Immunochromatographic strip, immunochromatographic kit, method of detection, and development liquid

Abstract

To provide a new immuno strip.SOLUTION: An immunochromatographic test strip for detecting or quantifying SARS-CoV-2-derived analyte contained in a specimen is provided. The test strip comprises: a determination part including capture ligand specifically coupled to the analyte; and a reaction part including upstream the determination part in a direction where the specimen develop, a labelled antibody obtained by labelling antibody specifically coupled to the analyte with a resin-metal composite body having a structure obtained by immobilizing a plurality of metal particles to resin particles. The antibody is antibody or its fragment coupled to protein composing the SARS-CoV-2-derived nucleocapsid. Epitope recognizing the antibody or its fragment is located at 208-th to 222-nd or 332-nd to 351-st of amino acid sequence represented by array number 1. The capture ligand is antibody or its fragment coupled to protein composing theSARS-CoV-2-derived nucleocapsid. Epitope recognizing the antibody or its fragment is located at first to 207-th or 374-th to 397-th of amino acid sequence represented by array number 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel immunochromatographic strip (test strip) and the like. [Background technology]

[0002] Immunoassays, also known as immunological assays, are methods for qualitatively and quantitatively analyzing trace components by utilizing specific antigen-antibody reactions. Antigen-antibody reactions are widely used in fields such as medicine due to their high sensitivity and selectivity. Various immunoassay methods are known based on their measurement principles, including enzyme immunoassays (EIA), radioimmunoassays (RIA), chemiluminescent immunoassays (CLIA), fluorescent immunoassays (FIA), latex agglutination assays (LIA, PA), immunochromatography (ICA), hemagglutination assays (HA), and hemagglutination inhibition assays (HI).

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

[0004] On the other hand, with regard to SARS-CoV-2 (the so-called novel coronavirus), an antibody or fragment thereof that binds to a protein constituting the SARS-CoV-2-derived nucleocapsid, characterized in that the epitope is located at positions 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1, has been proposed (Patent Document 1).

[0005] In addition, an antibody or a fragment thereof, or a combination thereof, which binds to a protein that constitutes the nucleocapsid derived from SARS-CoV-2, and the epitope recognized by the antibody or the fragment thereof, or the combination thereof, is located at positions 1 to 207 of the amino acid sequence represented by SEQ ID NO: 1, has been proposed (Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2022 / 004622 Brochure [Patent Document 2] Japanese Patent Application Publication No. 2023-128698 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel immunochromatographic strip (test strip) and the like. [Means for solving the problem]

[0008] The reactivity and specificity of an antigen and an antibody when they react to form a complex are important factors that determine the detection ability in immunoassays. Therefore, by selecting and using antigens and antibodies with excellent reactivity and specificity, the detection sensitivity of immunoassays can be increased. In other words, if the reactivity and specificity when an antigen and an antibody react to form a complex are insufficient, the sensitivity of the immunoassay cannot be sufficiently increased.

[0009] On the other hand, when detecting an antigen or antibody, the detection sensitivity can be increased by binding a labeling substance to the antibody, antigen, or complex. Therefore, the labeling ability of the labeling substance can also be said to be an important factor that determines the detection ability in immunoassays.

[0010] These points are no exception when it comes to the new coronavirus, and in particular, to detect trace amounts of new coronavirus antigens, such as in antigen tests, high detection capabilities of immunoassays are required.

[0011] As in Patent Document 1, antibodies (or fragments thereof, hereinafter sometimes simply referred to as antibodies, etc.) for detecting the novel coronavirus are currently being developed. However, according to the inventors' investigations, even with such antibodies, depending on the conditions such as the type of labeling substance and their combination (composition conditions), there is a risk that the detection sensitivity may be insufficient, or that although the detection sensitivity is sufficient, false negatives or false positives may occur. In particular, when a saliva sample is used for detection, the detection sensitivity is likely to be insufficient, or false negatives or false positives may occur.

[0012] In this situation, the present inventors have conducted extensive research and have found that, in an immunoassay (immunochromatographic strip), by combining a specific labeling substance (i.e., a metal-resin complex having a structure in which metal particles are immobilized on resin particles) with a specific antibody (i.e., an antibody whose epitope is located at positions 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1 (or at a specific one among positions 121 to 419)), in a specific manner (constituent requirement), it is possible to achieve sufficient detection sensitivity or suppress false positives (and even false negatives). Furthermore, the present inventors have discovered that by using a specific developing solution in the above-mentioned specific combination, sufficient detection sensitivity can be achieved even when using saliva samples, or false positives (and even false negatives) can be suppressed, and have completed the present invention.

[0013] That is, the present invention relates to the following inventions. [1] An immunochromatographic test strip (e.g., a lateral flow chromatographic test strip) for detecting or quantifying an analyte (an analyte derived from SARS-CoV-2) contained in a sample, a determination unit provided with (including) a capture ligand that specifically binds to the analyte (for example, a determination unit having a membrane and a capture ligand that specifically binds to the analyte immobilized on the membrane); a reaction section, located upstream of the determination section in the direction in which the sample develops, that is provided with (includes) a labeled antibody, which is an antibody that specifically binds to the analyte and is labeled 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 an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1. Test strips.

[0014] [2] An immunochromatographic test strip for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, a detection unit provided with a capture ligand that specifically binds to an analyte; a reaction section, located upstream of the determination section in the direction of sample development, which contains a labeled antibody, which specifically binds to the analyte and is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1. A kit (for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample) using (equipped with) a test strip.

[0015] [3] An immunochromatographic test strip (e.g., a lateral flow chromatographic test strip) for detecting or quantifying an analyte (an analyte derived from SARS-CoV-2) contained in a sample, a determination unit provided with (including) a capture ligand that specifically binds to the analyte (for example, a determination unit having a membrane and a capture ligand that specifically binds to the analyte immobilized on the membrane); a reaction section, located upstream of the determination section in the direction in which the sample develops, that is provided with (includes) a labeled antibody, which is an antibody that specifically binds to the analyte and is labeled 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 an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1. A test strip; Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 A kit (for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample) that uses (is equipped with, or includes) a developing solution (or specimen treatment solution) containing a surfactant (polyoxyethylene type surfactant) containing alkylphenyl ether (ALK).

[0016] [4] An immunochromatographic test strip (e.g., a lateral flow chromatographic test strip) for detecting or quantifying an analyte (an analyte derived from SARS-CoV-2) contained in a sample, a test strip including a determination part provided with (including) a capture ligand that specifically binds to an analyte (for example, a determination part including a membrane and a capture ligand that specifically binds to an analyte immobilized on the membrane); a labeled antibody in which an antibody that specifically binds to an analyte is labeled 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; Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 A kit using (including, combined with, or equipped with) a developing solution (or a specimen treating solution) containing a surfactant (polyoxyethylene surfactant) containing an alkylphenyl ether (alkylphenyl ether), the antibody (the antibody in the labeled antibody) is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1. A kit (for detecting or quantifying an analyte derived from SARS-CoV-2 in a sample).

[0017] [5] A method for detecting or quantifying an analyte (an analyte derived from SARS-CoV-2) contained in a sample (immunoassay method), a determination unit provided with (including) a capture ligand that specifically binds to the analyte (for example, a determination unit having a membrane and a capture ligand that specifically binds to the analyte immobilized on the membrane); The immunochromatographic test strip (or a kit using the test strip) includes a reaction zone provided with (including) a labeled antibody, which is an antibody that specifically binds to an analyte and is labeled 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, and is located upstream of the determination zone in the direction in which the sample develops, and the immunochromatographic test strip (or a kit using the test strip) includes the following steps (I) to (III); Step (I): A step of contacting (contacting in a reaction zone) an analyte contained in a sample (a sample, a sample containing the analyte) with a labeled antibody; Step (II): A step of contacting the complex containing the analyte and the labeled antibody formed in Step (I) with a capture ligand (contact in the test zone); Step (III): A step of measuring the color intensity of the resin-metal composite (color intensity resulting from light energy absorption due to localized surface plasmon resonance and electron transition); Including, The sample is a polyoxyethylene alkylphenyl ether (e.g., polyoxyethylene C 3―24 a developing solution (or a specimen treatment solution) containing a surfactant (polyoxyethylene surfactant) containing alkylphenyl ether; the antibody (the antibody in the labeled antibody) is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1. Method (immunoassay method).

[0018] [6] A method for detecting or quantifying an analyte (an analyte derived from SARS-CoV-2) contained in a sample (immunoassay method), An immunochromatographic test strip (or a kit using the test strip) including a determination unit provided with (including) a capture ligand that specifically binds to an analyte (for example, a determination unit having a membrane and a capture ligand that specifically binds to the analyte immobilized on the membrane), The following steps (I) to (III): Step (I): A step of contacting an analyte contained in a sample (sample, sample containing the analyte) with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte 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; Step (II): A step of contacting the complex containing the analyte and the labeled antibody formed in Step (I) with a capture ligand (contact in the test zone); Step (III): A step of measuring the color intensity of the resin-metal composite (color intensity resulting from light energy absorption due to localized surface plasmon resonance and electron transition); Including, The sample is a polyoxyethylene alkylphenyl ether (e.g., polyoxyethylene C 3―24 a developing solution (or a specimen treatment solution) containing a surfactant (polyoxyethylene surfactant) containing alkylphenyl ether; the antibody (the antibody in the labeled antibody) is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1. Method (immunoassay method).

[0019] [7] A developing solution (or specimen treatment solution) for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 a developing solution (or a specimen treatment solution) containing a surfactant (polyoxyethylene surfactant) containing alkylphenyl ether; a detection unit provided with a capture ligand that specifically binds to an analyte; a reaction section, located upstream of the determination section in the direction of sample development, which contains a labeled antibody, which specifically binds to the analyte and is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; A developing solution for use in combination with an immunochromatographic test strip, in which the capture ligand is an antibody or fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1.

[0020] [8] A developing solution (or specimen treatment solution) for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, Polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene C 3―24 a developing solution (or a specimen treatment solution) containing a surfactant (polyoxyethylene surfactant) containing alkylphenyl ether; a test strip including a detection zone having a capture ligand that specifically binds to an analyte; A developing solution for combining an antibody that specifically binds to an analyte with a labeled antibody that is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle, comprising: the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; A developing solution in which the capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or its fragment is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1.

[0021] [9] The test strip, kit, method, or developing solution according to any one of [1] to [8], wherein the sample contains saliva.

[0022]

[10] The polyoxyethylene alkylphenyl ethers are polyoxyethylene linear alkylphenyl ethers (e.g., polyoxyethylene linear C 3―24 The test strip, kit, method, or developing solution according to any one of [1] to [9], which contains a hydroxybenzoate (a hydroxybenzoate) containing ...

[0023]

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

[10] , wherein the antibody and the capture ligand are monoclonal antibodies.

[0024]

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

[11] , wherein the metal particles are silver, nickel, copper, gold, platinum or palladium, either alone or as an alloy.

[0025]

[13] In resin-metal composites, a first particle having a metal particle exposed outside the resin particle; a second particle entirely encapsulated in the resin particle; It contains The test strip, kit, method, or developing solution according to any one of [1] to

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

[0026]

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

[13] , wherein the resin particles are polymer particles having a substituent in their structure that can adsorb metal ions.

[0027]

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

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

[0028]

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

[15] , wherein the average particle size of the resin-metal composite is within the range of 30 to 1000 nm. [Effects of the Invention]

[0029] The present invention can provide a novel immunochromatographic strip (test strip), kit, detection (or quantification) method, developing solution, and the like. Such a strip (kit, method, and developing solution) can detect (or quantify) an analyte (SARS-CoV-2-derived analyte) contained in a sample. In particular, in one embodiment of the present invention, sufficient detection sensitivity can be achieved by combining a specific labeling substance with a specific antibody in a specific manner, particularly when a saliva sample is used. In addition, in another embodiment of the present invention, false positives (and even false negatives) can be reduced, particularly when a saliva sample is used. Therefore, even minute amounts of novel coronavirus antigens can be detected (quantified) using antigen tests, making this extremely useful. [Brief explanation of the drawings]

[0030] [Figure 1] 1 is a schematic diagram showing the cross-sectional structure of a metal-resin composite used in one embodiment of the present invention. [Figure 2] 1 is a cross-sectional view showing an immunochromatographic strip used in one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, and other publications and information referenced herein are incorporated herein by reference in their entirety.

[0032] <Immunochromatography Strips> The immunochromatographic strip (test strip) of the present invention can be used to detect or quantify an analyte (an analyte derived from SARS-CoV-2) contained in a sample, and in particular may be a lateral flow type chromatographic strip (test strip).

[0033] The sample (or specimen) may be any that contains an analyte derived from SARS-CoV-2, and is not particularly limited to, but examples include blood, plasma, serum, urine, semen, cerebrospinal fluid, saliva, oral mucosa, nasal discharge, sweat, tears, ascites, amniotic fluid, feces, blood vessels, organs (e.g., liver), tissues, cells, etc., and preferably saliva. The sample may be any sample containing the above-exemplified substances, and the above-exemplified substances may be used as they are, or may be a solution or extract of the above-exemplified substances.

[0034] The sample may be human or animal. The animal is not particularly limited as long as it can be infected with SARS-CoV-2, and examples include dogs, cats, etc.

[0035] The method for collecting the sample is not particularly limited, and any known method may be used, for example, a cotton swab may be used.

[0036] For example, saliva may be collected using the method described in the Manual for Specimen Collection and Transportation from Patients Suspected of 2019-nCoV (Novel Coronavirus) Infection (National Institute of Infectious Diseases, April 16, 2020). For example, saliva may be collected by placing 1 to 2 ml of saliva in a sterilized plastic tube, capping it, and sealing it with parafilm. Alternatively, saliva may be collected using a saliva collection kit such as Salivet (registered trademark), where saliva is absorbed into cotton and then separated and collected by centrifugation.

[0037] Components contained in the sample (components other than analytes derived from SARS-CoV-2) are not particularly limited, but include, for example, cations {e.g., monovalent cations [e.g., ions of alkali metals (e.g., sodium, potassium, etc.)], polyvalent (divalent or higher) cations [e.g., ions of alkaline earth metals (e.g., calcium, etc.)]}, digestive enzymes (e.g., amylase, etc.), etc. The sample may contain one or more of these components. The proportion of each component contained in the sample is not particularly limited.

[0038] Furthermore, in the present invention, even if the sample contains components that are (or may be expected to be) likely to cause false positives in the detection of SARS-CoV-2-derived analytes, the SARS-CoV-2-derived analytes can be efficiently detected or quantified. For example, although it is unclear, cations (especially polyvalent cations) have the effect of agglutinating particles, which is thought to be a cause of false positives. Furthermore, although it is unclear, digestive enzymes denature proteins, which is thought to be a cause of false positives due to nonspecific adsorption. The present invention can efficiently detect or quantify SARS-CoV-2-derived analytes even when the sample contains cations or digestive enzymes. Note that saliva typically contains cations, such as polyvalent cations, and digestive enzymes.

[0039] The immunochromatographic strip (test strip) of the present invention has at least a determination section equipped with (including) a capture ligand that specifically binds to an analyte, and may further have a reaction section equipped with (including) a labeled antibody (labeled substance).

[0040] The determination zone and reaction zone may be provided with (contain) a capture ligand and a labeled antibody, respectively, and there are no particular limitations on the form in which the capture ligand and the labeled antibody are present. For example, the determination zone and reaction zone may be a capture ligand and a labeled antibody, respectively, and the capture ligand and the labeled antibody may be added (contained) or immobilized (positioned), respectively.

[0041] Specific examples of the form of the reaction site (labeled antibody) include directly incorporating (attaching, adding) the labeled antibody into (forming a reaction site on) the membrane, or incorporating (attaching, adding) the labeled antibody into (forming a reaction site on) a fixing means provided (fixed) on the membrane [for example, a pad (a conjugate pad described below) formed from cellulose filter paper, glass fiber, nonwoven fabric, etc.].

[0042] As a specific form of the determination part (capture ligand), for example, the capture ligand may be directly immobilized on the membrane (forming the determination part), or the capture ligand may be immobilized on an immobilizing means provided on (fixed to) the membrane (forming the determination part).

[0043] The method of addition or immobilization can be appropriately selected depending on the form of existence, etc., and is not particularly limited. For example, a dispersion liquid containing a labeled antibody or a capture ligand may be added or immobilized (to form a determination zone or a reaction zone) by adhering or applying (impregnating) the membrane or immobilization means.

[0044] The reaction zone is located upstream (on the side) of the determination zone in the direction in which the sample develops (the direction in which the sample develops) [or the determination zone is located downstream (on the side) in the direction in which the sample develops].

[0045] As will be described in detail later, the present invention is characterized by the use of specific capture ligands and labeled antibodies.

[0046] The immunochromatographic strip may generally include a membrane, on which a determination zone (test zone, test line) and a reaction zone may be immobilized (positioned).

[0047] The membrane is formed of an inert material (a material that does not react with analytes, various ligands, etc.) that exhibits capillary action and is made of a microporous material that allows the sample to develop as soon as it is added.

[0048] Examples of membranes (membrane materials) include fibrous or nonwoven fibrous matrices, membranes, filter paper, glass fiber filter paper, cloth, cotton, etc., made of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc. Among these, membranes, filter paper, glass fiber filter paper, etc. made of cellulose derivatives or nylon are preferred, and more preferred are nitrocellulose membranes, mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate) membranes, nylon membranes, filter paper, etc.

[0049] The immunochromatographic strip may further include other portions (members, parts) as necessary, such as a support portion [a support portion (support) for supporting the membrane, such as a plastic support], a sample addition portion [a sample addition portion (sample addition pad, sample pad) located upstream of the determination portion and reaction portion for adding a sample containing an analyte, such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, or cotton cloth], a liquid absorption portion [a liquid absorption portion (absorption portion, liquid absorption (absorption) pad) located downstream of the determination portion and reaction portion, such as a pad made of an absorbent material such as cellulose filter paper, nonwoven fabric, cloth, or cellulose acetate], a conjugate portion [e.g., a conjugate portion (conjugate pad) interposed between the sample addition portion and the membrane], and a control portion (control line).

[0050] FIG. 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 includes a membrane 4. The membrane 4 is provided with, in order in the direction of sample development, a sample application section 2, a conjugate section (reaction section) 3, a test line (determination section) 5, a control line (control section) 6, and a liquid absorption section 7.

[0052] For easier operation, the test strip preferably includes a support 1 that supports the membrane 4. The support may be made of, for example, plastic.

[0053] The test strip may have a sample application section 2 for applying a sample containing an analyte. The sample application section 2 is a portion of the test strip for receiving the sample containing an analyte. The sample application section 2 may be formed on the membrane 4 upstream of the determination section 6 in the direction of sample development, either directly or via a conjugate section 3.

[0054] Here, a specific labeled antibody that specifically binds to the analyte is fixed to the conjugate section (conjugate pad) 3, and a specific capture ligand that specifically binds to the analyte is fixed to the test line 5 (forming a reaction section and a determination section).

[0055] The test strip may have a control section formed downstream of the test section in the direction of sample development, the control section comprising an immobilized capture ligand that specifically binds to the labeled antibody.

[0056] By measuring the color intensity at the control section (control line) 6 as well as the test line (judgment section) 5, it is possible to confirm that the sample applied to the test strip has developed and reached the reaction section and judgment section, and that the test has been performed normally.

[0057] The absorbent part 7 may be formed of a pad of absorbent material such as cellulose filter paper, nonwoven fabric, cloth, or cellulose acetate. The speed at which the development front of the added sample moves after it reaches the absorbent part 7 varies depending on the material and size of the absorbent part 7. Therefore, by selecting the material and size of the absorbent part 7, it is possible to set the optimum speed for detecting and quantifying the analyte.

[0058] [Antibodies or fragments thereof that bind to proteins that make up the SARS-CoV-2 nucleocapsid] In the present invention, the labeled antibody and capture ligand use an antibody or a fragment thereof that binds to a protein that constitutes the nucleocapsid derived from SARS-CoV-2.

[0059] The epitope recognized by the antibody or fragment thereof may be, typically, the epitope of the antibody or fragment thereof located at positions 1 to 419 of the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, sometimes referred to as "the antibody or fragment thereof of the present invention").

[0060] The epitope recognized by the antibody or fragment thereof of the present invention is preferably located at positions 1 to 222, 332 to 351, 374 to 397, or 398 to 419 of the amino acid sequence shown in SEQ ID NO:1.

[0061] In particular, in the present invention, it is preferable to select specific antibodies (or fragments thereof) for the labeled antibody and the capture ligand, respectively.

[0062] Specifically, it is preferable to select a labeled antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment is at positions 208 to 222 and / or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1.

[0063] Such antibodies or fragments thereof may recognize (recognize as an epitope) sequences other than those 208 to 222 and those 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1 (for example, sequences 1 to 207 and those 374 to 397), or may not recognize them, or may not particularly recognize them.

[0064] Furthermore, the capture ligand is preferably an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment is one of positions 1 to 207 (e.g., positions 1 to 50, 51 to 105, 106 to 160, 161 to 207) or 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1.

[0065] Such antibodies or fragments thereof may recognize (recognize as an epitope) sequences other than those 1 to 207 and 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1 (for example, sequences 208 to 222 and 332 to 351), or may not recognize them, or may not particularly recognize them.

[0066] Such antibodies or fragments thereof of the present invention specifically bind to proteins that constitute the nucleocapsid derived from SARS-CoV-2.

[0067] In this specification, "SARS-CoV-2" refers to a coronavirus belonging to the SARS-related coronaviruses whose outbreak was first confirmed near Wuhan, China in 2019, and is used interchangeably with "2019 novel coronavirus." SARS-CoV-2 is pathogenic to humans and causes acute respiratory disease (COVID-19). Like other coronaviruses, SARS-CoV-2 is composed of spikes, nucleocapsid, integral membrane proteins, envelope proteins, and RNA. The nucleocapsid binds to RNA to form NP, which is surrounded by lipid-bound spikes, integral membrane proteins, and envelope proteins to form the envelope.

[0068] The genome sequence of SARS-CoV-2 has been published under GenBank accession number MN908947.3.

[0069] In the present invention, SARS-CoV-2 also includes so-called mutant strains (for example, those with mutations in the spike protein such as N501Y, L452R, E484Q, D614G, P681H, Y145H, A222V, and E484K). Currently known (publicly known) mutant strains include the so-called alpha strain, beta strain, gamma strain, delta strain, kappa strain, lambda strain, mu strain, and omicron strain (e.g., BA.2 strain, BA.2.75 strain, BA.5 strain, and XBB.1.5), but the subject of the present invention includes not only these known mutant strains, but also currently unknown mutant strains and new mutant strains that will arise through mutation in the future.

[0070] In the present invention, the term "protein constituting the nucleocapsid" refers to a protein constituting a complex consisting of nucleocapsid and RNA, and is used interchangeably with "NP." The amino acid sequence of SARS-CoV-2-derived NP is represented by SEQ ID NO: 1.

[0071] In the present invention, the term "fragment of SARS-CoV-2-derived NP" refers to a protein fragment consisting of a region of the amino acid sequence of SARS-CoV-2-derived NP (SEQ ID NO: 1) that has low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequences of NP derived from other coronaviruses (the region from positions 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1). The fragment of SARS-CoV-2-derived NP has a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 2.

[0072] From the viewpoint of accurate, simple, and rapid detection of SARS-CoV-2-derived NP, it is preferable that the fragment of SARS-CoV-2-derived NP has a sequence identity of 95% or more to the amino acid sequence represented by SEQ ID NO: 2, particularly preferably 98% or more, and even more preferably 100%.

[0073] In one embodiment, the fragment of SARS-CoV-2-derived NP consists of the amino acid sequence shown in SEQ ID NO: 2. Since the fragment of SARS-CoV-2-derived NP corresponds to a region with low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequence of NP derived from other coronaviruses, the fragment can be prepared and used as an immunogen to obtain the antibody of the present invention or a fragment thereof according to standard methods.

[0074] Antibodies of the invention may be, for example, intact immunoglobulin molecules, preferably IgM, IgD, IgE, IgA, or IgG, more preferably IgG1, IgG2a, IgG2b, IgG3, or IgG4. Antibodies of the invention may also be modified and / or altered antibodies, such as chimeric and humanized antibodies.

[0075] The antibody of the present invention may be a monoclonal or polyclonal antibody, a modified or altered monoclonal or polyclonal antibody, or a recombinantly or synthetically produced or synthesized antibody. From the viewpoint of accurate, simple, and rapid detection of SARS-CoV-2-derived NP, the antibody of the present invention is preferably a monoclonal antibody.

[0076] The fragment of the antibody of the present invention may be a part of such an immunoglobulin molecule, such as a Fab fragment or a VL-, VH- or CDR-region, and the fragment of the antibody of the present invention can also specifically recognize and bind to NP derived from SARS-CoV-2 to the same extent as the antibody of the present invention.

[0077] Fragments of antibodies of the present invention can be antibody fragments and portions thereof, such as separated light and heavy chains, Fab, Fab / c, Fv, Fab', F(ab')2, etc. In one embodiment, a fragment of an antibody of the present invention is a Fab fragment, a Fab / c fragment, an Fv fragment, a Fab' fragment, or an F(ab')2 fragment.

[0078] The antibody or fragment thereof of the present invention may also be an antibody derivative such as a bifunctional antibody or an antibody construct such as a single chain Fv (scFv), a bispecific scFv or an antibody fusion protein.

[0079] For details of antibodies or fragments thereof, refer to Patent Document 1 (International Publication No. 2022 / 004622), Patent Document 2 (JP Patent Publication No. 2023-128698), etc., and they may be synthesized (manufactured) according to the methods described in those documents, or commercially available products may be used.

[0080] [Labeled antibody] In the present invention, a labeled antibody (label, labeling substance) is used, which is an antibody that specifically binds to an analyte and is labeled with a resin-metal complex (a composite of metal particles and resin particles).

[0081] (resin-metal composite) A resin-metal composite (metal-resin composite) is a composite containing metal particles and resin particles, and typically has a structure in which metal particles (plurality of metal particles) are fixed to resin particles.

[0082] 1 is a cross-sectional view showing an example of a metal-resin composite 100, and the metal-resin composite will be described with reference to this figure. The metal-resin composite 100 includes resin particles 10 and metal particles 20.

[0083] (Metal-resin composite structure) In a metal-resin composite 100 (or a metal-resin composite, hereinafter the same applies to those having the reference numerals shown in FIG. 1 ), metal particles 20 are dispersed or fixed in resin particles 10. In the example of FIG. 1 , it is preferable that in the metal-resin composite 100, some of the metal particles 20 are three-dimensionally distributed in the surface layer portion 60 of the resin particles 10, and some of the three-dimensionally distributed metal particles 20 are partially exposed to the outside of the resin particles 10, and the remaining part is encapsulated in the resin particles 10.

[0084] Here, the "surface layer" refers to the range from the surface of the resin particle 10 to 50% of the particle radius in the depth direction. Furthermore, the "three-dimensionally distributed" means that the metal particles 20 are dispersed not only in the plane direction of the resin particle 10 but also in the depth direction.

[0085] Here, the metal particles 20 may include one or more types selected from metal particles completely encapsulated in the resin particles 10 (hereinafter also referred to as "encapsulated metal particles 30" or "second particles"), metal particles having a portion embedded in 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 to the surface of the resin particles 10 (hereinafter also referred to as "surface-adsorbed metal particles 50").

[0086] The entire surface of the encapsulated metal particles 30 is covered with the resin that constitutes the resin particles 10 .

[0087] The partially exposed metal particles 40 are, for example, those in which 5% or more and less than 100% of their surface area is covered with the resin that constitutes the resin particles 10. From the viewpoint of durability as a labeling substance, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. The surface-adsorbing metal particles 50 are, for example, those in which more than 0% and less than 5% of their surface area is covered with the resin that constitutes the resin particles 10.

[0088] For example, when the metal-resin composite 100 is used in the present invention, an antibody is immobilized on the surface of the resin particle 10, the surface of the partially exposed metal particle 40, or the surface of the surface-adsorbed metal particle 50. In this case, for example, the antibody may be immobilized on the partially exposed metal particle 40 and the surface-adsorbed metal particle 50, but not on the internally encapsulated metal particle 30. However, because all of the metal particles 20, including the internally encapsulated metal particle 30, exhibit localized surface plasmon absorption, not only the partially exposed metal particle 40 and the surface-adsorbed metal particle 50 but also the internally encapsulated metal particle 30 contribute to improving the visibility of the labeled substance.

[0089] Furthermore, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a larger contact area with the resin particles 10 than the surface-adsorbed metal particles 50, and in addition, they have a strong physical adsorption force, such as an anchor effect, due to their embedded state, making them less likely to detach from the resin particles 10. Therefore, the metal-resin composite 100 can be used as a labeling substance with excellent durability and stability.

[0090] Furthermore, the amount of metal particles 20 (the 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 within a 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 is particularly excellent in visibility, visual determination, and detection sensitivity as a labeling substance. The amount of metal particles 20 supported is more preferably within a range of 15% to 70% by weight.

[0091] Furthermore, it is preferable that 10% to 90% by weight of the metal particles 20 are 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 more preferable that 20% to 80% by weight of the metal particles 20 are partially exposed metal particles 40 and surface-adsorbed metal particles 50.

[0092] It is also 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, which is preferable because it ensures a sufficient amount of antibody immobilized on the metal particles 20 and increases 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.

[0093] (resin particles) The resin particles (resin constituting the resin particles) 10 are not particularly limited, and known resins (e.g., polystyrene, polyvinyl alcohol, poly(sodium p-styrenesulfonate), melamine resin, urea resin, acrylic resin, cellulose, etc.) can be used. The resin particles 10 are preferably polymer particles whose structure includes a substituent capable of adsorbing metal ions. Nitrogen-containing polymer particles are particularly preferred. Nitrogen atoms in nitrogen-containing polymers are preferred because they have excellent visibility and easily 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 resulting metal particles 20 become encapsulated metal particles 30 or partially exposed metal particles 40.

[0094] Furthermore, polymers having functional groups such as carboxylic acid, such as acrylic acid polymers, can adsorb cationic metal ions, which are precursors of metal particles such as silver, nickel, copper, gold, platinum, and palladium, and are therefore capable of forming metal particles 20 of silver, nickel, copper, gold, platinum, and palladium, and are also capable of forming alloys with any of the above metals. Furthermore, when the polymer has a substituent in the side chain that adsorbs cationic metal ions, it can be used in a wide range of materials, such as phenolic resins, epoxy resins, cellulose resins, and melamine resins.

[0095] The nitrogen-containing polymer is a resin having nitrogen atoms in the main chain or side chain, such as polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, polyaniline, etc. Preferred are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. Furthermore, when a nitrogen atom is present in the side chain, a wide range of resins can be used, such as acrylic resins, phenolic resins, epoxy resins, cellulose resins, and melamine resins.

[0096] In addition, in the case of resin particles that do not have a substituent in their structure that can adsorb metal ions, such as unsubstituted polystyrene particles, metal particles can be fixed to the particle surface by irradiating the particle surface with ultrasound or ionizing radiation, metal plating the resin particle surface, or the like.

[0097] (metal particles) The structure of the metal particles 20 is not particularly limited, but those that exhibit color when immobilized on the resin particles 10 as a metal-resin composite 100 are preferred because this improves the detection sensitivity (visual determination) as a labeling substance.

[0098] For example, silver, nickel, copper, gold, platinum, and palladium can be used as the material of the metal particles 20. These metals can be used alone or in the form of a composite such as an alloy.

[0099] Gold, platinum, and palladium are preferred because they have excellent visibility and are easy to immobilize antibodies on, and these develop color due to absorption resulting from localized surface plasmon resonance. Gold and platinum are more preferred because of their excellent storage stability.

[0100] Furthermore, from the viewpoint of obtaining excellent color development when bound to an antibody, gold, platinum, a gold alloy, or a platinum alloy is the most preferred metal species.

[0101] Here, the term "gold alloy" refers to an alloy made of, for example, gold and metal species other than gold, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more of gold, while the term "platinum alloy" refers to an alloy made of, for example, platinum and metal species other than platinum, containing 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more of platinum.

[0102] Furthermore, the average particle diameter of the metal particles 20 measured by scanning electron microscope (SEM) observation is preferably within 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 be expressed, and sensitivity tends to decrease. The average particle diameter of the metal particles 20 is preferably within 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 within the range of 1 nm or more and less than 50 nm.

[0103] The average particle diameter of the metal-resin composite 100 is, for example, within a range of 30 to 1000 nm (e.g., 50 to 1000 nm, 100 to 1000 nm). If the average particle diameter is too small (e.g., less than 30 nm), for example, when gold particles or platinum particles are used as the metal particles 20, the amount of metal particles 20 supported tends to be small, resulting in weaker coloring than gold particles of the same size. On the other hand, if the average particle diameter is too large (e.g., greater than 1000 nm), when used as a labeled antibody, the pores of a chromatographic medium such as a membrane filter tend to clog, and dispersibility tends to decrease.

[0104] The average particle size 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. Here, the particle size of the metal-resin composite 100 means the value obtained by adding the particle size of the resin particles 10 to the length of the protruding portions of the partially exposed metal particles 40 or the surface-adsorbed metal particles 50, and can be measured by laser diffraction / scattering, dynamic light scattering, or centrifugal sedimentation.

[0105] The method for producing the metal-resin composite 100 is not particularly limited. For example, a solution containing metal ions is added to a dispersion of resin particles 10 produced by emulsion polymerization, causing the metal ions to be adsorbed onto the resin particles 10 (hereinafter referred to as "metal ion-adsorbed resin particles"). Next, the metal ion-adsorbed resin particles are 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) or the like as the solution containing the metal ions. Furthermore, when platinum particles are used as the metal particles 20, it is preferable to use an aqueous solution of chloroplatinic acid (HPtCl6) or the like as the solution containing the metal ions. Note that a metal complex may be used instead of the metal ions.

[0106] In addition, instead of water, hydrous alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, and t-butanol, or alcohols, or acids such as hydrochloric acid, sulfuric acid, and nitric acid may be used as the solvent for the solution containing metal ions.

[0107] If necessary, additives such as water-soluble polymers such as polyvinyl alcohol, surfactants, alcohols, ethers such as tetrahydrofuran, diethyl ether, and diisopropyl ether, polyols such as alkylene glycol, polyalkylene glycol, their monoalkyl ethers or dialkyl ethers, and glycerin, and various water-miscible organic solvents such as ketones such as acetone and methyl ethyl ketone may be added to the solution containing metal ions. Such additives are effective in accelerating the reduction reaction rate of the metal ions and controlling the size of the generated metal particles 20.

[0108] The reducing agent is not particularly limited, but examples thereof include sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, hydrazine sulfate, and Rochelle salt. Among these, sodium borohydride, dimethylamine borane, and citric acid are more preferred. A surfactant may be added to the reducing agent solution, or the pH of the solution may be adjusted, as necessary. The pH can be adjusted using a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide. Furthermore, the particle size of the metal particles 20 formed can be controlled by adjusting the reduction rate of the metal ions through the temperature of the reducing agent solution.

[0109] Furthermore, when reducing the metal ions in the metal ion-adsorbing resin particles to produce metal particles 20, the metal ion-adsorbing resin particles may be added to a reducing agent solution, or the reducing agent may be added to the metal ion-adsorbing resin particles, but the former is preferred from the viewpoint of ease of producing encapsulated metal particles 30 and partially exposed metal particles 40.

[0110] Furthermore, in order to maintain the dispersibility of the metal-resin composite 100 in water, a dispersant such as citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, or DISPERBYK184 (manufactured by BIGG-Chemie Japan) may be added. Furthermore, the dispersibility can be maintained by adjusting the pH with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.

[0111] (Mode of Labeled Antibody, Production Method, etc.) The labeled antibody is an antibody that specifically binds to an analyte and is labeled with a resin-metal complex (a composite of metal particles and resin particles).

[0112] Specifically, such a labeled antibody may be one in which the antibody is adsorbed (bound) (immobilized) onto the surface of a metal-resin complex (for example, resin particles and / or metal particles).

[0113] As mentioned above, the antibody that can be used here is an antibody or a fragment thereof that binds to the protein that constitutes the SARS-CoV-2 nucleocapsid. In particular, for labeled antibodies, it is preferable to use an antibody or a fragment thereof that binds to the protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment is at positions 208 to 222 and / or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1.

[0114] The labeled antibody can be produced by a conventional method, and a representative method will be explained below.

[0115] ·Antibody binding process First, the antibody may be mixed with the metal-resin complex to bind (adsorb, fix, attach) the antibody to the metal-resin complex (antibody binding step).

[0116] In such an antibody binding step, the antibody and the metal-resin complex may be mixed in the presence of a solvent [water or the like, particularly a buffer solution (binding buffer solution)].

[0117] The binding between the metal-resin composite and the antibody in this step may be a so-called physical bond, or the antibody may be bound to the surface of the metal particles and / or the surface of the resin particles in the metal-resin composite by physical interaction. Examples of the bond by physical interaction include electrostatic bonding, hydrogen bonding, hydrophilic-hydrophobic interaction, and van der Waals forces.

[0118] When a binding buffer is used, the type of binding buffer is not particularly limited, but examples include inorganic buffers such as boric acid, carbonates, and phosphates, and organic buffers such as Tris and Good's Buffer. The pH of the buffer is preferably about pH 5 to 10 to allow antibody binding.

[0119] In the antibody binding step, the antibody and the metal-resin complex are mixed in the presence of a solvent (particularly a binding buffer solution) and stirred thoroughly to obtain a labeled antibody dispersion.

[0120] In this step, for example, the antibody and the metal-resin complex may be bound in a solvent (particularly a binding buffer solution). Alternatively, the antibody may be dispersed in a solvent (particularly a binding buffer solution) and then the metal-resin complex added. Dispersion can be performed by known methods, but it is preferable to use a dispersing means such as ultrasonic treatment, stirring with a rotator, or a vortex mixer. The labeled antibody dispersion thus obtained can be separated into solid portions by solid-liquid separation means such as centrifugation.

[0121] The method for producing a labeled antibody of this embodiment can further include the following steps, if necessary.

[0122] Blocking process The blocking step is a step in which the labeled antibody is dispersed in a blocking buffer, and the labeled antibody obtained in the antibody binding step is dispersed in a blocking buffer to perform blocking, which suppresses nonspecific adsorption to the labeled antibody.

[0123] As the blocking buffer, it is preferable to use, for example, a solution of a protein that does not bind to the analyte. Examples of proteins that can be used in 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 ultrasonication or stirring with a rotator. In this way, a labeled antibody dispersion in which the labeled antibody is uniformly dispersed can be obtained. From this labeled antibody dispersion, only the labeled antibody can be separated as a solid portion using solid-liquid separation means such as centrifugation. If necessary, any steps such as a washing step, a preservation step, etc. will be carried out. The washing step and the preservation step will be described below.

[0124] Cleaning process The washing treatment is a process in which a washing buffer solution is added to the labeled antibody separated by solid-liquid separation means, and the labeled antibody is uniformly dispersed in the washing buffer solution. For dispersion, a dispersion method such as ultrasonic treatment is preferably used. The washing buffer solution is not particularly limited, but examples include Tris buffer (trishydroxymethylaminomethane buffer), glycinamide buffer, arginine buffer, etc., adjusted to a predetermined concentration within the pH range of 6 to 9. The pH of the washing buffer solution can be adjusted using, for example, hydrochloric acid, sodium hydroxide, etc. The washing treatment of the labeled antibody can be repeated multiple times as necessary.

[0125] Conservation treatment The preservation treatment involves adding a preservation buffer to the labeled antibody separated by solid-liquid separation means and uniformly dispersing the labeled antibody in the preservation buffer. Dispersion is preferably performed using a dispersing method such as ultrasonication. The preservation buffer may be, for example, a solution prepared by adding a predetermined concentration of an anti-aggregating agent and / or stabilizer to the washing buffer. Anti-aggregating agents include, for example, sugars such as sucrose, maltose, lactose, and trehalose, and polyhydric alcohols such as glycerin and polyvinyl alcohol. Stabilizers include, but are not limited to, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin. The labeled antibody can be preserved in this manner.

[0126] In each of the above steps, a surfactant or a preservative such as sodium azide or parahydroxybenzoic acid ester may be further used as needed.

[0127] When a reaction part (labeled antibody) is provided on the immunochromatographic strip, the form and method of forming the reaction part (labeled antibody) are not particularly limited and are as described above.

[0128] [Capture ligand] The capture ligand is an antibody (or fragment thereof) that specifically binds to the analyte.

[0129] As mentioned above, the antibody (or fragment thereof) that can be used here is an antibody or fragment thereof that binds to the protein that constitutes the SARS-CoV-2 nucleocapsid. In particular, the capture ligand may be an antibody or fragment thereof that binds to the protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment is located at positions 374 to 397 of the amino acid sequence shown in SEQ ID NO: 1 (particularly, it may be used in combination with the above-mentioned labeled antibody, i.e., an antibody or fragment thereof that binds to the protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment is located at positions 208 to 222 and / or 332 to 351 of the amino acid sequence shown in SEQ ID NO: 1).

[0130] The form and method of forming the determination part (capture ligand) are not particularly limited and are as described above.

[0131] <Kit and analyte detection method>

[0132] The kit of the present invention is not particularly limited as long as it uses an immunochromatographic strip (test strip) [the above-mentioned immunochromatographic strip (test strip)] (or a component of an immunochromatographic strip).

[0133] In one embodiment, the kit may include (combines) a test strip that does not include a labeled antibody (or a reaction site), and the labeled antibody. The labeled antibody may be in the form of a reagent (detection reagent) that includes the labeled antibody.

[0134] In one embodiment (another embodiment) of the kit, 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.

[0135] Furthermore, by using the immunochromatographic strip (test strip) (or kit) of the present invention, an analyte (an analyte derived from SARS-CoV-2) can be detected or quantified.

[0136] Therefore, the present invention also includes such detection or quantification methods (immunoassay methods).

[0137] Such a method is not particularly limited, and a conventional method can be used depending on the embodiment of the test strip (or kit), and may include, for example, the following steps (I) to (III). Step (I): A step of contacting (e.g., contacting in a reaction section) an analyte contained in a sample (a sample containing the analyte) with a labeled antibody. Step (II): A step of contacting the complex containing the analyte and the labeled antibody formed in step (I) with a capture ligand (for example, in a test zone). Step (III): A step of measuring the color intensity of the resin-metal composite (for example, the color intensity resulting from light energy absorption due to localized surface plasmon resonance and electron transition).

[0138] The sample used in step (I) may be the sample described above, or may be diluted with (or have added to) the developing solution (or specimen treatment solution) described below.

[0139] When a test strip (or kit) that does not contain a labeled antibody (or a reaction site) is used, step (I) may be performed inside or outside the test strip.

[0140] For example, after carrying out step (I) by contacting the analyte in the sample with the labeled antibody in the detection reagent, the sample may be applied to an appropriate portion of the test strip (a reaction portion not containing the labeled antibody, a sample addition portion, etc.), and steps (II) and (III) may be carried out sequentially.

[0141] According to such a method, when a sample (analyte) contains an analyte (antigen) derived from SARS-CoV-2, the analyte (analyte derived from SARS-CoV-2) can be detected or quantified.

[0142] In such samples, the concentration of the SARS-CoV-2-derived analyte (antigen) is not particularly limited, but may be a low concentration, such as a recombinant protein antigen concentration of 0.1 ng / ml or less, or a viral load of 500 copies / test or less. In the present invention, even such low concentrations can be efficiently detected or quantified.

[0143] [Developing solution] The developing solution (or specimen treating solution) may generally contain a surfactant, and may be an aqueous solution containing a surfactant. One or more surfactants may be used.

[0144] The surfactant is preferably a non-ionic surfactant. Examples of nonionic surfactants include polyoxyethylene (POE) surfactants (or surfactants having a polyoxyethylene chain) (e.g., ether surfactants, ester-ether surfactants, etc.).

[0145] Among nonionic surfactants such as POE-type surfactants, POE alkyl phenyl ethers may be preferred, from the viewpoint that immunochromatographic test strips can easily achieve sufficient detection sensitivity and can easily suppress false positives (and even false negatives).

[0146] In the POE alkyl phenyl ethers, the alkyl group is, 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.). The alkyl group may be a straight-chain alkyl group or a branched alkyl group. Representative examples of POE alkylphenyl ethers include POE octylphenyl ether, POE nonylphenyl ether, and POE dodecylphenyl ether.

[0147] In a POE surfactant (e.g., POE alkylphenyl ethers, etc.), the average number of moles of ethylene oxide added 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.), or may be, for example, 50 or less (e.g., 40 or less, 30 or less, 20 or less, etc.).

[0148] The HLB value of the polyoxyethylene surfactant is not particularly limited, but may be, for example, 5 to 30.

[0149] The concentration of the surfactant in the developing solution is not particularly limited, but may be, for example, 0.1 wt% or more, preferably 0.5 wt% or more, from the viewpoint of making it easier for the immunochromatographic test strip to achieve sufficient detection sensitivity and to suppress false positives (and even false negatives), or may be, for example, 5 wt% or less, preferably 2 wt% or less.

[0150] The developing liquid may contain other components in addition to the surfactant and water. The other components may be components commonly used in sample processing solutions. Other components include, but are not limited to, buffers, salts (sodium chloride), blocking agents (eg, BSA, sodium caseinate, etc.), preservatives, and the like. The developing solution may contain one or more other components.

[0151] The developing solution can be prepared by mixing a surfactant and water (and other ingredients).

[0152] As the buffer, known buffers may be used, such as Tris buffer, glycinamide buffer, arginine buffer, phosphate buffer, and the like. One or more buffers may be used.

[0153] The concentration of the buffer in the developing solution is not particularly limited, but from the viewpoint of pH stability, etc., 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.

[0154] The concentration of sodium chloride in the developing solution is not particularly limited, but may be, for example, 0.5 to 2 wt % (85 mM to 342 mM) from the viewpoint of having a composition similar to that of physiological saline.

[0155] Examples of blocking agents include bovine serum albumin (BSA) and milk-derived blocking agents (eg, casein, sodium caseinate, skim milk, Block Ace, etc.). One or more blocking agents may be used.

[0156] The concentration of the blocking agent in the developing solution is not particularly limited, but may be, for example, 0.1 wt% or more, preferably 0.2 wt% or more, from the viewpoint of making it easier for the immunochromatographic test strip to achieve sufficient detection sensitivity, or may be, for example, 2 wt% or less, preferably 1 wt% or less.

[0157] As the preservative, known preservatives may be used, such as sodium azide and Proclin (registered trademark). One or more preservatives may be used.

[0158] The concentration of the preservative in the developing solution is not particularly limited, but may be, for example, about 0.01 to 0.1 wt %.

[0159] The pH of the developing solution may be, for example, 7 to 9, from the viewpoint that the immunochromatographic test strip can easily achieve sufficient detection sensitivity.

[0160] The developer solution described above can be used to dilute a sample (a sample containing an analyte derived from SARS-CoV-2). A specimen solution can be prepared by diluting the sample with the developer solution (or by adding the developer solution to the sample).

[0161] That is, the present invention also encompasses a developing solution for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample. Such a developing solution may be for combination with the above-mentioned immunochromatographic test strip (a test strip containing a labeled antibody), or for combination with the above-mentioned immunochromatographic test strip (a test strip not containing a labeled antibody) and a labeled antibody. [Example]

[0162] Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. In the following examples and comparative examples, various measurements and evaluations were carried out as follows, unless otherwise specified.

[0163] <Antibody> The following antibodies were used:

[0164] Antibody B: Anti-coronavirus (SARS-CoV-2) antibody B, Kanto Chemical, product number 02126-67. The epitope recognized by this antibody is located at positions 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1.

[0165] Antibody D: Anti-coronavirus (SARS-CoV-2) antibody D, Kanto Chemical, product number 02128-67. The epitope recognized by this antibody is located at positions 374 to 397 of the amino acid sequence represented by SEQ ID NO: 1.

[0166] Antibody G1: An antibody produced according to the method described in paragraphs

[0051] to

[0065] of Patent Document 2, the epitope recognized by this antibody is located at positions 1 to 207 of the amino acid sequence represented by SEQ ID NO: 1.

[0167] Antibody G2: An antibody produced according to the method described in paragraphs

[0051] to

[0065] of Patent Document 2, the epitope recognized by this antibody is located at positions 1 to 207 of the amino acid sequence represented by SEQ ID NO: 1.

[0168] <Absorbance measurement of metal-resin composite particles> The absorbance of the metal-resin composite particles was measured by placing a 0.01 wt% (weight %) metal-resin composite particle dispersion (dispersion medium: water) in a quartz glass cell (light path length 10 mm) and using a spectrophotometer (Shimadzu Corporation, UV3600) to measure the absorbance at 570 nm for the gold-resin composite and at 400 nm for the platinum-resin composite.

[0169] <Measurement of solid concentration and metal loading amount> 1 g of the dispersion before concentration adjustment was placed in a porcelain crucible and dried for 3 hours at 70° C. The weight was measured before and after drying, and the solid content was calculated using the following formula. Solid content concentration (wt%)= [Weight after drying (g) / Weight before drying (g)] x 100 The sample after the drying treatment was further heat treated at 500° C. for 5 hours, and the weights before and after the heat treatment were measured, and the amount of supported metal was calculated using the following formula. Metal loading amount (wt%) = [Weight after heat treatment (g) / Weight before heat treatment (g)] x 100

[0170] <Measuring the average particle size of metal particles> A substrate was prepared by dropping a metal-resin composite particle dispersion onto a metal mesh with a carbon support film, and the area-average diameter of 100 random metal particles was measured from the image observed using a field emission scanning electron microscope (FE-SEM; Hitachi High-Technologies Corporation, SU-9000). This was used as the average particle diameter.

[0171] <Measurement of the average particle size of resin particles and resin composite particles> Measurement was performed using a disc centrifugal particle size distribution analyzer (CPS Disc Centrifuge DC24000 UHR, manufactured by CPS Instruments, Inc.) The measurement was performed with the resin particles or resin composite particles (metal-resin composite particles) dispersed in water.

[0172] [Production Example 1] <Synthesis of resin particles> Trioctylammonium chloride (1.3 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) were dissolved in 300 g of pure water, followed by the addition of 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g). The mixture was stirred under a nitrogen stream at 30°C for 50 minutes, then 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 stirred at 60°C for 3.5 hours to obtain resin particles A-1 with an average particle size of 349 nm. The resulting mixture was precipitated by centrifugation (9000 rpm, 40 minutes). The supernatant was removed, and the resulting mixture was redispersed in pure water and dialyzed to remove impurities. The concentration was then adjusted to obtain a 10 wt% resin particle dispersion B-1.

[0173] [Production Example 2] <Synthesis of platinum-resin composite particles> 85.0 g of pure water was added to 30.0 g of resin particle dispersion B-1, followed by 31.0 g of 7 wt% aqueous chloroplatinic acid solution and stirring at room temperature for 3 hours. This mixture was centrifuged (3000 rpm, 30 minutes) to precipitate the resin particles, and the supernatant was removed to remove excess chloroplatinic acid. The concentration was then adjusted to obtain 5 wt% platinum ion-adsorbing resin particle dispersion C-1.

[0174] Next, 70.5 g of C-1 was added to 4725 g of pure water, and 137 g of a 132 mM dimethylamine borane aqueous solution was added dropwise while stirring at 3°C. The mixture was then stirred at room temperature for 3 hours to obtain platinum-resin composite particles D-1 with an average particle size of 360 nm. D-1 was concentrated by centrifugation and then purified by dialysis to adjust the concentration, resulting in a 1 wt% platinum-resin composite particle dispersion (PtNCP) E-1. The absorbance of platinum-resin composite particles F-1 in E-1 was 1.75. The average particle size of the platinum particles in F-1 was 3.5 nm, and the platinum loading was 39.1 wt%.

[0175] <Saliva sample collection> The tip of the tongue was pressed against the roof of the mouth to collect saliva on the lower jaw. A cotton swab was immersed in the saliva for at least 40 seconds, and then rotated at least five times to collect saliva.

[0176] [Example 1] (Antibody binding process) 25 μg of antibody B {anti-novel coronavirus (SARS-CoV-2) antibody B, Kanto Chemical, product number 02126-67} was mixed with 0.45 ml of 50 mM HEPES (pH 7), and then 0.05 ml of 1 wt % platinum-resin composite particle dispersion E-1 was added. The mixture was stirred by end-over-end at room temperature for 2 hours to obtain labeled antibody dispersion J-1 containing antibody B labeled with platinum-resin composite particles F-1.

[0177] (Blocking process) Next, the labeled antibody dispersion J-1 was centrifuged at 3000 rpm for 5 minutes, and after removing the supernatant, 0.5 ml of a 5 mM Tris aqueous solution (pH 7) containing 1 wt% sodium caseinate was added to the sediment. The mixture was ultrasonically dispersed, and then further stirred by end-over-end stirring at room temperature for 1 hour to obtain labeled antibody dispersion K-1.

[0178] (Cleaning process) Next, the labeled antibody dispersion K-1 was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and 0.5 ml of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% of a surfactant was added to the sediment, followed by ultrasonic dispersion. This procedure was repeated three times as a washing treatment.

[0179] (Preservation treatment) Next, after cooling on ice, the mixture was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and 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 sediment, followed by ultrasonic dispersion to obtain labeled antibody dispersion L-1.

[0180] (Conjugate pad preparation) 0.12 ml of labeled antibody dispersion L-1 was centrifuged at 3000 rpm for 5 minutes, the supernatant removed, and 0.333 ml of an aqueous solution (pH 8.0) containing 5 wt% sucrose and 2.5 wt% BSA was added to the sediment and ultrasonically dispersed to obtain labeled antibody dispersion M-1. A glass fiber nonwoven fabric was uniformly impregnated with labeled antibody dispersion M-1 and then dried at 50°C for 1 hour to produce conjugate pad N-1. The volume of labeled antibody dispersion M-1 was adjusted so that the content of platinum-resin composite particles F-1 in conjugate pad N-1 was 3 μg per test evaluated by the immunochromatography method described below.

[0181] (Preparation of immunochromatographic strips) An immunochromatographic strip with the structure shown in Figure 2 was prepared. First, antibody D (anti-coronavirus (SARS-CoV-2) antibody D, Kanto Chemical, product number 02128-67) was applied to a 25 mm wide nitrocellulose membrane 4 to create a test line 5. Additionally, an anti-mouse IgG antibody was applied downstream of the test line 5 to create a control line 6. After drying the nitrocellulose membrane 4 at 50°C for 1 hour, a laminate film 1, a conjugate pad N-1 (conjugate pad 3), a sample pad 2 (glass fiber nonwoven fabric), and an absorbent pad 7 (cotton nonwoven fabric) were layered on top of each other, as shown in the cross-sectional diagram of the immunochromatographic strip in Figure 2. Finally, the membrane was cut to a width of 3.5 mm to create the immunochromatographic strip P-1.

[0182] (Preparation of developing solution) An aqueous solution (pH 7.5) containing 100 mM Tris, 150 mM NaCl, 0.5 wt % sodium caseinate, and 1.0 wt % surfactant NP-40 was prepared and used as developing solution 1.

[0183] (Evaluation by immunochromatography) Positive control (virus concentrations 9.5x10^6, 2.37x10^6, 0.59x10^6 copies / ml) and negative control (virus-free) sample solutions were prepared by diluting the SARS-CoV-2 cultured virus BA.2 strain using Developer 1. Furthermore, the BA.2 strain of cultured virus was diluted with the developing solution 1 to the above three concentrations to prepare specimen solution 1. 50 μl of each control sample solution was dropped onto sample pad 2 of Immunochromato Strip P-1. After 10 minutes, the color intensity of test line 5 was measured using an Immunochromato Reader (Hamamatsu Photonics C10066-10). The evaluation results by immunochromatography are shown in Table 1.

[0184] [Examples 2 to 3] The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 1, except that the specimens (cultured viruses) listed in Table 1 were used. The immunochromatographic evaluation results are shown in Table 1.

[0185] [Example 4] The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 1, except that the capture antibodies listed in Table 1 were used. The immunochromatographic evaluation results are shown in Table 1.

[0186] [Examples 5 to 6] The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 1, except that the specimens (cultured viruses) and capture antibodies listed in Table 1 were used. The immunochromatographic evaluation results are shown in Table 1.

[0187] [Table 1]

[0188] In Examples 1 to 6, sufficient immunochromatographic sensitivity was obtained, and false positives were suppressed. In particular, the results of Examples 1 to 3 showed that when antibody B was used as the labeled antibody and antibody D was used as the capture antibody, immunochromatography was highly sensitive, especially for the cultured virus strain BA.2. Furthermore, the results of Examples 4 to 6 showed that when antibody B was used as the labeled antibody and antibody G1 was used as the capture antibody, immunochromatography was highly sensitive, particularly for the cultured virus strains BA.2.75 and BA5.

[0189] [Example 7] The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, and immunochromatographic strip preparation were carried out in the same manner as in Example 1, except that the saliva sample obtained above was used as the sample.

[0190] (Evaluation by immunochromatography) Sample solution 2 was prepared by diluting PCR-tested coronavirus-positive saliva samples (Ct values ​​18.99, 21.23, 23.09) and coronavirus-negative saliva samples 20 times using developer solution 1. 50 μl of each sample solution 2 was dropped onto sample pad 2 of immunochromatographic strip P-1. After 10 minutes, the color intensity of test line 5 was measured using an immunochromatographic reader (Hamamatsu Photonics C10066-10). The evaluation results by immunochromatography are shown in Table 2.

[0191] [Examples 8 to 9] The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, immunochromatographic strip preparation, and immunochromatographic evaluation were performed in the same manner as in Example 7, except that the capture antibodies shown in Table 2 were used. The immunochromatographic evaluation results are shown in Table 2.

[0192] [Example 10] Developing solution 2 was prepared in the same manner as developing solution 1, except that Tween 20 was used instead of NP-40. The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, and immunochromatographic strip preparation were carried out in the same manner as in Example 7, except that developing solution 2 was used as the developing solution. In addition, immunochromatographic evaluation was performed in the same manner as in Example 7, except that a novel coronavirus-positive saliva sample (Ct value 18.99) and a novel coronavirus-negative saliva sample were evaluated. The immunochromatographic evaluation results are shown in Table 2.

[0193] [Example 11] Developing solution 3 was prepared in the same manner as developing solution 1, except that polyoxyethylene (20) cetyl ether was used instead of NP-40. The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, and immunochromatographic strip preparation were carried out in the same manner as in Example 7, except that developing solution 3 was used as the developing solution. In addition, immunochromatographic evaluation was performed in the same manner as in Example 7, except that a novel coronavirus-positive saliva sample (Ct value 18.99) and a novel coronavirus-negative saliva sample were evaluated. The immunochromatographic evaluation results are shown in Table 2.

[0194] [Reference examples 1~3] The binding step, blocking step, washing treatment, preservation treatment, conjugate pad preparation, and immunochromatographic strip preparation were carried out in the same manner as in Example 7, except that the labeled antibodies and capture antibodies shown in Table 2 were used. In addition, immunochromatographic evaluation was performed in the same manner as in Example 7, except that a novel coronavirus-positive saliva sample (Ct value 18.99) and a novel coronavirus-negative saliva sample were evaluated. The immunochromatographic evaluation results are shown in Table 2.

[0195] [Table 2]

[0196] Although the embodiments of the present invention have been described in detail above for the purpose of illustration, the present invention is not limited to the above-described embodiments. [Industrial Applicability]

[0197] According to the present invention, a novel immunochromatographic strip and the like can be provided. [Explanation of symbols]

[0198] 1...Support (laminate film), 2...Sample pad, 3...Conjugate pad, 4...Membrane (nitrocellulose membrane, etc.), 5...Test line, 6...Control line, 7...Absorbent pad, 10...Resin particle, 20...Metal particle, 30...Encapsulated metal particle, 40...Partially exposed metal particle, 50...Surface-adsorbed metal particle, 60...Surface layer, 100...Metal-resin composite

Claims

1. An immunochromatographic test strip for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: a detection unit provided with a capture ligand that specifically binds to an analyte; a reaction section, located upstream of the determination section in the direction of sample development, which is provided with a labeled antibody, which specifically binds to the analyte and is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1. Test strips.

2. An immunochromatographic test strip for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: a detection unit provided with a capture ligand that specifically binds to an analyte; a reaction section, located upstream of the determination section in the direction of sample development, which is provided with a labeled antibody, which specifically binds to the analyte and is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1. A kit using test strips.

3. An immunochromatographic test strip for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: a detection unit provided with a capture ligand that specifically binds to an analyte; a reaction section, located upstream of the determination section in the direction of sample development, which is provided with a labeled antibody, which specifically binds to the analyte and is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1. A test strip; A kit using a developing solution containing a surfactant including polyoxyethylene alkylphenyl ethers.

4. An immunochromatographic test strip for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, a test strip including a detection zone having a capture ligand that specifically binds to an analyte; a labeled antibody obtained by labeling an antibody that specifically binds to an analyte with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; A kit using a developing solution containing a surfactant including polyoxyethylene alkylphenyl ethers, the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1. kit.

5. A method for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: a detection unit provided with a capture ligand that specifically binds to an analyte; and a reaction zone provided upstream of the test zone in the direction of sample development with a labeled antibody, the labeled antibody being an antibody that specifically binds to an analyte, and the labeled antibody being an antibody that is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle. The immunochromatographic test strip is used, and the following steps (I) to (III) are carried out: Step (I): contacting an analyte contained in a sample with a labeled antibody; Step (II): contacting the complex comprising the analyte and the labeled antibody formed in step (I) with a capture ligand; Step (III): A step of measuring the color intensity of the resin-metal composite; Including, the sample contains a developer containing a surfactant containing polyoxyethylene alkylphenyl ethers, the antibody (the antibody in the labeled antibody) is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1. method.

6. A method for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: An immunochromatographic test strip is used, which includes a test zone provided with a capture ligand that specifically binds to an analyte. The following steps (I) to (III): Step (I): A step of contacting an analyte contained in a sample with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; Step (II): contacting the complex comprising the analyte and the labeled antibody formed in step (I) with a capture ligand; Step (III): A step of measuring the color intensity of the resin-metal composite; Including, the sample contains a developer containing a surfactant containing polyoxyethylene alkylphenyl ethers, the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; The capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1. method.

7. A developing solution for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: A surfactant containing polyoxyethylene alkylphenyl ethers is included, a detection unit provided with a capture ligand that specifically binds to an analyte; a reaction section, located upstream of the determination section in the direction of sample development, which is provided with a labeled antibody, which specifically binds to the analyte and is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle; the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; A developing solution to be combined with an immunochromatographic test strip, wherein the capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1.

8. A developing solution for detecting or quantifying an analyte derived from SARS-CoV-2 contained in a sample, comprising: A surfactant containing polyoxyethylene alkylphenyl ethers is included, An immunochromatographic test strip including a test portion having a capture ligand that specifically binds to an analyte; A developing solution for combining an antibody that specifically binds to an analyte with a labeled antibody that is labeled with a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle, comprising: the antibody is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid, and the epitope recognized by the antibody or the fragment thereof is located at positions 208 to 222 or 332 to 351 of the amino acid sequence represented by SEQ ID NO: 1; A developing solution in which the capture ligand is an antibody or a fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid, and the epitope recognized by the antibody or fragment thereof is located at positions 1 to 207 or 374 to 397 of the amino acid sequence represented by SEQ ID NO:

1.

9. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the sample comprises saliva.

10. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the polyoxyethylene alkylphenyl ethers include polyoxyethylene linear alkylphenyl ether.

11. The test strip, kit, method, or developing solution according to any one of claims 1 to 8, wherein the antibody and the capture ligand are monoclonal antibodies.

12. 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, or an alloy thereof.

13. In the resin-metal composite, a first particle having a metal particle exposed outside the resin particle; second particles entirely encapsulated in the resin particles; It contains 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 three-dimensionally distributed in the surface layer portion of the resin particles.

14. 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 a substituent in their structure capable of adsorbing metal ions.

15. 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 within the range of 1 to 100 nm.

16. The 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 within the range of 30 to 1000 nm.

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