as needed. The washing and preservation processes are described below.

[0110] • Cleaning process The washing process involves adding a washing buffer to the labeled antibody separated by a solid-liquid separation means, and uniformly dispersing the labeled antibody in the washing buffer. For dispersion, it is preferable to use a dispersion method such as sonication. The washing buffer is not particularly limited, but for example, a predetermined concentration of Tris buffer (tris-hydroxymethylaminomethane buffer), glycinamide buffer, or arginine buffer adjusted to a pH range of 6 to 9 can be used. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. The washing process of the labeled antibody can be repeated multiple times as needed.

[0111] • Preservation process The preservation process involves adding a preservation buffer to the labeled antibody separated by a solid-liquid separation means, and uniformly dispersing the labeled antibody in the preservation buffer. For dispersion, it is preferable to use a dispersion method such as sonication. As the preservation buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregating agent and / or stabilizer to the washing buffer can be used. As anti-aggregating agents, for example, sugars such as sucrose, maltose, lactose, and trehalose, or polyhydric alcohols such as glycerin and polyvinyl alcohol can be used. As stabilizers, there are no particular limitations, but for example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin can be used. In this way, the preservation process for labeled antibodies can be carried out.

[0112] In each of the above steps, surfactants and preservatives such as sodium azide and parahydroxybenzoic acid esters may be used as needed.

[0113] Furthermore, when a reaction site (labeled antibody) is provided on an immunochromatographic strip, the form and method of forming the reaction site (labeled antibody) are not particularly limited and are as described above.

[0114] [Capture Ligand] A capture ligand is an antibody (or a fragment thereof) that specifically binds to the LRG.

[0115] Here, as the antibody (or fragment thereof), an antibody or fragment that binds to LRG can be used, as described above, but in particular, as the capture ligand, Anti-Human LRG(50A1)Mouse IgG may be used.

[0116] Furthermore, the morphology and formation method of the detection unit (capture ligand) are not particularly limited and are as described above.

[0117] <Kit and LRG detection method>

[0118] The kit of the present invention may use an immunochromatographic strip (test strip) [the above-mentioned immunochromatographic strip (test strip)] (or components of an immunochromatographic strip), and is not particularly limited.

[0119] In one embodiment, the kit may include (combine) a test strip that does not contain the labeled antibody (and furthermore, the reaction part) and the labeled antibody. The labeled antibody may also be in the form of a reagent (detection reagent) containing the labeled antibody.

[0120] One embodiment (or other embodiment) of the kit may include (combine) a test strip (the test strip) and other components (components for detection, such as a developing solution), and preferably may include a developing solution.

[0121] Furthermore, LRG can be detected or quantified by using the immunochromatographic strip (test strip) (or kit) of the present invention.

[0122] Therefore, the present invention also includes such detection or quantification methods (immunoassay methods).

[0123] Such methods are not particularly limited, and conventional methods can be used depending on the form of the test strip (or kit), but may include, for example, the following steps (I) to (III). Step (I): A step of bringing the LRG contained in the sample (the sample containing the LRG) into contact with the labeled antibody (for example, in contact in the reaction section). Step (II): A step of bringing the complex formed in Step (I), which includes the LRG and the labeled antibody, into contact with a capture ligand (for example, in the detection unit). Step (III): Step for measuring the color intensity of the resin-metal composite (e.g., color intensity derived from light energy absorption due to localized surface plasmon resonance and electronic transitions).

[0124] The sample used in step (I) may be the sample described above, or it may be a sample diluted with the developing solution (or sample processing solution) described later (or to which the developing solution has been added).

[0125] Furthermore, if a test strip (or kit) is used that does not contain a labeled antibody (or reaction site), step (I) may be performed inside or outside the test strip.

[0126] For example, after performing step (I) by bringing the LRG in the sample into contact with the labeled antibody in the detection reagent, the sample may be applied to an appropriate part of the test strip (a reaction area that does not contain the labeled antibody, a sample addition area, etc.) and steps (II) and (III) may be performed sequentially.

[0127] Using this method, if a sample contains LRG, LRG can be detected or quantified.

[0128] Step (III) may also include: Step (III-1): a step of contacting a labeled antibody and / or complex with a capture ligand in the control unit; Step (III-2): a step of measuring the color intensity caused by the labeled substance in the determination unit and the control unit, respectively; and Step (III-3): a step of quantifying the amount of LRG contained in the sample based on the measured color intensity.

[0129] In step (III), the color intensity can be measured using a measuring device such as an immunochromatograph. The principle of measuring the color intensity in the measuring device is not particularly limited. For example, an immunochromatograph irradiates light into a determination unit and a control unit and measures the color intensity from the attenuation of the reflected light.

[0130] In step (III), the amount of LRG contained in the sample can be quantified based on the measured color intensity. It is preferable to estimate the amount of LRG contained in the sample after correcting the color intensity in the determination unit by dividing it by the sum of the color intensities in the determination unit and the control unit.

[0131] When using a test strip with two control lines in the control section, the correction can be performed based on the following formula (1), where TL is the color intensity of the test line in the determination section, CL1 is the color intensity of control line 1 (6 in Figure 2), and CL2 is the color intensity of control line 2 (7 in Figure 2). TL / (TL+CL1+CL2) Equation (1)

[0132] Furthermore, the above correction can be performed based on the following formula (2) when using a test strip with n control lines (where n is an integer). TL / (TL+CL1+CL2+...CLn) Equation (2)

[0133] Since the color intensity of the test line and the color intensity of the control line are in a constant proportional relationship, by performing corrections using the above equations (1) and (2), it is possible to offset variations in the concentration of the developed LRG-labeled antibody conjugate and labeled antibody, as well as variations in the development speed, thereby improving quantitative accuracy.

[0134] In step (III-3), reference standard data showing the relationship between the color intensity in the detection unit or the correction value obtained by formula (2) above and the amount of LRG can be used. Examples of reference standard data include calibration curves, tables, and formulas showing the relationship between color intensity and LRG concentration. Reference standard data may be those prepared in a general manner, or they may be prepared by conducting preliminary experiments based on standard reagents with known LRG concentrations before the immunoassay.

[0135] In the method of the present invention, the initial total amount of labeled antibody used in step (I) is used in step (II) The total amount of labeled antibody in the LRG-labeled antibody conjugate captured by the capture ligand in step (III) (capture yield) is preferably 90% or higher, more preferably 95% or higher, and even more preferably 98% or higher. A capture yield of 90% or higher allows for accurate determination of the relationship between the color intensity of the LRG-labeled antibody conjugate captured on the test line and the color intensity based on the total amount of labeled antibody deployed on the test strip, enabling highly accurate quantification by correcting based on formula (1) or (2) above.

[0136] In such samples, the concentration of LRG is not particularly limited, but it may be a low concentration, for example, 1 ng / ml or less. In this invention, even at such low concentrations, detection or quantification can be performed efficiently.

[0137] Since the method of the present invention can detect LRG, it can detect or diagnose symptoms or diseases in which an increase in LRG in the blood is expected (or related to an increase in LRG in the blood). Examples of such diseases include Kawasaki disease. Therefore, the method of the present invention can be used for the detection or diagnosis of Kawasaki disease.

[0138] [Developing solution] The developing solution (or sample processing solution) may typically contain a surfactant, and may be an aqueous solution containing a surfactant. One or more types of surfactants may be used.

[0139] The surfactant is preferably a nonionic surfactant. Examples of nonionic surfactants include polyoxyethylene (POE) type surfactants (or surfactants having polyoxyethylene chains) (e.g., ether type surfactants, ester-ether type surfactants, etc.).

[0140] Among nonionic surfactants such as POE-type surfactants, from the viewpoints of easily achieving sufficient detection sensitivity and easily suppressing false positives (and even false negatives) for immunochromatographic test strips, POE alkylphenyl ethers and the like are preferred.

[0141] In the POE alkylphenyl ethers, the alkyl group may be, for example, C 3―24 alkyl (e.g., C 4―24 alkyl, C 5―22 alkyl, C 6―22 alkyl, C 7―20 alkyl, C 8―20 alkyl, etc.) and the like. The alkyl group may be a linear alkyl or a branched alkyl. Representative examples of POE alkylphenyl ethers include, for example, POE octylphenyl ether, POE nonylphenyl ether, POE dodecylphenyl ether, and the like.

[0142] In the POE-type surfactant (e.g., POE alkylphenyl ethers and the like), the average added mole number of ethylene oxide may be, for example, 5 or more (e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc.), and may be, for example, 50 or less (e.g., 40 or less, 30 or less, 20 or less, etc.).

[0143] The HLB value of the polyoxyethylene-type surfactant is not particularly limited, and may be, for example, 5 to 30 or the like.

[0144] In the developing solution, the concentration of the surfactant is not particularly limited, but from the viewpoints of easily achieving sufficient detection sensitivity and easily suppressing false positives (and even false negatives) for immunochromatographic test strips, the concentration may be, for example, 0.1 wt% or more, preferably 0.5 wt% or more, and may be, for example, 5 wt% or less, preferably 2 wt% or less.

[0145] The developing solution may contain other components besides the surfactant and water. Other components may be those commonly used in sample processing solutions. Other ingredients, though not limited to those mentioned above, may include buffering agents, salts (sodium chloride), blocking agents (e.g., BSA, sodium caseinate, etc.), and preservatives. The developing solution may contain one or more other components.

[0146] The developing solution can be prepared by mixing a surfactant and water (and other components).

[0147] As a buffering agent, known buffering agents may be used, such as (Tris) buffer, glycinamide buffer, arginine buffer, phosphate buffer, etc. One or more types of cushioning material may be used.

[0148] In the developing solution, the concentration of the buffer is not particularly limited, but from the viewpoint of pH stability, it may be, for example, 10 mM or more, preferably 30 mM or more, or for example, 200 mM or less, preferably 100 mM or less.

[0149] In the developing solution, the concentration of sodium chloride is not particularly limited, but from the viewpoint of having a composition similar to physiological saline, it may be, for example, 0.5 to 2 wt% (85 mM to 342 mM).

[0150] Examples of blocking agents include bovine serum albumin (BSA) and milk-derived blocking agents (e.g., casein, sodium caseinate, skim milk, Block Ace, etc.). Blocking agents may be used one or more types.

[0151] In the developing solution, the concentration of the blocking agent is not particularly limited, but from the viewpoint of ensuring that the immunochromatographic test strip can easily achieve sufficient detection sensitivity, it may be, for example, 0.1 wt% or more, preferably 0.2 wt% or more, or for example, 2 wt% or less, preferably 1 wt% or less.

[0152] As a preservative, known preservatives may be used, such as sodium azide and Procline®. One or more types of preservatives may be used.

[0153] In the developing solution, the concentration of the preservative is not particularly limited, but for example, it may be around 0.01 to 0.1 wt%.

[0154] Other additives to the developing solution may include polyethylene glycol, phospholipid polymers, chelating agents, sugars, alcohols, and HAMA blockers, for the purpose of sensitizing and inhibiting nonspecific reactions.

[0155] In the developing solution, the concentration of the additive is not particularly limited, but for example, it may be around 0.01 to 10 wt%.

[0156] The pH of the developing solution may be, for example, 7 to 9, from the viewpoint of ensuring that the immunochromatographic test strip can easily achieve sufficient detection sensitivity.

[0157] The developing solution described above can be used to dilute the sample (a sample containing LRG). By diluting the sample with the developing solution (or by adding the developing solution to the sample), a sample solution can be prepared.

[0158] In other words, the present invention also includes a developing solution for detecting or quantifying LRG contained in a sample. Such developing solutions may be intended for use with the immunochromatographic test strips (test strips containing labeled antibodies) described above, or for use with the immunochromatographic test strips (test strips without labeled antibodies) and labeled antibodies described above.

[0159] The test strips, kits, and developing solutions of the present invention can be used for the detection or diagnosis of symptoms or diseases in which an increase in LRG in the blood is expected (or is related to an increase in LRG in the blood). Examples of such diseases include Kawasaki disease. Therefore, the test strips, kits, and developing solutions of the present invention can be used for the detection or diagnosis of Kawasaki disease. [Examples]

[0160] Next, the present invention will be specifically described with reference to examples, but the present invention is not limited in any way by these examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are performed as described below.

[0161] <Antibody> The following antibodies were used. ·Antibody A: Anti-Human LRG (48A1) Mouse IgG, Immunobiology Institute, product number 99912-100 ·Antibody B: Anti-Human LRG (50A1) Mouse IgG, Immunobiology Institute, product number 99912-099 • Antibody C: Anti-Influenza Virus Type A Nucleoprotein, Mouse-Mono (InA108), High Test, Catalog Number 3IN5-InA108 • Antibody D: Anti-Influenza Virus Type A Nucleoprotein, Mouse-Mono (InA224), High Test, Catalog Number 3IN5-InA224

[0162] <Absorbance measurement of metal-resin composite particles> The absorbance of metal-resin composite particles was measured using a spectrophotometer (Shimadzu Corporation, UV3600) with a dispersion of 0.01 wt% (weight%) of metal-resin composite particles (dispersion medium: water) placed in a quartz glass cell (optical path length 10 mm). The absorbance at 570 nm for gold-resin composites and 400 nm for platinum-resin composites were measured.

[0163] <Measurement of solid content concentration and metal load amount> One g of the dispersion before concentration adjustment was placed in a porcelain crucible and dried at 70°C for 3 hours. The weight was measured before and after drying, and the solid content concentration was calculated using the following formula. Solid content concentration (wt%)= [Weight after drying (g) / Weight before drying (g)] × 100 Furthermore, the samples after the above drying treatment were subjected to further heat treatment at 500°C for 5 hours, and the weights before and after heat treatment were measured. The amount of metal supported was then calculated using the following formula. Metal load (wt%) = [Weight after heat treatment (g) / Weight before heat treatment (g)] × 100

[0164] <Measurement of average particle size of metal particles> A substrate was prepared by dropping a metal-resin composite particle dispersion onto a metallic mesh with a carbon support film. Images were observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000). The area-average diameter of 100 arbitrary metal particles was measured from these images and defined as the average particle diameter.

[0165] <Measurement of average particle size of resin particles and metal-resin composite particles> The average area diameter of 100 arbitrary resin particles and metal-resin composite particles was measured from images of substrates prepared by dropping resin particles and metal-resin composite particles onto a metallic mesh with a carbon support film, using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000). This was then used to determine the average particle diameter.

[0166] [Example 1] <Synthesis of resin particles> Trioctylammonium chloride (0.60 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) were dissolved in 300 g of pure water. Then, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by stirring at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.25 g) dissolved in 18.00 g of pure water was added dropwise, and the mixture was stirred at 60°C for 3.5 hours to obtain resin particles A-1 with an average particle size of 425 nm. The particles were precipitated by centrifugation (9000 rpm, 40 minutes), the supernatant was removed, and the mixture was dispersed again in pure water. Impurities were then removed by dialysis. Subsequently, the concentration was adjusted to obtain a 10 wt% resin particle dispersion B-1.

[0167] [Example 2] <Synthesis of resin particles> Trioctylammonium chloride (1.70 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) were dissolved in 300 g of pure water. Then, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by stirring at 60°C for 30 minutes. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.25 g) dissolved in 18.00 g of pure water was added dropwise, and the mixture was stirred at 60°C for 3.5 hours to obtain resin particles A-2 with an average particle size of 365 nm. The particles were precipitated by centrifugation (9000 rpm, 40 minutes), the supernatant was removed, and the mixture was dispersed again in pure water. Impurities were then removed by dialysis. Subsequently, the concentration was adjusted to obtain a 10 wt% resin particle dispersion B-2.

[0168] [Example 3] <Synthesis of gold-resin composite particles> 30.0 g of resin particle dispersion B-2 was mixed with 78.0 g of pure water, then 42.9 g of 7 wt% aqueous chloroauric acid solution was added, and the mixture was stirred at room temperature for 3 hours. The resin particles were precipitated from this mixture by centrifugation (3000 rpm, 30 minutes), and excess chloroauric acid was removed by removing the supernatant. Subsequently, the concentration was adjusted to obtain 5 wt% gold ion adsorbent resin particle dispersion C-3.

[0169] Next, 65 g of C-3 was added to 4725 g of pure water, and while stirring at 3°C, 15 g of 528 mM aqueous dimethylamine borane solution was added dropwise. The mixture was then stirred at room temperature for 3 hours to obtain gold-resin composite particles D-3 with an average particle size of 379 nm. D-3 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt% gold-resin composite particle dispersion (AuNCP) E-3. The absorbance of gold-resin composite particles F-3 in E-3 was 1.36. The average particle size of the gold particles in F-3 was 21 nm, and the gold load was 48.3 wt%.

[0170] [Example 4] <Synthesis of platinum-resin composite particles> 30.0 g of resin particle dispersion B-2 was mixed with 85.0 g of pure water, then 31.0 g of 7 wt% chloroplatinic acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. The resin particles were precipitated from this mixture by centrifugation (3000 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. Subsequently, the concentration was adjusted to obtain 5 wt% platinum ion adsorbed resin particle dispersion C-4.

[0171] Next, 70.5 g of C-4 was added to 4725 g of pure water, and while stirring at 3°C, 137 g of a 132 mM aqueous dimethylamine borane solution was added dropwise. The mixture was then stirred at room temperature for 3 hours to obtain platinum-resin composite particles D-4 with an average particle size of 381 nm. D-4 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt% platinum-resin composite particle dispersion (PtNCP) E-4. The absorbance of platinum-resin composite particles F-4 in E-4 was 1.64. The average particle size of the platinum particles in F-4 was 3.5 nm, and the platinum load was 37.5 wt%.

[0172] [Example 5] <Synthesis of platinum-resin composite particles> 30.0 g of resin particle dispersion B-1 was mixed with 85.0 g of pure water, then 31.0 g of 7 wt% chloroplatinic acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. The resin particles were precipitated from this mixture by centrifugation (3000 rpm, 30 minutes), and excess chloroplatinic acid was removed by removing the supernatant. Subsequently, the concentration was adjusted to obtain 5 wt% platinum ion adsorbed resin particle dispersion C-5.

[0173] Next, 70.5 g of C-5 was added to 4725 g of pure water, and while stirring at 3°C, 137 g of 132 mM dimethylamine borane aqueous solution was added dropwise. The mixture was then stirred at room temperature for 3 hours to obtain platinum-resin composite particles D-5 with an average particle size of 442 nm. D-5 was concentrated by centrifugation, purified by dialysis, and the concentration was adjusted to obtain a 1 wt% platinum-resin composite particle dispersion (PtNCP) E-5. The absorbance of platinum-resin composite particles F-5 in E-5 was 1.72. The average particle size of the platinum particles in F-5 was 3.6 nm, and the platinum load was 38.6 wt%.

[0174] [Example 1] (Antibody binding process) After mixing 12.5 μg of antibody A {Anti-Human LRG (48A1) Mouse IgG, Institute of Immunobiological Laboratories, product number 99912-100} with 0.45 mL of 50 mM HEPES aqueous solution (pH 7), 0.05 mL of 1 wt% platinum-resin composite particle dispersion E-5 was added, and the mixture was inverted and stirred at room temperature for 2 hours to obtain labeled antibody dispersion J-1 containing antibody A labeled with platinum-resin composite particles F-5.

[0175] (Blocking process) Next, labeled antibody dispersion J-1 was centrifuged at 3000 rpm for 5 minutes, and after removing the supernatant, 0.5 mL of 50 mM HEPES aqueous solution (pH 7) containing 1 wt% sodium caseinate was added to the settled sediment, and after ultrasonic dispersion, the mixture was further stirred by inversion at room temperature for 2 hours to obtain labeled antibody dispersion K-1.

[0176] (Cleaning process) Next, the labeled antibody dispersion K-1 was centrifuged at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant was added to the settled sediment and ultrasonically dispersed. This procedure was repeated three times to complete the washing process.

[0177] (Preservation process) Next, after cooling with ice, the mixture was centrifuged at 3000 rpm for 5 minutes. After removing the supernatant, 0.5 mL of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant and 10 wt% sucrose was added to the settled sediment and ultrasonically dispersed to obtain labeled antibody dispersion L-1.

[0178] (Conjugate pad creation) 0.12 ml of labeled antibody dispersion L-1 was centrifuged at 3000 rpm for 5 minutes, and after removing the supernatant, 0.333 ml of 5 mM Tris aqueous solution (pH 8.5) containing 5 wt% sucrose and 2.5 wt% sodium caseinate was added to the settled sediment and ultrasonically dispersed to obtain labeled antibody dispersion M-1. Labeled antibody dispersion M-1 was uniformly impregnated into a glass fiber nonwoven fabric and dried at 50°C for 1 hour to prepare conjugate pad N-1. At this time, the volume of labeled antibody dispersion M-1 was adjusted so that the content of platinum-resin composite particles F-5 in conjugate pad N-1 was 3 μg per test of evaluation by the immunochromatographic method described later.

[0179] (Preparation of immunochromatographic strips) An immunochromatographic strip with the structure shown in Figure 2 was prepared. First, antibody B {Anti-Human LRG (50A1) Mouse IgG, Immunobiological Laboratories, product number 99912-099} was applied to a 25 mm wide nitrocellulose membrane 4 to create test line 5. Anti-mouse IgG antibody was also applied downstream of test line 5 to create two control lines 6 and 7. After drying the nitrocellulose membrane 4 at 50°C for 1 hour, a laminate film 1, conjugate pad N-1 (as a conjugate pad 3), sample pad 2 (glass fiber nonwoven fabric), and absorbent pad 8 (cotton nonwoven fabric) were laminated as shown in the cross-sectional view of the immunochromatographic strip in Figure 2. Finally, the strip was cut to a width of 3.5 mm to create immunochromatographic strip P-1.

[0180] (Preparation of developing solution) An aqueous solution (pH 7.5) containing 50 mM Tris, 150 mM NaCl, 0.5 wt% sodium caseinate, 1.0 wt% NP-40, and 0.09% sodium azide was prepared and used as developing solution 1.

[0181] (Evaluation by immunochromatography) By diluting LRG antigen (Recombinant Human LRG (Immunobiological Laboratories, product number 27769S-0.5ML)) with developing solvent 1, sample solutions for strongly positive control (antigen concentration 10 ng / ml), weakly positive control (antigen concentration 1 ng / ml), and negative control (antigen-free) were prepared. 50 μl of the sample solution was dropped onto sample pad 2 of immunochromatographic strip P-1. After 30 minutes, the color intensity of test line 5 was measured using an immunochromatographic reader (Hamamatsu Photonics C10066-10). The results of the immunochromatographic evaluation are shown in Table 1.

[0182] [Reference example 1] As shown in Table 1, in Reference Example 1, the antibody binding process, blocking process, washing process, storage process, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 1, except that the antibody used on the particle side and the antibody coated on the membrane test line were swapped. Table 1 shows the results of the immunochromatographic evaluation.

[0183] [Example 2, Example 3, Comparative Example 1] As shown in Table 1, the antibody binding process, blocking process, washing process, storage process, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 1, except that Example 2 used gold-resin composite particle dispersion E-3 instead of platinum-resin composite particle dispersion E-5, Example 3 used platinum-resin composite particle dispersion E-4, and Comparative Example 1 used Au colloid (Tanaka Kikinzoku Kogyo, Au colloid solution-SC, particle size 20 nm). Table 1 shows the results of the immunochromatographic evaluation.

[0184] [Comparative Example 2] As shown in Table 1, the antibody conjugation process, blocking process, washing process, storage process, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Comparative Example 1, except that a 5 mM Tris aqueous solution (pH 9.5) containing 1 wt% BSA was used as the conjugation buffer and a 5 mM Tris aqueous solution (pH 9.5) containing 1 wt% BSA was used as the blocking buffer. Table 1 shows the results of the immunochromatographic evaluation.

[0185] [Reference examples 2 and 3] As shown in Table 1, the antibody binding, blocking, washing, storage, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 1, except that antibody C (Anti-Influenza Virus Type A Nucleoprotein, Mouse-Mono (InA108), Hi-Test, Catalog No. 3IN5-InA108) and antibody D (Anti-Influenza Virus Type A Nucleoprotein, Mouse-Mono (InA224), Hi-Test, Catalog No. 3IN5-InA224) were used instead of antibody A and antibody B. For the immunochromatographic evaluation, the FluA antigen (Recombinant Influenza A virus NP (Signalway Antibody, Product No. AP71235)) was used instead of the LRG antigen. The results of the immunochromatographic evaluation are shown in Table 1.

[0186] From the test results of Examples 1-3, Comparative Examples 1-2, and Reference Examples 1-3, it can be seen that by using gold-resin composite particles or platinum-resin composite particles as particles (labeling substances), and using antibody A as the antibody bound to the particles and antibody B as the antibody coated on the membrane, the color intensity of the test line in positive samples in immunochromatographic evaluation is increased, and false positives in negative samples are suppressed. This indicates that the sensitivity of immunochromatography is improved compared to when other particles or combinations of antibodies are used. Furthermore, among Examples 1-3, the conditions in Example 1 showed the highest immunochromatographic sensitivity, and the best combination was found to be platinum-resin composite particle E-5, with antibody A bound to the particles and antibody B coated on the membrane.

[0187] [Table 1]

[0188] [Example Test] Immunochromatographic evaluation was repeated nine times under the same conditions as in Example 1. For strongly positive and weakly positive samples, the color intensity of test line 5 (TL), control line 6 (CL1), and control line 7 (CL2) after 30 minutes was measured using an immunochromatograph reader. TL / (TL+CL1) and TL / (TL+CL1+CL2) were calculated as correction calculations using the color intensity of the control lines to evaluate reproducibility and variability in repeated tests. The measurement results are shown in Table 2.

[0189] [Table 2]

[0190] Using the results in Table 2, the calculations for Examples 4 to 6 below were performed.

[0191] [Example 4] The mean value and coefficient of variation (CV) were calculated using the TL value after nine repeated measurements without performing any correction calculations. The CV value was calculated as standard deviation / mean value. The calculated values ​​are shown in Table 3.

[0192] [Example 5] As a correction calculation using the CL value, the average value of TL / (TL+CL1) and the coefficient of variation (CV) were calculated, and the effect of the correction calculation on reducing variability was evaluated. The results are shown in Table 3.

[0193] [Example 6] As a correction calculation using the CL value, the average value of TL / (TL+CL1+CL2) and the coefficient of variation CV value were calculated, and the effect of the correction calculation on reducing variability was evaluated. The results are shown in Table 3.

[0194] The evaluation results from Examples 4-6 confirmed that variability is reduced by correction calculations using CL values. The variability reduction effect was higher with correction calculations using two control lines, demonstrating the variability reduction effect of using multiple control lines.

[0195] [Table 3]

[0196] Although embodiments of the present invention have been described in detail above for illustrative purposes, the present invention is not limited to the above embodiments. [Industrial applicability]

[0197] According to the present invention, novel immunochromatographic strips and the like can be provided. [Explanation of Symbols]

[0198] 1...Support (packing sheet), 2...Sample addition section (sample pad), 3...Conjugate section (reaction section, conjugate pad), 4...Membrane (nitrocellulose membrane, etc.), 5...Test line (detection section), 6...Control line 1, 7...Control line 2, 8...Liquid absorption section (absorption pad), 10...Resin particles, 20...Metal particles, 30...Encapsulated metal particles, 40...Partially exposed metal particles, 50...Surface adsorbed metal particles, 60...Surface layer, 100...Metal-resin composite

Claims

1. An immunochromatographic test strip for detecting or quantifying leucine-rich α2-glycoprotein (LRG) contained in a sample, A determination unit equipped with a capture ligand that specifically binds to LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, and The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. Test strip.

2. An immunochromatographic test strip for detecting or quantifying LRG contained in a sample, A determination unit equipped with a capture ligand that specifically binds to LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, and The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. A kit that uses test strips.

3. An immunochromatographic test strip for detecting or quantifying LRG contained in a sample, A determination unit equipped with a capture ligand that specifically binds to LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, and The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. Test strip and A kit using a developing solution containing a surfactant including polyoxyethylene alkylphenyl ethers.

4. This is an immunochromatographic test strip for detecting or quantifying LRG contained in a sample. A test strip comprising a determination unit equipped with a capture ligand that specifically binds to LRG, A labeled antibody is obtained by labeling an antibody that specifically binds to LRG with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, A kit using a developing solution containing a surfactant including polyoxyethylene alkylphenyl ethers, The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. kit.

5. A method for detecting or quantifying LRG contained in a sample, A determination unit equipped with a capture ligand that specifically binds to LRG, Using an immunochromatographic test strip, the following steps (I) to (III) are performed, with the sample unfolding in the direction of the test strip, and the reaction section comprising a labeled antibody, in which an antibody that specifically binds to LRG is labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, located upstream of the detection section; Step (I): A step of contacting the LRG contained in the sample with the labeled antibody. Step (II): A step of contacting the complex formed in Step (I), which contains the LRG and the labeled antibody, with a capture ligand. Process (III): Process for measuring the color intensity of the resin-metal composite. Includes, The sample contains a developing solution containing a surfactant including polyoxyethylene alkylphenyl ethers, The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. method.

6. A method for detecting or quantifying LRG contained in a sample, An immunochromatographic test strip is used, which includes a detection unit equipped with a capture ligand that specifically binds to LRG. The following steps (I) to (III); Step (I): A step of contacting the LRG contained in the sample with an antibody that specifically binds to the LRG, which is labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles. Step (II): A step of contacting the complex formed in Step (I), which contains the LRG and the labeled antibody, with a capture ligand. Process (III): Process for measuring the color intensity of the resin-metal composite. Includes, The sample contains a developing solution containing a surfactant including polyoxyethylene alkylphenyl ethers, The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. The capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. method.

7. A developing solution for detecting or quantifying LRG contained in a sample, Contains surfactants including polyoxyethylene alkylphenyl ethers, A determination unit equipped with a capture ligand that specifically binds to LRG, Upstream of the detection unit in the direction in which the sample unfolds, the reaction unit includes a labeled antibody that specifically binds to LRG, labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, and The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. A developing solution for use with an immunochromatographic test strip, wherein the capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody.

8. A developing solution for detecting or quantifying LRG contained in a sample, Contains surfactants including polyoxyethylene alkylphenyl ethers, An immunochromatographic test strip, comprising a determination unit equipped with a capture ligand that specifically binds to LRG, and A developing solution for combining an antibody that specifically binds to LRG with a labeled antibody that is labeled with a resin-metal composite having a structure in which multiple metal particles are immobilized on resin particles, The antibody is Anti-Human LRG (48A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody. A developing solution in which the capture ligand is Anti-Human LRG (50A1) Mouse IgG or an antibody having the same antigen recognition site as said antibody.

9. The kit, method, or developing solution according to any one of claims 3 to 8, wherein the polyoxyethylene alkylphenyl ethers include polyoxyethylene linear alkylphenyl ethers.

10. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the metal particles are silver, nickel, copper, gold, platinum, or palladium in elemental form or an alloy thereof.

11. In resin-metal composites, A first particle having a portion of the metal particle exposed outside the resin particle, A second particle whose entire body is enclosed within a resin particle, It includes, The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein at least some of the first particles and the second particles are distributed three-dimensionally on the surface layer of the resin particles.

12. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the resin particles are polymer particles having substituents in their structure that are capable of adsorbing metal ions.

13. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the average particle size of the metal particles is in the range of 1 to 100 nm.

14. A test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the average particle size of the resin-metal composite is in the range of 30 to 1000 nm.

15. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the reaction section has a plurality of control lines spaced apart in the direction in which the sample develops.

16. The test strip, kit, method, or developing solution according to claim 15, having two to five control lines.

17. A test strip, kit, or developing solution according to any one of claims 1 to 4, 7, and 8 for the detection or diagnosis of Kawasaki disease.

18. The method according to claim 5 or 6 for diagnosing Kawasaki disease.

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